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ANY

THE JOURNAL

OF

COMPARATIVE NEUROLOGY

EDITORIAL BOARD

Henry H. Donaupson ApoLF MEYER

The Wistar Institute Johns Hopkins University
J. 3. JoHNSTON OuiverR S. STRONG

University of Minnesota Columbia University

C. JuDsoNn HERRICK, University of Chicago
Managing Editor

VOLUME 27

1916-1917

PHILADELPHIA, PA.

THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY

COMPOSED AND PRINTED AT THE
WAVERLY PRESS
By tHe Witiiams & WILKINS CoMPANY
BaAvtTimorE, Mp., U.S. A.

CONTENTS

1916-1917

No. 1. DECEMBER, 1916

De DUCATI ON Pane eee teehee nse Vn NARS oer \cldcire Oat Week ay yeep Ss HUNG GSC Suontieee ace ouctea® One
SURARISA 1Bssonigs (CVAGin Sleiselbn IGE NGG ogo ooo bce roe ooo eae maoG oo eso gC
F. L. Lanpacre. The cerebral ganglia and early nerves of Squalus acanthias. Thirteen

W.M. Smatutwoop Aanp Ruru L. Puinuirs. Nuclear size in the nerve cells of the bee
dunmeethemliteccy.clets (One siourees ee. tree ats sc dtens sree aia ahs of ai Sea) <usie cy coneapepecoe

Henry H. Donatpson. A revision of the percentage of water in the brain and in the
sSpinalecord of ptheyalbinograt: + (One wchanrts 11 4..cceeuia so 2 elec ie «ete beueereeee s

No. 2. FEBRUARY, 1917

GerorGE E. Nicuouus. Some experiments on the nature and function of Reissner’s
HET aM yatVe ME OUTESE.... ase Rte emis Mee coe ey Che Se te Se hk on eae

CarouineE M. Hott. Studies on the olfactory bulbs of the albino rat—in two parts.
I. Effect of a defective diet and of exercise. Il. Number of cells in bulb. Four
| OUEEH EC oan pe Sek EN ie acts ec a BoM RES PNM RP aC as A ie RE a UR a

Now 3: APR:

C. U. Artins Kaprrers. Further contributions on neurobiotaxis. IX. An attempt to
compare the phenomena of neurobiotaxis with other phenomena of taxis and tropism.
The dynamic polarization of the neurone. Six figures

Davip H. Dotiey. Further verification of functional size changes in nerve cell bodies
byrhesuse of the polar planimeter4 “Three fisuress- 0.0.2.1... 56a Jalen. ae.

ELIzABETH CAROLINE CrosBy. The forebrain of Alligator mississippiensis. Forty-six

M. J. GReenNMAN. The number, size and axis-sheath relation of the large myelinated
fibers in the peroneal nerve of the inbred albino rat—under normal conditions, in
disease and after stimulation

No. 4. JUNE

Davenport Hooker. Studies on regeneration in the spinal cord. II. The effect
of reversal of a portion of the spinal cord at the stage of the closed neural folds
on the healing of the cord wounds, on the polarity of the elements of the cord and
on the behavior of frog embryos. Nine figures

1

117

201

403

1V CONTENTS

Simon H. Gace. Glycogen in the nervous system of vertebrates. Ten figures (one
plate) o.c aes peatk d eee Nee yest ener ey RE = ela? oo Ae SP ve ee 451
Davipson Buack. The motor nuclei of the cerebral nerves in phylogeny: a study of
the phenomena of neurobiotaxis. Part I. Cyclostomi and Pisces. Forty-two
TOUTES en roee erent Ci chs, hae ay eee aE te ee SN ee eae to RON 2 Gear 8 oe Rae Oe 467
EnizaABpetH Hopkins Dunn. Primary and secondary findings in a series of attempts
to transplant cerebral cortex in the albino rat. Hight figures................ OD

THIS VOLUME OF

THE JOURNAL OF COMPARATIVE NEUROLOGY

IS DEDICATED TO THE MEMORY OF

SUSANNA PHELPS GAGE

WHOSE CONTRIBUTIONS TO COMPARATIVE NEUROLOGY HAVE WON
AN HONORABLE PLACE IN THE PERMANENT STRUCTURE OF
THE SCIENCE, AND WHOSE PERSONALITY COM-
MANDED THE ESTEEM AND AFFECTION OF
ALL HER ASSOCIATES

SUSANNA PHELPS GAGH, PH.B.
DECEMBER 26, 1857: OcTOBER 5, 1915

Mrs. Gage was born and spent her childhood and early youth
in Morrisville, the county seat of Madison County, New York.

The father, Henry S. Phelps, was also born in Madison
County, but his father, John, and grandfather, Elijah Phelps,
came from New England where their ancestors were among the
earliest English settlers. The grandfather was a soldier of the
Revolutionary War. The father, John Phelps, died before the
children had reached manhood. This made it necessary for the
boy, who was to be the father of the subject of this sketch, to
play the part of a man early in life.

A part of his boyhood and youth were spent in Cortland
County in and near Cortland, but his mature life was lived in
the village of Morrisville where he was a leading merchant, and
a citizen respected and trusted by the community.

The mother, Mary Austin Phelps, was a native of Cortland
County, and like the father was of New England descent, with
Scottish as well as English blood. Her father was a prosperous
farmer and mill owner in the rich Cortland valley and could
give his children more of the comforts of life and especially the
advantages of the best education then available for young
women. But, as was the custom in those days, the education
was not alone in books but in the real work of the household and
the art of conducting a home, which could come only with a
perfect familiarity with all the work which makes such a home
possible. She early became a teacher, and had a genius for
starting young people on the road to learning; and it was a
sound start she gave them; no shirking was permitted and no
slipshod work accepted. She taught several years (1847-1854)
in the South, and saw that region from the inside in what seemed
its most prosperous period of slavery and chivalry.

5

6 SIMON HENRY GAGE

Mrs. Gage’s parents were then these two people with their
sound heredity and wholesome youthful training, and their
rather broad experiences. Naturally they were interested in
and upholders of the church, the schools, and all the other en-
terprises for the betterment of the community. While these
interior villages of the country could not all have colleges or
Seminaries for higher learning, they could have the lyceum on
whose stage came some of the best men of the country; and
her parents did their full share in supporting this institution.

With such a father and mother it seemed perfectly natural
that the child should have a sound character, and not at all sur-
prising that there was strong love of learning and a taste for
the finer things of life. While of course the father and mother
wished all good things for their daughter, they were not so
fatally unwise as to neglect the homely duties in education and
training, for they knew full well that the finer things of life
come only through the portals of labor and service; they knew
also that the labor was not the only purpose of life, only a
means to an end.

Morrisville, the birthplace and home, is in one of the beauti-
ful valleys so common in New York State. The surrounding
country is a rich farming region with its hills and upland pla-
teaus. On the north not far away are Utica and Syracuse,
and to the south is Binghamton. In the neighboring villages of
Hamilton and Clinton are Colgate and Hamilton colleges.

The home was in the midst of the village just across the
street from the school house and the churches, and the father’s
store was only a short walk down the street. Higher up on the
street stood the court house and other county buildings. ‘This
main street was a part of the once famous Cherry Valley Turn-
pike. Before her eyes then as she grew up were the physical
representations of transportation in the turnpike; law and goy-
ernment in the county buildings; education in the school house,
and the spiritual life in the churches. While the village in the
valley seemed in security and peace as if surrounded and pro-
tected by the everlasting hills, those same hills gave opportunity
for the wide view and the call that comes from mountain tops

SUSANNA PHELPS GAGE rf

to the larger world, and she loved to go where she could see
these wide views; and her subsequent career showed that she
heard also the call to the larger world beyond the hills.

The early education was like that of practically all children
in the more settled parts of New York State between 1860 and
1870,—the village school, with its variety of teachers, young
men aspiring to the ministry or the law for the winter terms and
young women also preparing for the real work of life in the sum-
mer. The difference, and it was a fundamental difference, lay
in the fact that the mother, with her fine instincts for teaching
and sound training, supplemented the regular school work and
saw to it that there was a thoroughness in the elements which
should lay the foundation for any attempts which time or cir-
cumstance might make possible; and in the mother’s mind was
the hope for some college training such as women were just com-
ing into the possibility of having at that time.

As the years advanced and the eager zest of the young woman
for learning and the better things of life manifested themselves
with her growth, the college decided on for her was Vassar, and
' preparation for entrance was undertaken at the then famous
Cazenovia Seminary. There she came in contact with some of
the high-minded advanced teachers who gave uplift in those
days to so many young men and women and showed the beauty
and the possibilities in the intellectual and spiritual life. She
was especially inspired by the teacher of Latin, Isaac N. Cle-
ments, later the head of the school. This man had had the
stern training of the Civil War and knew life and its savagery
as well as the blessed side represented by the gospel, of which
he was one of the ministers. -In the home of his one-time
pupil he recently told the husband and son of the enthusiasm,
fullness of life, and intellectual vigor of his former student.

As stated above, Vassar College had been in mind for the
young woman and was the choice of the mother. But the
father had been stirred by the accounts he had heard of Cornell
University. The father especially could understand and appre-
ciate the splendid promise of the new institution and was cap-
tivated by the dreams of Ezra Cornell for the education of the

8 SIMON HENRY GAGE

young men and women of the country; and the broad scope of |
that education as outlined by Mr. Cornell and President White
appealed to him who knew from his own experience in the world
the need of something in addition to a knowledge of the ancient
classics. Henry W. Sage had just built and given Sage College
for housing the women students of the University so that pro-
tection as well as education seemed cared for. The father who
had known almost pioneer life did not hesitate because the
institution was young, and perhaps a little rough; he knew by
experience the fundamental virtues residing in youth and
roughness. His wishes, aided by the adventurous spirit of the
daughter, prevailed, and he came with her to the University
which she entered in the autumn of 1875 As good fortune
would have it, when their carriage drove up to the entrance of
the newly finished Sage College, President White was there.
For as was his custom, he had been looking at this building,
as he always did at all growing buildings, to see if it was all
ready for the fine young women he felt sure would come to it
to receive the instruction already given men. He welcomed
the father and daughter and went into the building with them
and saw to it that food and a room were provided for the first
Sage College student.

The course pursued in college included a further study of
Latin, much English literature, and a large amount of historical
study. In the historical study two subjects seemed of para-
mount interest, Roman history and American constitutional
history. She also never failed to make the most of every op-
portunity to hear the lectures of Goldwin Smith on English his-
tory, and those of Andrew D. White on modern European history.

With these classical, literary, and historical studies came
studies in modern science, among which physics and biology
took the strongest hold upon her. She was the first woman to
take laboratory work in physics in Cornell University. The
facilities for laboratory work were limited, the only space being
under the raised seats of the lecture room. But as with all her
other teachers, Prof. Wm. A. Anthony, the founder of the de-
partment of physics at Cornell, believed so much in the ability
and earnestness of his aspiring pupil that he gave her space and

SUSANNA PHELPS GAGE 9

opportunity for laboratory work in his own cramped quarters.
Later he was often a guest in her home and seemed to rejoice
at the sacrifice he had made to give her opportunity to work
out for herself some of the fundamental things in physics. In
passing it may be said that she never had any trouble in convine-
ing her teachers that it was worth while giving her a chance to
do things. The animated face and honest gray eyes kindling
with enthusiasm were her passport everywhere.

It was not to be in physics that she was to do her intellectual
life work, however, but in the zoological side of biology. The
teaching staff in zoology and comparative anatomy at that time
was presided over by Prof. Burt G. Wilder for the vertebrate
side, and Prof. J. H. Comstock for the invertebrates, in which
entomology was the major interest. She took all the courses
offered by the two departments.

In 1881 occurred her marriage to Asst. Prof. Simon Henry
Gage. This gave opportunity for work and investigation in
biology. She made the most of her chance, entering at once
with full enthusiasm into the work of her husband, making
drawings for his papers, and wall diagrams for his courses, and
many also for Professor Wilder’s courses. But the naturally
independent mind could not long be satisfied as a mere helper;
there was a desire to undertake some original work on her own
account. .

At that time the form and relations of the fibers of striated
muscles were not well understood, and especially were they mis-
understood with small animals like mice and small birds. So
it became her first published scientific work to show what the
relations of the fibers really were in the small animals and later
in laboratory animals generally. So fundamental and con-
vincing was her work, and so clear the drawings accompanying
the text that the veteran KOlliker revising his Histology for
the sixth time, declared in his introduction that the literature
is now so large that he can only refer to the ‘ Allergewichte,’ and
gave her as authority for the statement with which he closes
the discussion of the form and length of the fibers in striated or
skeletal muscles (KGlliker’s Gewebelehre, Sechste Auflage, Bd.
I, p. 371). And ina letter from Dr. Minot who was giving some

10 SIMON HENRY GAGE

lectures in embryology at Cornell he says, concerning the muscle
work: “I was greatly interested in your wife’s muscle prepara-
tions which are convincing as to the great accuracy of her
observations.”

-As shown by the list of her publications at the end, her inde-
pendent work covered a considerable range, and she joined in
the preparation of papers upon a variety of subjects. But her
main work was in neurology, for as a student, and especially as
a member of Prof. Burt G. Wilder’s seminary, the nervous sys-
tem grew in fascination for her. It is not known by many,
perhaps, that as assistant to Louis Agassiz, Dr. Wilder was
urged by that great man to interest himself in the nervous sys-
tem. Finally the wisdom of Agassiz’ advice appealed strongly
to him and as early as the college year 1870-1871, Dr. Wilder
gave a special course in comparative neurology. In 1875 this
course in neurology became fully established in Cornell. As
stated in the introduction to the Wilder Quarter Century Book:
“Tt isin this course of neurology perhaps more than in any
other that is realized [in him] the picture drawn by Agassiz, in
his address at the inauguration of Cornell University, of the
teacher going before his class with his own thoughts and as an
elder brother inspiring his pupils to the most enthusiastic
effort.”

Certain it is that the inspiration for her was masterful, and
with growing maturity of thought, the phylogeny, development
and morphology of the nervous system was to her the supreme
question in biology.

Of her twenty-six independent publications, ten were upon
neurology. These were her most important papers and they
comprised 65 per cent of the total number of pages written by
her. An estimate of the value of her papers would hardly be
in place here. It may be said, however, that her writing has a
clearness and directness that make her meaning unmistakable.
She did not hope to have all agree with her, but she did hope
that all could understand exactly what she meant. She had
no patience with the oracular form of writing which could be
interpreted in almost any way, and to which discoveries made
long afterward could be referred.

SUSANNA PHELPS GAGE 11

From her own standpoint, no pains were too great to make
sure that the interpretation she had made of structure or of
ideal plan of structure and relationship were the true ones. As
with all investigators endowed with the divine gift of imagina-
tion, some of the cherished images had to be broken when the
facts discovered on fuller study showed that they were false,
but never was an iconoclast more merciless than she when once
convinced that the images were false. Furthermore, her work
won her the respect of her fellow investigators in her own and
in other countries. She was welcomed in all biological labora-
tories and given every facility. No single laboratory can hope
to be adequately equipped in embryologic and anatomic series
of all forms and stages and prepared by all the standard methods;
hence the need of making use of the facilities of many labora-
tories to gain the broadest view on some fundamental questions.
Fortunately there is the spirit of helpfulness in the laboratories,
and any worthy worker is given the needed facilities without
stint. She was freely accorded such facilities. Most often she
used the rich collections of Dr. Minot at the Harvard Medical
School, and of Dr. Mall at the Johns Hopkins Medical School.’
Indeed, one of her most important papers was based on a three
weeks embryo loaned to her for more than a year by Dr. Mall.

In 1911 it became possible for her to carry out a long hoped-
for visit to Europe to see the places where history had been
made, to enjoy the art, and most of all, to see some of the lab-
oratories from which have originated so much of the funda-
mental work in modern science. Fortunately the meeting of
the Anatomische Gesellschaft was held soon after her arrival in
Leipzig and the British Association for the Advancement of
Science a few months later. These meetings gave her a chance
to see with her own eyes the methods and work of two of the
great foreign societies, and to compare them with those of the
societies of her own country with which she was so familiar.'
She became a member of the German Anatomical Society; and

1 Mrs. Gage was a fellow of the American Association for the Advancement of
Science, a member of the American Anatomical Association, the American So-
ciety of Zoologists, the American Microscopical Society and the Anatomische
Gesellschaft.

'2 SIMON HENRY GAGE

later in visiting some of the universities the distinguished men
whom she had met showed her every courtesy in their labora-
tories and gave her opportunity to study the choicest among
their series. Among the experiences most cherished were those
connected with the laboratory of Golgi in Italy and of Ariéns-
Kappers in Holland. Golgi not only showed her some of the
specimens already mounted, but mede for her before her own
eyes some of his incomparable preparations.

In speaking of college studies above, it was stated that among
those which gave her the greatest pleasure and inspiration was
American history; and a few years later while staying with her
father during a period of family bereavement, she read aloud to
him Irving’s life of Washington. She was keen in perceiving the
breadth and foresight of Washington in all educational matters
and was especially interested in his desire for a national univer-
sity, for the founding of which he gave a generous share of his
private fortune.

Her own intimate knowledge of the need for research oppor-
tunities in our country, if it were to become a real and a wise
‘leader among the nations of the earth, led her to ponder deeply
on such an institution, the crown of our educational system,
which has not yet been realized in our land. It seemed to her
that the women of the nation, who were coming into the sun-
light of opportunity in university education, if they only knew
of this great hope and desire of Washington and the urgent
need, would rally with the same enthusiasm as that which filled
her mind and heart and the greatest of all our American univer-
sities would arise in Washington City—an institution which
would represent the highest and best in the university idea, and
which, being the offspring of the whole country, would realize
Washington’s dream and hopes far beyond what he could have
imagined.

She helped to found the George Washington Memorial Asso-
ciation to bring about the hoped-for result, and served it for sev-
eral years as secretary. From 1895 to 1905 she urged in spoken
words and in writing the fulfilment of this dream of Washing-
ton with a zeal and eloquence worthy of success. Perhaps her

SUSANNA PHELPS GAGE 13

point of view may best be seen by a quotation from her paper
before the University Convocation held in the senate chamber
at Albany in 1897. The subject of the paper was the need of a
national university for the common schools, and the quotation
follows the description of the vitalizing power with which a
university teacher had conducted a common school exercise in a
difficult subject.

But we can not spare from their present work of investigation the
few men and women in the country who are specialists, to preach their
gospel in the highways and hedges. We need thousands of young men
and women who shall go to these specialists and receive such training
that they can give living inspiration to the children. We need more
and more places where this supreme training can be acquired, we need
finally a national university where in quiet thoughtfulness the newest
questions may be solved and the oldest be reconsidered. The knowl-
edge gained here at the very surface of the known, will then penetrate
through college and high school and common school to the living units
of the great democratic mass, that each may live a life richer in the
things of the spirit. He at the very outside not only gives to him
within but some. day he helps the pupil now standing at his side to
mount upon his shoulders and push a little farther into the hitherto
unknown universal knowledge.

Like so many of the splendid dreams of noble men and women
she did not see even the beginnings of a National University
realized any more than did Washington. Apparently it must
wait, but that it would come true sometime she never doubted.

While the great public service she hoped to render in helping
to make real Washington’s plan for higher education seemed to
fail, she found it possible to aid her native village by the gift
to it of her childhood home for a library. And now in her father’s
house is one of the best libraries of any village in the state;
and it is greatly appreciated by the people of the surrounding
region as well as by the villagers.

In the bookplate and the bronze tablet which she designed
for the library, the bittersweet was used for decoration. ‘This
came about naturally from the fact that for many years a mag-
nificent bittersweet vine was a distinguishing feature of her
father’s house, and the clusters of red fruit gave it in winter the
appearance of cheer that the abundant flowers around the house

14 SIMON HENRY GAGE

lent in summer. In the bookplate is this sentiment going with
the decoration and expressing what books had been to her:
“The bitter and the sweet of all the past shall strengthen us.’’

Doubtless as the years pass away many a boy and girl will
find in some book in that library the intellectual and spiritual
food which they long for and which will show them the way they
are seeking to a career of noble living, and of service to their day
and generation comparable with that of the gracious woman
who made the library possible.

It was always a pleasure to her to attend the seminaries in
the different biological groups in the university and there was
ever a word of commendation and cheer for the young workers
who were trying to walk alone. They loved to talk over their
work and plans with her, and none ever did so without having
his purpose strengthened.

It was not only in the laboratories and seminaries that she
met the students, but they were welcomed in her home. And
from the testimony of many who have found high success those
home experiences added to life and its aspirations what could
not be given by the laboratory alone. She was much sought
after not only by the students but the young people in the
teaching staff of the university who found in her the abounding
sympathy and enthusiasm of youth combined with the wisdom
that comes only from maturity and experience; and none who
sought help ever went away empty handed but all gained from
her new courage and enthusiasm, new faith that life was worth
living, and that something worth while could be accomplished in
the world.

As expressed by her friend of thirty-five years, Mrs. Anna
Botsford Comstock, in the Cornell Alumni News of November
4, 1915:

Mrs. Gage’s personality made a lasting impression upon all who
met her. She had great charm of manner, deep earnestness, a vigor-
ous and quaint originality of thought and expression, a fine sense of
humor, keen sympathies, and above all the power of briiging cheer
to all with whom she came in contact. Her merry musical laugh was

so much a part of her that even those of us most bereft must be com-
forted by the memory of it. Her character gave a firm and broad basis

SUSANNA PHELPS GAGE 15

for her attractive personality. She had high ideals, unswerving hon-
esty and singleness of purpose, and great power of helpfulness to the
person or cause that might be in need. She had a genius for friendship
for her heart was loyal and loving. Her strong mental fiber and keen ,
intellect rendered her friendship as stimulating as it was comforting.

On Saturday, Sunday, and Monday, the 2d, 3d, and 4th of
October, 1915, the son, Henry Phelps Gage, Ph.D., visited the
home and during that time explained and demonstrated to his
mother the daylight glass he had succeeded in producing, and
showed its use for microscopic work in demonstrating the most
delicate colors and tints equal to what could be accomplished
by daylight, and also the use of a large lamp with the daylight
glass for matching delicate colors of silks, and for water color
work. What parent is there who cannot imagine the happi-
ness that came to the mother’s heart to see realized in the ac-
complishment of her son some of the dreams that the long-before
laboratory work in physics of her own youth gave rise to.

On Sunday there was an automobile ride along Cayuga
Lake and on the return was one of the gorgeous sunsets that
Ithaca is famed for. We did not know then as we gazed upon it
that one of our number was so soon to enter into that glory.

After four years of failing health, death came suddenly and
painlessly on the morning of October 5, 1915.

In the home at Ithaca were simple funeral services, a part of
which were some of the heart-sustaining and soul-uplifting
hymns she loved so well, played upon the university chimes.

As long mutually agreed upon, the body was cremated; and
in the childhood home which she had given for the village
library, with the books looking down from the shelves, and in
the presence of life-long friends, some fitting words were spoken
over the ashes. These now rest in the village cemetary beside
those of the father and mother and brother who had preceded
her.

The sky that she looked up to with such joy in childhood
looks down upon the quiet resting place. The encircling hills,
from which in youth she looked forth with such enthusiasm
and high courage to the world of work and service, shall hold
forevermore their guardianship over the beautiful valley.

1887

1888

1890

1891

1904

1892

1892

1893

1895

1896

SIMON HENRY GAGE

PUBLICATIONS OF SUSANNA PHELPS GAGE

The commonwealth of mind. The Cornell Review, June, vol. 6, pp. 346-
351. This paper was given as class essayist at the graduation of her
class in 1880. It is an argument and an appeal for the fundamental
democracy of the mind in human beings, and this remained her cher-
ished belief throughout life.

Ending and relation of the muscular fibers in the muscles of minute ani-
mals (mouse, mole, rat, and English sparrow). Abstr. Proc. Amer.
Soe. Micro, pp. 1-2.

Form, endings, and relation of striated muscular fibers in the muscles of
minute animals (mouse, shrew, bat, and English sparrow). The
Microscope, vol. 8, August, pp. 225-237; 257-272, 5 pl. It is in these
two papers that the author expounds the true form and relationship
of the fibers of skeletal muscle in small animals.

The intramuscular endings of fibers in the skeletal muscles of the domestic
and laboratory animals. Proce. Am. Soe. Microscopists, 138th meet-
ing, pp. 132-138, 1 pl.

A review. The evolution of sex, by Prof. Patrick Geddes and J. Arthur
Thompson. The Nation, vol. 52, May 14, p. 407.

The story of little red-spot. Boys and Girls, April, pp. 11-16, 1 fig. The
author says of this story: ‘‘The scientific name of Red-Spot is Diemyc-
tylus viridescens . . . . The photoengraving on p. 11 is taken
from the colored lithographic plate drawn by the present author for
an article by Prof. S. H. Gage in the American Naturalist, December,
1891, where he tells the same story in scientific language. The present
author when making the drawings studied red-spots in their homes
and wrote the story for her little son.’ Anna Botsford Comstock,
the nature study expert, says: “‘It is one of the most charming science
stories for children in our literature.”

Evolution and the training of children. Abst. Kindly Light, vol. 1, no.
(i), Te ey 2eyonall:

A reference model. Proc. Amer. Soc. of Micros. (Rochester meeting),
pp. 154-155, 1 fig, vol. 14, 1892, printed July, 1893.

The brain of Diemyctylus viridescens from larval to adult life, and com-
parisons with the brain of Amia and Petromyzon. Wilder Quarter
Century Book, pp. 259-313, 8 pl.

with Anna Botsford Comstock, editors and authors. A tribute
to Henry W. Sage from the women graduates of Cornell University.
Ithaca, N. Y., May 30, 84 pp. Illustrated by Anna Botsford Com-
stock. This is the fullest and best discussion cf co-education in a great
university, and the only adequate account of it in Cornell University.

Lines on the engraving ‘‘Two incarnations in stripes,’ by Anna Botsford
Comstock. Illustrated Buffalo Express, March 1, p. 5.

1895-1896 Comparative morphology of the brain of the soft-shelled turtle

(Amyda mutica) and the English sparrow (Passer domesticus). Proc.
Amer. Mic. Soc., vol. 17, pp. 185-238, 5 pl. Abstr. Am. Month. Mis.
Journ., vol. 17, Jan., pp. 4-7.

SUSANNA PHELPS GAGE ile

1896 Modification of the brain during growth. Amer. Assoc. Adv. Sei., August
24, 1896. See Proc. Abstr., Amer. Naturalist, October, pp. 836-837.
Science N. S., vol. 4, October 22, 1896, pp. 602-603.

1896 The brain of the embryo soft-shelled turtle. Trans. Amer. Mic. Soc.,
vol. 18, pp. 282-286.

1897 Washington and the national university. The New Unity, June. Re-
printed, Active Interests, December, 1897, pp. 15-23. With bibliog-
raphy and plea for George Washington Memorial, pp. 6-7.

1897 The need of a national university in its relation to the common school.
Proc. 35th University Convocation (Albany), June, pp. 313-319.

1898 A Washington memorial university. The Outlook, February 26, pp. 521-
524.

1898 Relation of a national university to the graduate departments of existing
universities. Address given at a meeting of the George Washington
Memorial Association, December 15, pp. 15-27. Papers of 1897 and
1898 cannot be read without admiration for her breadth of view and
grasp on the educational conditions of our country. Her devoted
patriotism and sympathy with the ideals of Washington are shown in
every paragraph.

1899 Notes on the chick’s brain. Abstr. Amer. Assoc. Adv. Sci. Proc., vol.
48, p. 256.

1902-1903 An unusual attitude of a four weeks human embryo. Comparisons
with the mouse. Abstr. Proc. Am. Assoc. Adv. Science, vol. 52, p.
458. Science N.S., vol. 17, 1903, p. 254.

1904 The mesonephros of a three weeks human embryo. Proc. Assoc. Am.
Anat., March, p. VI. in Am. Jour. Anat., vol. 3, 1904.

1904-1905 Total folds of the forebrain, their origin and development to the
third week in the human embryo. Proc. Assoc. Amer. Anat. in Am.
Journ. Anat., vol. 4, no. 2, 1905, p. IX.

1905-1906 Relations of the total folds of the brain tube of human embryos to
definitive structure. Proc. Assoc. Am. Anat., 20th Ses. in Am. Jour.
Anat., vol. 5, pp. [X—X.

1905 A three weeks human embryo with especial reference to the brain and
the nephric system. Am. Jour. Anat., vol. 4, no. 4, pp. 409-443, 5 pl.
The embryo for this study was loaned by Dr. F. P. Mall, and he ex-
pressed himself satisfied with the results gained from its study. This
is one of Mrs. Gage’s most important papers, and illustrates well her
method and thoroughness of work.

1906 The notochord of the head in human embryos of the third to the twelfth
week and comparisons with other vertebrates. Abstr. Proc. Am.
Assoc. Adv. Sci., vol. 56, pp. 277-278. Science N.8., vol. 24, Septem-
ber 7, 1906, pp. 295-296.

1907 The method of making models from sheets of blotting paper. Anat.
Record, no. 7, of Am. Jour. of Anat., vol. 7, no. 3, November 10, pp.
166-169. Read, Assoc. Am. Anat., December, 1905. Abstr. Am.
Jour. Anat., vol. 5, 1905-1906, p. XXIII, Demonstr, 7th Internat.
Zool. Cong., August, 1907. These models combine the good features
of the French papier maché and the German wax-plate models, and
represent her inventive turn of mind, and ability to adapt means to
ends.

18 SIMON HENRY GAGE

1907 Changes in the form of the forebrain of human embryos during the first
8 weeks. Read, Seventh International Zool. Cong., August. Printed
in Proc. Seventh International Zool. Cong., 1910, 2 pp. 3 figs.

PAPERS IN COLLABORATION WITH S. H. GAGE

As stated in the text above, Mrs. Gage entered enthusiastically into the origi-
nal work in biology and for a long time made most of the drawings to illustrate
her husband’s papers. She alsobecame partner, in a broader sense, in the papers
named below:

1885-1887 Aquatic respiration in soft-shelled turtles (Aspidonectes and Amyda).
A contribution to the physiology of respiration in Vertebrates. Proc.
Amer. Assoc. Adv. Sci., vol. 34, Ann Arbor meeting, August, 1885,
pp. 316-318. Amer. Naturalist, 1886, pp. 233-236. Science, Septem-
ber 11, 1885, p. 225. Scientific Amer. Supplement, to November 14,
1885, p. 8230. Biologisches Centralblatt, Bd. 6, 1886-1887, pp. 213-214.

1886 Amceboid movements of the cell-nucleus in Necturus. Science, vol. 7,
p. 146. :

1888 Combined aerial and aquatic respiration. Science, vol. 7, 1886, p. 394.
See note in Ref. Handbook Med. Sci., vol. 6, p. 197.

1886 Pharyngeal respiratory movements of adult Amphibia (Diemyctylus)
under water. Science, vol. 7, p. 395.

1889 Staining and permanent preservation of histological elements isolated hy
means of caustic potash (KOH) and nitric acid (HNO;). Proc. Amer.
Soe. of Mier., vol. 11, pp. 34-45. Gage (Dr. Simon H., et Suzanne P.).
Coloration et conservation permanentes des éléments histologique
isolés par la potasse ou l’acid nitrique. Jour. de Micrographie, t. 15,
1890, pp. 43, 102.

1890 Changes in the ciliated areas of the alimentary canal of the Amphibia
during development, and the relation to the mode of respiration.
Abstract, Proc. Amer. Assoc., Adv. Sci., vol. 39, pp. 337-3388.

1908 Sudan III deposited in the egg and transmitted to the chick. Science,

N.S., vol. 28, pp. 494-495.

1909 Coloration of the milk in lactating animals and staining of the growing
adipose tissue in the suckling young. The Anat. Record, vol. 3,
April, pp. 203-204. i

1898 The life history of the toad. Teachers Leaflet, No. 9, for use in the public
schools, pp. 79-98, 8 figs. Prepared by the College of Agriculture,
Cornell University, I. P. Roberts, Director. Issued under Ch. 67,
Laws of N. Y., 1898. 2d revised ed., 1904. pp. 185-206, 14 figs. (In
bound volume of Cornell Nature Study Leaflets.) Abbreviated by
Susanna Phelps Gage from Leaflet No. 9, Series G. Nature Study
and published by the League for Social Service of N. Y. City, Geo.
Leighton Tolman Donation. Spring of 1899. The Sanitary Home,
vol. 3, May, 1901, p. 57+.

THE CEREBRAL GANGLIA AND EARLY NERVES OF

SQUALUS ACANTHIAS

F. L. LANDACRE
Department of Anatomy, College of Medicine of the Ohio State University

THIRTEEN FIGURES

CONTENTS
See RETO MEMCLEOE ry. Shes asta hn este oe ta a eas ne es 5s) ra de 20
2. The nervus olfactorius and nervus terminalis................0.0: 6.2.22: 22
See AROCUNIS MANE ITONG © a, 22-5 on ashes os ame mines cree ein gees cletes 24
Aa thetG asseriant Sango: s..28. ses. Ses. cee fe cect ee de ve hema oe we aaaieees 26
5 Ramustopithalmicus superiicialis: Vis.) 0S.c). ve oh ci hslss Se aaes pons eae 26
(Fo eRe frvtbrollll Pei ila, Aaa ean Inde irc ci Ae ee Oe Rigen Caer oe 28
PER RTAUTS OTAT TAC TNNUE SETS” Vises oop isc rs ns foe, eatneic aaa ooep sl Se ss Yahav, fe ee Sesh eysteu gl om eam wae 29
Sothe dorsal lateralis ganglion of WIT. 0% oe. eek sas. noe Wee a ee 30
9) Raniusophthalmicussuperiierlis VIL. . (32.22% y 0. Soc hd Sete eo tw ae 30
I; TReneaOi [cere T Ie As eee aocitcs om ciacicic.o c om O acne ABO erro core rmaein 31
ifs dhe aeustico-lacialis canslionic complex. . go o.55 06 o<< <6 soe tens eae os Soo 32
iP er sUCITOLY SAnewon AMG Came ns - oc, sameness 5 cc ate fee Shia oa oe «sae 33
fo he penteula Lest AN eli OM.) 200 ae sd <-. e's 4 seo ine vos le Rice a este siauetal close Meee ee 33
14. Ramus palatinus and ramus pretrematicus.................+++0.eeese eee 34
ie heswenural lateraliseVikk Sangam... .o6-cctanes <del. . 5 sales sain 42 eee 35
LO REUNEUS NY OMVANATDULALISs. Seg cree + «apne me Pherae seit eyecare ROPER 2: 35
if. bhesdossopharyngeal ganglion and root. -2::1-.0.. 04.0. ore one ees gee 37
ioe Pamuc supratemiporaliss DX 2 752042 Ws fake ae eae 3 eed reece eias dean ss 38
19. Ramus pharyngeus and ramus pretrematicus [X...................-- ooo
POE MUEUTIEUS A OSSOpla by NPAUS. oo faces tein oni hen aieatets Se es cL yienats orisha)» © afoot 39
Pee hery Meise m mele: cadet kn eta Sens oe he che eee ee ee oe ene 40
PIPPMRAR INE: WA PISEROM EBL Miiery ts Case de tht create cee Wane aire nee No tave nero sh cig agent 4]
Deamibie: Meulae ether ean POI rs in. aciskenes =.0ia ards aye Sep Aged s eeB Roe PRS 42
PEL EIS URE CULATISM ones cs Set Neste kaon s atte whee or Searels a osha atelnn s Dis /tte sh 42
Pom ter eaTHAbON, Wa UCLA et. ssc. een ciiec eis mee aA eoe + at tee ake wires e oe oe eile 44
Ae aL aMetre SUPrALeMPOFAls Waste .'.\. 2k. fea RL oh ete blbele eS 45
Zea crmAneHOn, tA henale ha fastd 7 bots Mode oid sles Pah ed ate Sie: Ae sla Tees Pas 46
a PPeEDGC ED BE TTel Mal LEGUNS MER No Whe te wate ko lah teeth ies fea, <a tcalayeipha aie ais aa 46
eM MEET aR LEE ACD N gone eh iS arars ote vais ste steyahnle: tie age 6S sgeicpols ra, Wyse wd eo 47
SOs Seconauancnunind amt lybenales Xe. caaceiclece oe foe e eo ieierree t= 48
Slevin vis cenalice xpUOu aaah solids os thd: be Ser sdetiola b desia amen 48
Bae HaLsG AMEE ane Md BTS Nar 2c. acy) oo) eis vnckbecios) Ge aoe ts pq qa0> “Roe 49
Soe ECONG pL NtetGucm DD PAMGNsaNIG SRAMee ee. 2 oc. cters, Jlerde clase eine cel aipereia\s ore cit = ERR 49
19

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, No. 1

20 F. L. LANDACRE

34. Third truncus'branchitahis ext. 4-eeeeeee ee oe oe oe ee eee 50
35. Hourthitruncusibranchia lis exer ea eee eee eee 50
36. “The ‘ganglion visceralle Xs. at seer ac Gee Aarne sonic «nate eee See BS 50
37; Ramis svisceralis ac ttle eee a eee ea ee ee eee 51
Sunimary fang discussioncstnd. rose. c us See So ee nee oe ee 51
iteratureiciteden tere. sect. Sees oe Pea EEE eee eee 55

1. INTRODUCTION

Up to the present time no detailed analysis of the cerebral
nerves of the shark has been published. A voluminous litera-
ture covering almost every other phase of the anatomy and em-
bryology exists, but for some reason an analysis of the cerebral
nerves of the shark such as we now have for a number of fishes
and amphibians and reptiles does not exist. The present study
of the embryonic ganglia and early nerves is not offered, as, in
any sense, a substitute for such an analysis of specimens old
enough to show complete medullation of the nerves. The author
has attempted an analysis similar to those made on Ameiurus
(10), Lepidosteus (712), and Rana (12), m which it was found
that there was a particularly favorable condition of the ganglia
in that they were well isolated, and a development of the chief
nerves sufficient to enable one to identify them with certainty
when their composition was known in detail in the adult.

The present study has the disadvantage of not being preceded
by a careful analysis of older specimens but, like the previous
studies mentioned, has the advantage of presenting a very simple
condition of the various ganglia and, in cases where the nerves
are pure or contain only one component, the morphological rela-
tions of the ganglia and nerves make their identification a simple
matier. On the other hand, mixed nerves and very immature
nerves present greater difficulties and in these cases where the
various components could not be traced definitely to their dis-
tribution they have been identified provisionally.

The amount of attention given to the description of nerves in
a paper devoted ostensibly to the description of ganglia would
be unwarranted if the exact composition of the nerves were
known. In the absence of this information the nerves had to
be followed with the greatest care and their description is in-

GANGLIA AND NERVES OF SQUALUS 21

cluded in the body ot the paper. The general morphological
relations of ganglia are fairly safe criteria for their identifica-
tion provided one is sure of their presence; otherwise, the dis-
tribution of the fibers must be known to identify a ganglion with
certainty.

The differences in size in the cells of different ganglionic com-
ponents sometimes found in other types and particularly in
mature types do not seem to exist in the material studied; such
differences as exist are those between older and younger gan-
glion cells rather than between different components. How-
ever, I do not believe any serious oversight has been made
unless it is in the failure to find a general cutaneous component
in the seventh and ninth nerves.

The number of nerves described is small compared with the
adult, of course, but those present in the stage described are the
chief nerves. While this study should have followed and not
preceded that of older material, it is hoped that it will help to
fill the gap in our knowledge of our most generalized vertebrate
and it will certainly serve as a foundation for the study of the
origin of the cerebral] ganglia on a component basis. The effort
to describe the origin of these ganglia, en masse, is entirely futile
in the author’s opinion. It must be done with a thorough
knowledge of all the ganglia involved. This is true whether
they arise as discrete ganglia having different sources of origin
or whether they differentiate out of a common primordium.

The author is under obligation to Dr. H. V. Neal for most of
the material, which was fixed in vom Rath’s fluid and mounted
unstained. This material was supplemented by younger ma-
terial stained in Delafield’s haematoxylin and orange G. The
embryos ranged in length from 18 to 30 mm. after fixation and
several specimens of each length, except the 30 mm. embryo,
were examined. The plot and drawings were made from a 22
mm. embryo. The terms anterior, posterior, dorsal ,and ven-
tral are used in the body of the paper to indicate the relative
positions of structures on the plot and not their true position
in the adult which is sometimes quite different.

22 F. L. LANDACRE
2. THE NERVUS OLFACTORIUS AND NERVUS TERMINALIS

The connection between the olfactory pit and forebrain,
which has no olfactory bulb at this time, consists of a thick
cellular and fibrous mass which probably represents both the
above mentioned nerves. The main portion of this connection,
that lying more dorsal in position in the plot (fig. 1, n.olf.),
consists of a dense mass of medium sized cells extending from
the brain wall back to the epithelium of the olfactory capsule,
where, just before coming into contact with the brain, it breaks
up into three or four masses of cells where it is connected with
the brain. The anterior end of this mass is solid and does not
show the loose character of the posterior end. There are iso-
lated strands of fibers in this mass aside from those mentioned
below but their connec‘ion with neither the brain wall nor the
olfactory epithelium could be made out with certainty. These
strands of fibers are identified as olfactory fibers.

In addition to the main dorsal portion described above, there
are two strands of cells on the anterior end of the nerve lying
ventral 1o the main strand containing definite fiber bundles
(fig. 1, N.Ter.). The entrance of these fibers into the brain
wall at a point more ventral and median than the main mass of
cells is easily made out and the fiber bundles can also be traced
through the connecting cellular mass to the olfactory epithe-
lum. These two strands in the specimen plotted are repre-
sented by only one strand in four other specimens of about the
same age and there is only one strand of cells and fibers on the
opposite side of the same specimen. Both strands are accom-
panied by a limited number of round cells lying in the position
indicated by Locy (’99, ’05) as the location of the ganglion of the
nervus terminalis, namely near its entrance into the brain wall.
In the 30 mm. embryo the ganglion of the n. terminalis while
small is well defined and well isolated. There can be no doubt
as to the identification of these as the nervus terminalis on
account of the point of entrance into the brain wall and the
greater degree of development. lLocy (’99, ’05) gives a rather
full account of the history of this nerve, but his account shows

GANGLIA AND NERVES OF SQUALUS 29

both the olfactory and terminalis nerves to be much more de-
veloped and better isolated at 25 mm. than in my specimen of the
same age. In fact, one would infer from his description that
at 16 mm. the connections of the olfactory and terminalis nerves
were more definite than in the 22 mm. embryo plotted in this
paper.

At the posterior end of the connecting mass in all specimens
examined there are two strands of cells which detach them-
selves trom the main mass and come into contact with the olfac-
tory epithelium ai a point more ventral and posterior to the
chief mass. These masses in none of my earlier specimens con-
tain fibers and the strands are absent in a 30 mm. specimen.

This connecting mass appears to be, on first examination, in
a rather undifferentiated condition as indicated by the small
proportion of fibers, and the very large number of cells. An-
other interpretation however is possible, namely, that the large
mass of cells represents the beginning of the close fusion be-
tween. the olfactory capsule and olfactory bulb characteristic of
Squalus in addition to early sheath cells of the n. terminalis and
n. olfactorius The olfactory capsule contains at all points ex-
cept the anterior border a well defined basement membrane.
At the anterior border this membrane is lacking and the cells
of the capsule mingle with those of the mass of cells connecting
the capsule with the brain wall. Younger embryos, in which the
connecting mass is not so large, present the appearance of a
migration of cells from the capsule to what I have designated the
connecting mass. However, my series of embryos is not suffi-
ciently large to determine definitely the origin and fate of this
mass of cells. It seems from a comparison with the conditions
in Amia, Ameiurus, and Lepidosteus (Brookover, 1908, 1910,
1911) entirely too large to be the ganglion of the nervus termi-
nalis and I am inclined to interpret it as the beginning of the
fusion of the capsule with the bulb plus early sheath cells, as
indicated above. The connecting mass is much smaller in
younger embryos and shows a decided increase in size in a 23
mm. embryo as compared with the 22 mm. embryo plotted.

24. F, L. LANDACRE

3. THE PROFUNDUS GANGLION

The profundus or mesocephalic ganglion (figs. 1 and 2, G.Pro.)
lies Just posterior to the mid-dorsal border of the eye. It is
slightly crescent-shaped with the convexity on the dorsal sur-
face. The root of the ganglion extends dorsally and slightly
caudad until it comes into contact with the anterior surface of
the Gasserian ganglion. The root from the ganglion to the
point of contact with the Gasserian contains a rather large
amount of cells and the relations of the fiber bundles are not
easy to determine definitely. There seems to be little doubt,
however, that the fibers from the profundus ganglion run on
the anterior surface of the Gasserian ganglion, curve forward
and upward and enter the brain wall through the most anterior
of the three roots shown in figure 1 (Rt.Pro.). The point of
entrance is just opposite the dorsal border of the spinal V
column, which they enter.

The relations of the remaining two divisions of the portio
minor are not so clear. The second division does not enter the
descending V tract but enters slightly mesial to that tract
and as a well isolated bundle of fibers passes to a more mesial
position. This root in the specimen plotted is evidently a
visceral motor root and presumably comes from the motor com-
ponent of the ramus mandibularis V after running through the
Gasserian ganglion. The third root in the portio minor is sen- .
sory and enters the spinal V tract. An examination of a number
of other specimens ranging from 18 mm. to 23 mm. shows that
there are in the older specimens several more small roots in the
portio minor, all of which seem to be sensory, since they enter
the spinal V tract. In all of the specimens there are two roots
constantly present, the most anterior (the one identified as the
root of the profundus), and the second of the three plotted in
figure 1, which is identified as the visceral motor root of V. In
the younger specimens these two roots are the only ones pres-
ent, so that the remaining roots of the older specimens and the
third root of my plot are in all probability accessory sensory
roots of either the profundis or of the Gasserian, but from which
ganglion they come I am unable to determine.

GANGLIA AND NERVES OF SQUALUS 25

Neal (’98, p. 233) has discussed the relation of the various
divisions of the portio minor and my results agree substan-
tially with his rather than with those of Mitrophanow, ’93. The
mode of entrance of the profundus root in Lepidosteus (Land-
acre, 12) reinforces the identification of the most anterior root
as that of the profundus since in Lepidosteus the root of the
profundus enters well forward and entirely distinct from that
of the Gasserian. There can be little doubt in my opinion that
the second root is visceral motor. In Neal’s paper he does not
describe three roots in detail but figures them (Neal, ’98, p. 234,
fig. K.).

From the anterior end of the ganglion in the specimen plotted
extends a mass of cells from which no fibers pass out, the ramus
ophthalmicus profundus leaving the ganglion near its mid-ven-
tral border. This forward extension of the ganglion is evidently
the remains of the structure which Neal identifies as a persistent
connection of the ganglion with the ectoderm (Neal, ’98, p.
234, fig. K.) and which Scammon (’11, pp. 54, 55, figs. 11 and
12) identifies as the utrochlea process, i.e., the remains of the
connection of this ganglion with the neural crest. This process
gives the profundus ganglion a curious shape in contrast with
nearly all other ganglia, in which the nerves practically always
arise from the free end of the ganglion. In this case, as men-
tioned above, the profundus nerve arises near the mid-ventral
border of the ganglion. The proximal portion of the profundus
wnerve is difficult to follow in the 22 mm. embryo on account of
its being compressed between the mesial wall of the orbit and
the primordia of the eye muscles and adjacent blood vessels.
After it reaches a point at the level of the dorsal border of the
lens its course 1s easily followed. It forms a gentle curve ceph-
alad and ventral, more than half of its course being mesial to
the eye. In a 30 mm. specimen the whole course of the nerve
is well isolated.

The profundus nerve, aside from its large size and length as
compared with the r. oph. sup. V, has the relation usual in elas-
mobranchs and ganoids. Only two small twigs (figs. 1, Pro. 1,
Pro. 2) seem to be given off before the nerve reaches its most

26 F. L. LANDACRE

peripheral point of distribution, which is to the skin at a point
ventral to the point of entrance of the olfactory nerve into the
brain wall. All these run to the ectoderm. The size of this
nerve in the specimen plotted as compared with the r. oph. sup.
V is much more like the 18 mm. embryo plotted by Seammon
(11) than the 20 mm. embryo plotted by the same author. In
the 20 mm. embryo plotted by Scammon the r. oph. sup. V is
much longer than the r. oph. prof. V.

4. THE GASSERIAN GANGLION

The Gasserian ganglion (figs. 1 and 3, G.Gass.) is very large
and lies just posterior to the dorsal half of the eye. It is placed
diagonally in the head with its long axis nearly in the trans-
verse plane but with its proximal end slightly anterior to its
distal end. It extends ventrally and slightly caudad from its
proximal end so that its distal end lies at the level of the dorsal
border of the lens. It comes into contact on its dorsal and an-
terior surface with the root of the profundus ganglion and on
its ventral and lateral surface with the dorsal lateralis ganglon
of VII. On its mesial surface it comes into contact with the m.
rectus externus of the eye. Viewed from the lateral surface the
ganglion is partly concealed by the dorsal lateralis ganglion of
VII and by the r. oph. sup. VII and r. buccalis VII.

The ganglion is forked at its distal extremity where the two
chief rami arise, but its form is not modified by the exit of the
r. oph. sup. V. It is of nearly uniform thickness throughout’ |
its length. Excepting the two small roots entering with the
portio minor mentioned above, the fibers passing proximally
from this ganglion form a single compact and relatively massive
root whose fibers pass into the spinal V tract.

5. RAMUS OPHTHALMICUS SUPERFICIALIS V

Of the three rami arising from the Gasserian ganglion at this
stage, the most anterior one, the r. oph. sup. V (figs. 1 and 2,
R.O.S.V.), is much the smallest. It arises on the anterior sur-
face of the proximal end of the ganglion slightly dorsal and

GANGLIA AND NERVES OF SQUALUS 27

lateral to the point of contact of the root of the profundus with
the Gasserian. From its point of exit it pursues a course ceph-
alad parallel to the longitudinal axis of the body and slightly
ventral and mesial to the r. oph. sup. VII and parallel with that
nerve to a point approximately over the anterior end of the
lens, where it comes into-contact with the superior oblique muscle
of the eye. This contact is at the point of entrance of the
trochlear nerve into this muscle and the sensory nerve could
not be traced beyond this point in the specimen plotted, conse-
quently no mass of cells at the growing point of the nerve could
be identified such as Neal (14, plate 7, figs. 55 and 56) figures. ©

There is, however, a mass of cells apparently not belonging
to the muscle but slightly detached from it and containing a
few large cells which may be the mass figured by Neal but
seems rather to be the primordium of the sympathetic ganglion.
This nerve is evidently in a much less mature condition than in
the 25 mm. specimen figured by Neal. It may be mentioned
incidentally that in my specimen the trochlearis does not show
the two well defined rami which he figures in plate 7, figures
54 and 55. These two terminal rami in the specimen plotted
are quite small. Otherwise my findings agree with those of
Neal and I have nothing to add to his very thorough descrip-
tion of the eye muscle nerves. Ther. oph. sup. V gives off one
small twig about the middle of its course which runs close to the
ectoderm but could not be traced into it with certainty. There
is, of course, owing to the small size of this ramus no anasto-
mosis with r. oph. sup. VII, as in the adult.

The small size of the r. oph. sup. V in Squalus as compared
with the large r. oph. prof. at this stage furnishes a basis for an
interesting phylogenetic comparison of these nerves, in embryos
of different types. In Lepidosteus at approximately the same
stage (Landacre, ’12, fig. 1) the two nerves are equal in size.
In embryos of urodeles (Coghill, ’16, figs. 1 to 4), the ophthal-
mic ramus comes from the profundus ganglion and is identified
by him as r. oph. prof. In Anura (Landacre and McLellan,
12) only one ophthalmic ramus comes from V and this comes
from the profundus portion of the fused profundus and Gasseri-

28 F. L. LANDACRE

an ganglion. Apparently the ophthalmicus profundus has sup-
planted largely the r. oph. sup. V in Amphibia. In higher
forms the ophthalmic nerve seems to come from the Gasserian
ganglion, in which case the r. oph. sup. V. has supplanted the
r. oph. profundus.

6. RAMUS MAXILLARIS V

The ramus maxillaris V (figs. 1 and 4, R.Mz.V) arises from
the ventral end of the Gasserian ganglion and pursues a course
directly ventral to the position of the third lateral line organ
innervated by the r. bucecalis VII, where it gives off a number
of twigs to the ectoderm. From this point it turns slightly
cephalad and before reaching its most distal point of distribu-
tion gives off several small twigs (fig. 1, S./—4), all of which run
to the ectoderm. The extreme end of this nerve breaks up into
a number of twigs which could not in some cases be traced to
the ectoderm and present the appearance of the tip of a growing
nerve.

The r. maxillaris is accompanied throughout its whole course
by the r. buccalis which lies more lateral in position, but the
two nerves are quite separate except at the level of the third
lateral line primordium mentioned above where there are two
fibrous connections between the two nerves (not shown in fig. 1)
in which the fibers seem to pass from the r. maxillaris to the r.
bucealis. The R. Mx. V. seems to be purely general somatic
sensory. It has no connection at any point with the ganglion
or nerves of visceral VII. There is a possibility that lateral
line fibers enter it through the two anastomoses mentioned
above, in which case it would contain a few lateral line or special
somatic fibers. The appearance of the anastomoses do not
favor this view and there are no lateral line primordia on the
course of the r. maxillaris beyond the anastomoses. There is,
further, an anastomosis (fig. 1, R.Com.) between the r. mandib-
ularis V and the r. maxillaris V which will be described under
the r. mandibularis V.

GANGLIA AND NERVES OF SQUALUS 29

7. RAMUS MANDIBULARIS V

The r. mandibularis V (figs. 1 and 4, R.Md.V) arises from the
distal and ventral end of the Gasserian ganglion slightly pos-
terior to the origin of the r. maxillaris V. Its general course is
ventral and posterior. This nerve, like the r. maxillaris, is
large and easily followed at this stage. The first twig given off
(fig. 1, Mo.1) runs dorsal and mesial to enter the primordium
of the mandibular muscles. This primordium lies on the mesial
side of the nerve throughout its whole course and all the motor
twigs run mesially to enter it, while the sensory twigs have a
lateral direction. The second twig (fig. 1, S./) runs dorsally and
laterally to the ectoderm and is general somatic sensory. The
third twig (fig. 1, R.Com.) runs ventrally and slightly laterally
and joins the r. maxillaris V, as mentioned above.

There can be little doubt from the character of the connec-
‘tion of this anastomosing branch that its fibers run from the
mandibularis to the maxillaris and that, since the maxillaris
supplies neither lateral line organs nor muscles, the fibers are
somatic sensory and destined for the ectoderm, although they
could not be followed after entering the r. maxillaris. The
fourth (fig. 1, Mo.2) and fifth (fig. 1, Mo.3) twigs are motor and
enter the primordium of the mandibular muscles. Their course
after leaving the main nerve is ventral and mesial. The sixth
twig (fig. 1, S.2) is sensory and runs to the ectoderm. The
seventh (fig. 1, Mo.4) is motor and arises nearly opposite the
sixth. It runs medially and enters the primordium of the
mandibular muscle. The remaining four twigs (fig. 1, S.3-6)
seem to be sensory; at leas: they do not enter the primordium
of the muscle, since they extend beyond the distal extremity
of the muscle. Neither do they enter the ectoderm but disap-
pear near the ectoderm and, like the terminal twigs of the r.
maxillaris, have the appearance of growing nerves. ‘There can-
not be much doubt that they are somatic sensory fibers.

30 F. L. LANDACRE

8. THE DORSAL LATERALIS GANGLION OF VII

This large ganglion (figs. 1 and 4, G.L.VIID.) is triangular in
form with the r. oph. sup. VII arising from its anterior angle,
the r. bucealis arising from its ventral angle and the root of the
ganglion representing the third somewhat truncated angle. It
lies lateral to the Gasserian ganglion, which it conceals in part
from the lateral view and comes into contact with the distal
and ventral end of it where the r. max. V andr. mand. V. arise.
There is, however, no fusion at this stage. On its posterior
and dorsal border it comes into contact with the anterior end
of the VIII ganglion. This point of contact consists of a rather
close fusion in the specimen plotted, but in a 20 mm. embryo
the line separating the two masses of cells is quite distinet and
the two roots can be identified up to the point where they enter
the brain wall.

The root of the dorsal lateral line ganglion of VI! (fig. 1,
Rt.L.VIID.) is massive and enters the brain as the most ante-
rior division of the large root, of which the root of the auditory
ganglion and those of the remaining ganglia of VII compose the
posterior division. ‘These relations are not so evident in the
specimen plotted as in a 20 mm.’ embryo where there is less
fusion. However they can be made out after seeing them in the
younger specimen.

9. RAMUS OPHTHALMICUS SUPERFICIALIS VII

The r. oph. sup. VII (figs. 1, 2 and 3, R.O.S.VIZ) runs from
the anterior angle of the dorsal lateralis VII ganglion and forms
a great semicircle curving around the anterior border of the eye
and terminating at a point nearly ventral to the middle ot the
eye and near the olfactory capsule. Its position is always quite
near the ectoderm. It is a pure lateral line nerve and supplies
fibers to two large primordia of lateral line organs (fig. 1, L.1
and 1.2). The first of these lies dorsal to the eye and the nerve
gives off three well defined twigs, the most posterior of which
divides as it enters the primordium. This primordium evi-
dently represents the most posterior organs of the supraorbital

GANGLIA AND NERVES OF SQUALUS 31

line. The second primordium lies ventral to the anterior border
of the eye and just anterior to the nasal capsule. There are
six or seven twigs given off to this primordium which represents
the anterior supraorbital lateral line organs.

10. RAMUS BUCCALIS VII

The ramus buccalis VII (figs. 1 and 4, R.B.VII), a pure lat-
eral line nerve, arises from the ventral angle of the dorsal later-
alis VII ganglion. It pursues a course directly ventral and
slightly posterior to that of the r. max. VII, which it conceals
partially from the lateral view. At the point of exit of this
ramus from the ganglion and in the angle formed by the r.
buccalis and r. oph. sup. VII on the anterior. border of the gan-
glion arise two short twigs which run laterally to a primordium
of a lateral line organ (fig. 1, Z.1). This is apparently the
-primordium of the most posterior organs of the infraorbital line.
When the r. buccalis reaches the level of the ventral border of
the lens it gives off a twig to a primordium of lateral line organs
(fig. 1, 1.3), after which it runs slightly cephalad and ventral
to end under the posterior border of the eye. Near its termina-
tion it gives off a twig to a second primordium of lateral line
organs (fig. 1, £.4). Beyond this point it becomes an extremely
delicate twig and disappears while in contact with the ecto-
derm. While there are no primordia of lateral line organs be-
yond this point, the relation of the terminal ramus to the ecto-
derm leaves no doubt that it is lateralis in type.

At the opposite posterior border of the ganglion and at a
slightly more dorsal level near the point of contact between the
dorsal lateralis ganglion with the auditory ganglion arises a
second twig (fig. 1, R.O.), the first three divisions of which in-
nervate a lateral line primordium (fig. 1, L.2) which is located
where the supraorbital and infraorbital lines will probably join.
This cannot be stated definitely, of course, since the lateral line
organ primordia are discontinuous at this stage, as in Amelurus
(Landacre, ’10). It would be interesting to follow the history
of these primofdia in a close series in view of the author’s hypo-
thesis based on a study of Lepidosteus (Landacre and Conger,

a2 F. L. LANDACRE

13) that lateral line primordia arise from discontinuous areas
rather than from a continuous area on the ectoderm. After
supplying three twigs to the lateral line primordium mentioned
above, the ramus continues beyond this point and disappears
near the anterior border of the spiracular gill cleft. The nature
of this terminal twig could not be determined. This nerve is
identified provisionally as the r. oticus VII.

11. THE ACUSTICO-FACIALIS GANGLIONIC COMPLEX

These ganglia are rather closely fused, especially at their
proximal ends, in the specimen plotted, but with the aid of a
20 mm. embryo the relations seem to be intelligible. The genic-
ulate (fig. 1, G.Gen.) and ventral lateralis VII (fig. 1, G.L.VII
V) form the ventral portion of a V-shaped mass, of which the
auditory ganglion (fig. 1, G.Aw.) forms the dorsal arm. The
apex of the V projects cephalad and is formed by the point of
union of these two masses near their roots. The apex of the V
is In contact with the dorsal lateralis VII ganglion on its posterior
surface. The ventral arm of the V extends caudad and slightly
ventral, while the dorsal arm formed by the auditory is approxi-
mately horizontal. The most anterior member of the group is
the geniculate, which lies on the ventral and anterior border
of the ventral arm of the V. The ventral lateralis VII les
slightly dorsal and lateral to the geniculate partly concealing the
geniculate from a lateral view. The VIII ganglion is partly
concealed by the auditory vesicle.

The root of this complex enters the brain along with that of
the dorsal lateralis VII ganglion and occupies a position posterior
and mesial to the root of that gariglion. Reading from posterior
to anterior the first root encountered, that of the auditory (fig.
1, Rt.Aud.), lies lateral to the two succeeding roots and enters
in conjunction with that of dorsal lateralis VII. The next root
(fig. 1, Rt.Gen.) encountered is that of the geniculate accom-
panied by the motor fibers of the r. hyomandibularis. The third
root (fig. 1, Rt..VII V) encountered is that of the ventral
lateralis VII. These last two roots mentioned leave the proxi-
mal end of the combined ventral lateralis VII and geniculate

GANGLIA AND NERVES OF SQUALUS 33

ganglia in the reverse order, 1.e., the root of theventral lateralis
is lateral and somewhat posterior in position and in their course
from the ganglion to the brain wall they cross so that the root
of the ventral lateralis ganglion enters more anterior than that
of the geniculate and motor portion of the r. hyomandibularis.
The sensory and motor fibers of the combined geniculate and
motor root could not be followed separately, since they form a
compact bundle.

12. THE AUDITORY GANGLION AND RAMI

The auditory ganglion (figs. 1 and 5, G.Auw.) will be treated
first, since its relations are much simpler than those of the re-
mainder of the complex. The ganglion is well isolated through-
out most of its extent, especially in the 20 mm. stage, and its
ventral and anterior boundary, which is the one in contact
with the remainder of the complex, can be recognized up to the
point where it comes into contact with the posterior border of
the dorsal lateralis VII; from this point the two masses of enter-
ing fibers are distinct but their accompanying cells cannot be
distinguished. The most anterior ramus arising from the audi-
tory ganglion, enters the auditory vesicle on its ventral and
lateral border, while the more posterior rami enter the vesicle
on the posterior and mesial border. The undeveloped condi-
tion of the auditory vesicle makes it difficult to identify these
rami definitely, since the sensory areas of the auditory vesicle
are not differentiated. The more anterior ramus seems to be
connected with the saccular portion and thetwo posterior rami
with the utricular portion.

13. THE GENICULATE GANGLION

The geniculate ganglion (figs. 1 and 5, G.Gen.), the most an-
terior ganglion of the VII and VIII complex, as mentioned
above under the general discussion of the VII and VIII com-
plex, lies ventral and slightly mesial tothe ventral lateralis VII.
The ganglion can be identified throughout its whole extent,
although it is in contact with the ventral lateralis VII. At the

34 F. L. LANDACRE

point of exit of the ramus hyomandibularis, however, the fibers
from the geniculate and from the ventral lateralis VII fuse into
a compact trunk in which the different components cannot be
identified. The roots of these two ganglia, as mentioned above
can be distinguished.

In form the geniculate ganglion is roughly triangular with
the root representing the dorsal angle, the origin of the ramus
-palatinus and the ramus pretrematicus representing the ven-
tral angle and the r. hyomandibularis representing the posterior
angle. In the specimen plotted no distinction could be made out
between general visceral cells and special visceral cells derived
from the epibranchial placode. In a 20 mm. embryo, however,
Reed (716) was able to identify these cells and there is, further,
in the specimen plotted a slight contact between the genicu-
late ganglion and the ectoderm at the point at which the pla-
code proliferated cells which were added to the ganglion.

14. RAMUS PALATINUS AND RAMUS PRETREMATICUS

These two nerves arise from the ventral angle of the genicu-
late ganglion where it rests on the anterior face of the spiracular
gill cleft. Just at the point of emergence of the larger r. pala-
tinus, a small twig, the ramus pretrematicus (fig. 1, R.Pr.VIJ)
or ramus prespiracularis VII, arises and immediately divides
running caudad along the anterior wall of the spiracular cleft.
Beyond this point the r. palatinus (figs. 1 and 5, R.Pal.VIT)
passes nearly ventral in direction, dividing into two twigs neither
of which reaches the endoderm of the pharynx. Both, however,
pass in a mesial and ventral direction and come into close rela-
tion with the endoderm. While several of the finer divisions
of these twigs can be identified last in the loose mesenchyme
near the pharyngeal endoderm, there is every reason, from the
behavior of these nerves in other types and the absence of
muscle primordia in their vicinity, for identifying them all as
visceral sensory nerves.

At the posterior angle of the ganglion there is given off, at
the point where the geniculate ganglion joins the ventral lateral
line ganglion, a ramus which immediately fuses so closely with

GANGLIA AND NERVES OF SQUALUS 30

fibers from the lateral line ganglion forming the ramus hyo-
mandibularis that the two components are indistinguishable.
Consequently the ramus hyomandibularis willbe described sepa-
rately, since it contains not only visceral sensory fibers from
the geniculate but lateral line fibers and motor fibers as well.

15. THE VENTRAL LATERALIS VII GANGLION

The ganglion identified as ventral lateralis VII (figs. 1 and
5, G.L.VII V.) occupies the position with reference to the
geniculate and auditory usually held in the embryos of fishes
and amphibians (Landacre, ’14) but is surprisingly large in pro-
portion to the single lateral line primordium innervated by the
r. hyomandibularis into which all the fibers from this ganglion
pass. This disproportion may be explained on the basis that,
while the r. hyomandibularis innervates in the adult at least
five lateral line organs in the hyomandibular line, only one has
appeared at this stage. The ventral lateralis VII is approxi-
mately as large as the geniculate and rather closely fused with
it, although, as stated in discussing the geniculate, it can at all
points in the contact be distinguished from it except in the
r. hyomandibularis, and further the root of the lateral line
ganglion can be traced into the brain where it enters with that
of the dorsal lateralis VII. The ventral lateralis ganglion is
crescent-shaped with the convexity on the ventral side and no
pure lateral line rami leave it at this time so that the evidence
for its identity except as presented above is not so definite as
for the other members of the acustico-facial complex of ganglia.

16. TRUNCUS HYOMANDIBULARIS

The truncus hyomandibularis (fig. 1, R.Hyo.VII) is a mixed
nerve quite compact in structure and easy to follow but some-
what difficult to analyze. It arises from the posterior fused
ends of the geniculate and ventral lateralis VII. It pursues a
course slightly ventro-caudad to a point where it gives off a
motor twig (fig. 1, Mo.1) to the primordium of the hyoid muscu-
lature, then turns directly ventral. I+ gives off next three twigs

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 1

36 F. L. LANDACRE

(figs. 1, S.1, 2 and 3) which run toward but do not reach the
endoderm. ‘They are certainly not motor and are apparently
visceral sensory. Beyond this point there is given off a twig
which runs slightly cephalad and dorsal to end on a primordium
of a lateral line organ (fig. 1, L.1). Opposite the lateral line
twig is given off a long motor twig (fig. 1, Mo.2) which runs
ventral and caudad and after giving off several motor twigs
enters the extreme end of the primordium of the hyoid muscles.
Between the lateral lme and the motor twigs arise two large
twigs (figs. 1, S.4 and 5) which run directly to the ectoderm.

The ectoderm at this point is slightly thickened but not
sufficiently differentiated to enable one to determine positively
whether the thickening is that of a lateral line primordium or of
gustatory organs. It has more the appearance of early gusta-
tory organs and I have identified these twigs as visceral sensory,
although so far as their appearance and mode of termination is
concerned, aside from the slight thickening of the ectoderm,
they might be general somatic sensory. ‘The evidence against
this view rests on the absence of any recognizable somatic sen-
sory ganglion on this nerve at this time and the absence of any
connecting ramus from the Gasserian ganglion. This is said to
be present in the adult.

In view of the fact that there are said to be not only general
somatic fibers in the VII which may come from the Gasserian
ganglion but that in certain types such as Amblystoma (Land-
acre, 714, note on p. 603) there are fibers of this character in the
VII and, further, that Norris (’13) has deseribed a general cu-
taneous ganglion on the VII, a careful search was made in the
type plotted for such a ganglion, especially in view of the diffi-
culty of determining ‘the character of the fibers mentioned
above. No isolated ganglionic mass aside from those already
described could be identified either on the 22 mm. or on older
specimens. However, the late differentiation of the general
cutaneous ganglia and the small size of their cells, making them
hard to distinguish from the indifferent cells found on the roots
of all nerves, render it unsafe to say that there are no such cells
or fibers in the VII nerve. This interesting point must be

GANGLIA AND NERVES OF SQUALUS BE

settled on older material than that at my disposal. From the
material at hand the evidence seems to be against such a view.
If they are found in other vertebrate types they should certainly
be expected in such a generalized type as the shark.

17. THE GLOSSOPHARYNGEAL GANGLION AND ROOT

The glossopharyngeal ganglion is elongated in its dorso-
ventral axis, extending from the middle of the medulla ventrally
and slightly caudad nearly to the level of the roof of the pharynx.
It contains two easily recognizable divisions; the proximal is
the lateralis IX ganglion (figs. 1 and 6, G.L.JX) and the distal
and ventral division is the visceral division or ganglion petro-
sum (figs. 1 and 6, G.V.JX). The proximal division extends
from the point of contact with the medulla to the point of ori-
gin of the ramus supratemporalis IX (figs. 1 and 6, R.St.1X).
The two ganglionic masses are in contact at this point and can-
not be distinguished with certainty but throughout the re-
mainder of the extent of the lateralis ganglion the visceral gan-
glion is represented by a fibrous root apparently not accom-
panied by ganglion cells. A short distance ventral to the origin
of the ramus supratemporalis the lateral line ganglion cells
cease and it could not be determined with certainty that no
lateral line fibers entered the truncus glossopharyngeus. No
lateral line primordia are innervated, however, by that nerve
beyond those mentioned below and presumably no lateral line
fibers enter it at this stage.

The root of the lateral line IX (fig. 1, Rt.L.7X+X) passes
dorsally, mesial to the posterior end of the auditory capsule,
along with visceral sensory and motor fibers of the. truncus
glossopharyngeus. In that part of their course between the
proximal end of the ganglion and the medulla both the lateral
line root and the visceral sensory and motor roots are fibrous
and form a compact bundle. Unless, however, the lateral line
fibers change their position in this region of the root, the vis-
ceral fibers, both motor and sensory, enter at a somewhat more
ventral level where they join a more mesial column than the

38 F, L. LANDACRE

lateral line fibers. The lateral line fibers join those of the
lateral line root of X and enter at a somewhat more dorsal
level, passing into a well defined column in contact with the
limiting membrane of the lateral wall of the medulla.

The ganglion petrosum or visceral IX (figs. 1 and 6, G.V.LX), »
as mentioned above, begins at the point of origin of the ramus
supratemporalis IX and extends ventrally and caudally to the
dorsal border of the gill pocket. Its distal end is still in con-
tact with the epibranchial placode (fig. 7, G.V.IX-+ Pl) and
cells are evidently being added to the ganglion in the specimen
plotted. From the distal end of the ganglion extends cau-
dally a large mass of cells (fig. 1, G.P.JX) closely in contact with
the ectoderm, which is apparently not yet fully incorporated
into the ganglion giving it a curious form. ‘The same appear-
ance is presented by the visceral portion of the VII ganglion
in a 26 mm. embryo. This mass will probably be incorporated
with the remaining cells to give the slender spindle-shaped gan-
glion of the adult. Throughout the whole extent of the petro-
sal ganglion the visceral motor component of the truncus glosso-
pharyngeus can be followed, but in the root of the ganglion,
motor and sensory fibers are so closely fused that they cannot
be separated. They enter the medulla somewhat more ventral
than the lateral line root but at the same anterior-posterior level
(fig. 1, Rt.Vis.IX).

18. RAMUS SUPRATEMPORALIS IX

The ramus supratemporalis IX (figs. 1 and 6, R.St.LX) arises
from the distal end of the lateralis IX ganglion, from which
point jt runs directly lateral then curves slightly posterior and
then runs dorsal and slightly anterior. The first twig is given
off a short distance from its exit from the ganglion and ends on
a small primordium of a lateral line organ (fig. 1, L.1). A sec-
ond small twig (not named on figure 1), arises at the same point
runs slightly more dorsal and comes quite close to the ectoderm
but does not enter it. The ectoderm is not modified at this
point and the nature of this twig could not be identified. It re-

GANGLIA AND NERVES OF SQUALUS 39

sembles a general cutaneous nerve but the absence of any iso-
lated general somatic ganglion argues against this view. It is
more probable that it is a special visceral sensory nerve such as
accompanies the ramus supratemporal IX in Menidia (Herrick,
99). The visceral sensory ganglion on IX is so situated that
fibers from that ganglion could readily enter the ramus supra-
temporalis. From the point of origin of these two twigs the
ramus supratemporalis curves dorsal and cephalad to end on the
primordium of a lateral line organ (fig. 1, L.2) situated almost
directly lateral to the proximal end of the ductus endolymphat-
icus.

19. RAMUS PHARYNGEUS AND RAMUS PRETREMATICUS Ix

These two rami arise together as one nerve from the middle
of the anterior border of the ganglion petrosum. The first
twigs to be given off are the pretrematic rami (figs. 1 and 6,
R.Pr.IX) which curve caudad and end on the epithelium of the
gill bar. The second ramus or ramus pharyngeus (figs. 1 and 6,
R.Ph.IX) turns ventral and mesial and after pursuing a much
longer course comes into direct contact with the endoderm of the
roof of the pharynx. All these rami are evidently visceral
sensory.

20. TRUNCUS GLOSSOPHARYNGEUS

The truncus glossopharyngeus (figs. 1 and 7, R.PO.LX) arises
from the distal end of the ganglion petrosum and is a combined
sensory and motor root containing visceral sensory and visceral
motor fibers so closely combined that they cannot be distin-
guished. This nerve runs ventral and slightly caudad to the
level of the floor of the pharynx. The first twig given off is
sensory, arises quite close to the ganglion and runs to the endo-
derm of the gill bar. The third twig (fig. 1, Mo.1) is motor
entering the primordium of the branchial musculature as do all
the motor twigs of this nerve. The second, fourth and fifth
(figs. 1, S.2, 3 and 4) are sensory and run to the epithelium of the
gill bar. The sixth twig (fig. 1, Mo.2) seems to be motor, as
does also the seventh and terminal twig. However, the muscle

40 F, L. LANDACRE

primordia are at this time poorly developed and one or more
of these twigs may be visceral sensory. No lateral line organ
primordia are present at any point innervated by the truncus
glossopharyngeus and all its sensory fibers seem to be visceral
sensory.

21. THE VAGUS GANGLIA

The vagus ganglion is irregular in form. It extends in the
longitudinal axis from the point of entrance of the IX caudad to
the level of the third spinal ganglion. Its dorsal portion is thin
from mesial to lateral but is continuous from the point of en-
trance of the IX to a point directly over the dorsal border of the
second true gill slit, from which point it extends caudally as a
narrow strand of cells which is continuous with the first spinal
ganglion. This proximal portion of the ganglion, which con-
tains root fibers and the primordium of the somatic sensory or
jugular X ganglion, is continued ventrally by five branchial
ganglia. The anterior branchial ganglia are well isolated but the
posterior ones are somewhat more fused. The proximal portion
of each of the first three branchial ganglia is chiefly lateral line
plus root fibers and will be designated as lateralis ganglia X,,
X., X;. The distal portion of all five is visceral sensory and
will be designated as visceral ganglia X,, Xs, X3, Xu, Xs.

The lateral line rami arise from the dorsal and lateral borders
of the proximal or lateral line portions, while from the distal or
visceral portions arise the pretrematic, posttrematic and motor
rami. All the branchial ganglia extend from their proximal
ends in a ventral and caudal direction and all the visceral sen-
sory ganglia of X are still in contact with their respective epi-
branchial placodes and are still receiving cells from these sources
as in the 20 mm. embryo (Reed 716). The proximal portion of
the first’ two branchial ganglia (including the lateralis ganglion
X, and the jugular ganglion associated with branchial X, and
X,) and nearly all of the lateralis ganglion X. (including of
course all roots of X) lie mesial to the primordium of the somatic
musculature. The distal portiens of visceral X, and X, and
X, lie lateral to the muscle primordium while the lateral line
ganglion X; and the remainder of visceral X, and X; lie ven-

GANGLIA AND NERVES OF SQUALUS 41

tral to this muscle primordium. The epibranchial portions of
X, and X; which are still attached to the ectoderm are more
lateral in position and not directly under the muscle primordium.
The muscle primordium is pierced by the proximal end of the
visceral ganglion of branchial Xo».

22. THE VAGUS ROOTS

The analysis of the vagus roots in detail beyond the number
and point of attachment is very difficult and sometimes impos-
sible in a 22 mm. embryo.

The first root of X (fig. 1, Rt.L.IJX-+X) is a lateral line root
and enters along with the lateral line root of LX just dorsal to
the visceral sensory and motor roots of IX. Posterior to this
most anterior root are three roots much alike in appearance.
These three roots arise anterior to the level of the point of origin
of the r. supratemporalis X. The second, third and fourth
roots arise from the thicker anterior portion of the X ganglion,
while the first or lateralis root, joins the brain wall only after
a rather long course cephalad as is usually the case with the
lateral line root of X which connects the X ganglion with the IX.

Each of the second, third and fourth roots is round in trans-
verse section, contains a rather large number of cells on the
anterior face of the root, and as it enters the brain wall, divides
into two divisions one of which turns slightly dorsally and the
other ventrally and mesially. Owing to the very minute size
of the general cutaneous rami of X the dorsal division is identi-
fied provisionally as visceral sensory and the more ventral divi-
sion as visceral motor. The more dorsal fibers do not enter the
tract which the lateral line fibers of IX and X from the first
root enter, but do enter the same column entered by the more
ventral or visceral root of IX. Caudally of the first four roots
there are twelve to fifteen roots (not named on figure 1) arising
from the more attenuated caudal portion of the ganglion, all of
which show the same composition as the second, third and fourth.
They are slightly smaller and each root on entering the medulla
divides into a dorsal and a ventral branch. The dorsal division
becomes progressively smaller in the more posterior roots, and

42 F, L. LANDACRE

some of the posterior roots on the opposite side from that
plotted, are made up exclusively of the ventrally directed roots.
These roots are identified provisionally, for reasons given above,
the dorsal as visceral sensory, and the ventral as visceral motor.

23. THE JUGULAR XTH GANGLION

The mass of cells identified as the jugular or general cutaneous
ganglion of X (figs. 1 and 7, G.J.X.) is situated in the proximal
portion of the X complex. It les lateral to the proximal at-
tenuated fibrous root and extends from the level of the anterior
end of the lateralis ganglion on branchial X,, to the middle of
the lateralis ganglion on X,. It lies dorsal to both these ganglia
where it comes into contact with them and, except for the fibrous
bundle running between X, and X,, it forms the ventral bound-
ary of the Xth complex in this region. This ganglion is com-
posed of small cells and is apparently in a very immature con-
dition, as is the same ganglion in Amelurus and Lepidosteus
(Landacre, ’10 and 712) in approximately the same stage of
development. No root fibers from this ganglion could be
identified.

The mass of cells described above as the jugular ganglion is
found in a 20 mm. embryo, but is better defined in a 25 mm.
embryo, where it is well isolated from the ganglia lateralis Xy
and X, with which it is in contact on its posterior and ventral
surface. In a 30 mm. embryo this mass of cells is not isolated
but seems to be fused with lateralis X;. If this interpretation is
correct, it has migrated, between the 25 mm. stage and the 30
mm. stage, from a position mesial to the somatic muscle pri-
mordium to a position lateral to this primordium. This is
equivalent to a migration from an intracranial to an extra-
cranial position.

24, RAMUS AURICULARIS

The ramus auricularis (figs. 1, 9, 10 and 11, R.Aur.) or ramus
cutaneous dorsalis vagi has not been identified with certainty.
There are certainly no well defined nerves of this character
arising from the dorsal and proximal portion of the Xth gan-

GANGLIA AND NERVES OF SQUALUS 43

glionic complex which is the usual place of origin of this nerve
in embryos. All the embryos at my disposal from 20 mm. to
30 mm. have been examined repeatedly with the greatest care
and no nerves pass dorsally to the ectoderm from the proximal
portion of the ganglion. ‘This is true of the 25 mm. embryo,
where the mass of cells identified above as the jugular ganglion
is best isolated.

There are, however, in the 22 mm. and 25 mm. embryos sev-
eral minute processes arising from the posterior and dorsal bor-
der of the root of X which could not be followed farther than
the width of one ganglion cell but present the appearance of
very immature nerves. They are constant in neither number
nor position and vary in both on opposite side of the same
embryo and are not included in the reconstruction in figure 1.

In the 30 mm. embryo a nerve arises apparently from the root
of X near the first spinal ganglion but not from it and runs,
cephalad passing along the ventral border of the anterior end of
the primordium of the somatic musculature then runs to the
ectoderm of the mid-dorsal region of the head where it ends
just posterior to the area supplied by the most dorsal rami of
supratemporalis IX and X. ‘This nerve is very small and could
not be located on the opposite side of the same specimen. It
has the usual distribution of a r. auricularis In embryos except
that it arises too far posterior.

There is in addition in all embryos from 22 mm. to 30 mm. in
length a nerve (figs. 1, 9, 10 and 11, R.Aur.) running out with
the lateral line ramus arising from the ganglion lateralis X»._ It
arises with the lateral line nerve but soon separates from it and
pursues a course caudad parallel to it but slightly more ventral
in position than the lateral line nerve to a point near the third
spinal ganglion, when it turns dorsal and is distributed to the
ectoderm. Its relation to the lateral line nerve is not entirely
clear since it seems to form anastomoses with it but some of the
fibers of this nerve are distributed to the ectoderm at points
where there are no lateral line primordia at the stages studied.

The fibers seem to pursue a course from the distal end of the
jugular ganglion proximally along the dorsal border of lateralis

44 F, L. LANDACRE

X;. This nerve is identified provisionally as the ramus auricu-
laris. Both the jugular ganglion and its nerve are surprisingly
small and there seems to be a large amount of ectoderm on the
posterior portion of the head devoid of general cutaneous in-
nervation at the stages studied. Both the nerves identified as
supratemporal rami were carefully examined for general cu-
taneous fibers without success. The morphology of the r. au-
ricularis X has been treated fully by Herrick (99, p. 267-273).
A more detailed description of this nerve requires older. material
than that at my disposal.

25. THE GANGLION LATERALE Xi

There are three lateral line ganglia on the Xth nerve occupy-
ing the proximal portions of the first four branchial ganglia
which will be designated in the description as Laterale X4, X»,
“and X; respectively. The most anterior or laterale X, (figs. 1
and 7, G.L.X,) is situated on the proximal portion of branchial
X,. It extends from the anterior end of the jugular ganglion
ventrally and caudally along the root fibers of branchial X,
almost to the proximal end of the ganglion viscerale X,. It
does not at this stage come into contact with that ganglion,
there being a short fibrous root of viscerale X; containing no
cells. On its proximal end it is in contact with the jugular
ganglion to which it is ventral in position.

Throughout the whole length of the lateralis X, ganglion the
root of the ganglion viscerale X, and motor X le mesial to it.
This ganglion is compact and nearly round in transverse sec-
tion except at the origin of two lateral line rami, where a large
mass of cells projects laterally toward the ectoderm making the
ganglion triangular in form. Its cells are large and readily
distinguished from those of the jugular X, with which it is in
contact dorsally. The cells of this lateral line ganglion are not
readily distinguished from those of the visceral ganglion, but
this produces no confusion here since these two ganglia are not
in contact.

GANGLIA AND NERVES OF SQUALUS 45
26. RAMUS SUPRATEMPORALIS VAGI

The ramus supratemporalis X (figs. 1 and 7, R.St.X) arises
near the middle of the lateral surface of the ganglion laterale X,
from a prominent mass of cells that extends from the ganglion
nearly to the ectoderm. Immediately after its origin the ramus
divides into two twigs, posterior and anterior, the posterior
nerve running nearly directly caudad and the anterior larger
twig curving dorsal and cephalad. The anterior twig innervates
the primordia of two lateral line organs, of which the proximal
one (fig. 1, L.2) lies directly over the point of origin of the com-
bined ramus and at the dorso-ventral level of the attachment
of the roots of X to the medulla. The other organ (fig. 1, L.1)
lies farther dorsal and anterior at the level of the entrance of
the second root of X and at a dorso-ventral level of the dorsal
border of the auditory vesicle. The posterior twig supplies a
long primordium of lateral line organs (fig. 1, 1.3), to which it
gives off two twigs, indicating that there will be at least two
organs derived from this primordium in the adult. This lat-
eral line primordium lies directly over the visceral ganglion of
branchialis X, and just ventral to the level of the posterior
roots of X. It is placed diagonally to the long axis of the body
with the anterior end more dorsal. .

In a 20 mm. embryo this primordium and the more posterior
of the two innervated by the anterior twig are continuous, while
the most anterior lateral line primordium is not present. The

rapid appearance of these primordia at this time renders diffi-
cult the exact identification of the organs as belonging to the
main head lateral line or as being accessory. The occipital
lateral line commissure is not yet formed and these organs, in-
cluding the one innervated by supratemporalis IX, are identi-
fied as the last four organs of the head posterior to the junction
of infraorbital and supraorbital lines, the primordium inner-
vated by the most proximal and posterior twig of dorsal lateralis
VII being considered as the point of future junction of supra-
orbital and infraorbital lines. The primordium innervated by

46 F. L. LANDACRE

the posterior twig of lateralis X, may belong to the main body
line. It les much more ventral in position than those inner-
vated by the anterior twig.

27. THE GANGLION LATERALE X,

The lateralis ganglion (figs. 1 and 8, G.L.X,) associated with
the second branchial ganglion of X resembles that on the first
branchial ganglion of X in its relations to other members of
the complex. It is, however, longer and placed parallel to the
long axis of the body. It is not so definitely confined to its
branchial ganglion as that on X,, since its posterior end is con-
tinuous with lateralis X;. Its proximal and anterior end is
mesial to the somatic muscle primordium and is in contact with
jugular X, to which it hes ventral. Throughout its whole
course it lies lateral to the fibrous motor and sensory root of the
remaining visceral ganglia of X. Its posterior and distal end
lies lateral to the somatic muscle primordium. On its extreme
distal and posterior end its cells are continuous with those of
lateralis X; and throughout the posterior half of its extent it is
in contact with the visceral ganglion of branchial X;, to which
it lies dorsal. Throughout the proximal portion of this contact
the two ganglia are closely fused and, owing to the similarity in
size of their cells, indistinguishable. However, throughout the
greater portion of the contact the combined ganglia are indented
both on the mesial and on their lateral surfaces, indicating the
line of separation between them. Near its posterior end this
ganglion gives rise to the first ramus lateralis X.

28. FIRST RAMUS LATERALIS X

Owing to the position of the lateral line primordium inner-
vated by the lateral line ramus arising from this ganglion, it is
identified as the first primordium of the main body line and its
nerve as the first lateral line ramus of X, rather than as a homo-
logue of the more anterior ramus supratemporalis X. The
study of this nerve in much older material, however, may show
it to be homologous to a supratemporal ramus.

GANGLIA AND NERVES OF SQUALUS 47

This nerve (figs. 1, 9, 10 and 11, R.L.X.1) arises near the
posterior end of ganglion laterale X, accompanied by the nerve
identified as ramus auricularis. Immediately after its exit from
the ganglion it gives off a small twig to the lateral line primor-
dium. Posterior to this twig at least two more twigs are given
off to the same primordium which extends caudad to the level
of the first spinal ganglion. Posterior to this point the ter-
minal twig of this nerve can be followed, but it does not end
on a lateral line primordium. There is nothing in its relation
to the ectoderm, other than the absence of lateral line primordia
posterior to this point, by which to identify it. The extremely
immature condition of the general cutaneous rami of X make
it difficult to be certain of its identity. The only other possi-
bility apparently is that it might be a visceral sensory twig
destined for terminal buds on the ectoderm. This can be de-
termined, however, only on older material and it is indicated on
the plot as a lateral line nerve.

29 THE GANGLION LATERALE X;

This ganglion (figs. 1, 9, 10 and 11, G.L.X;) shows many of
the characteristics of lateralis X,. It is longer and placed
slightly more diagonally in the body with its anterior end more
dorsal than its posterior end. The ganglion is round in trans-
verse section and lies throughout its whole extent lateral to the
primordium of the somatic musculature.

At its anterior and proximal end it is in contact and con-
tinuous with lateralis X» on its dorsal surface, while on its ven-
tral surface it is contact for a short distance with the visceral
ganglion of X». The remainder of its dorsal surface is free,
but on its ventral surface it is in contact first with the visceral
ganglion of X;, and at its posterior end for a short distance with
the visceral ganglion of X,. Its ganglion cells are much larger
than the visceral ganglion cells and their identification is easy.
The fibrous motor and sensory roots of the complex posterior to
this point lie on the ventral portion of lateralis X; instead of on
the mesial surface, as in the case of the two preceding lateral line
ganglia.

AS F, L. LANDACRE

30. SECOND AND THIRD RAMI LATERALES X

The lateral line trunk (figs. 1, 12 and 13, R.L.X.2) for the
body lateral line organs arises by two rami from the dorsal and
lateral surface, near the posterior end of lateralis X;, the gan-
glion cells continuing caudad for a short distance beyond the
second of the two twigs which make up this ramus. They arise
near together and on the side plotted remain distinct but on the
opposite side of the same embryo after a short course as separate
twigs combine into a single ramus which retains nearly its origi-
nal size back to the level of the middle of the yolk stalk, where
my series ends.

From the anterior twig several small branches run to thicken-
ings of the skin which were identified as primordia of lateral line
organs.

31. THE GANGLIA VISCERALIA X; TO X,

The visceral sensory portions of the branchial ganglia of X
(fig. 1, G.V.X-. to X;; figs. 8 to 12, G.V.X, to X; + Fl) are all
similar in form with the exception that the first three are much
better isolated than the remaining two. All give rise to pharyn-
geal and pretrematic and posttrematic rami. All these visceral
ganglia are still attached to the epibranchial placodes of their
respective gills and all are still receiving cells from the ecto-
derm. The attachment of the last three is much more inti-
mate and more extensive in proportion to the size of the gan-
glion than that of the first two. The last two ganglia are much
more poorly defined with more uneven borders than the anterior
ones which are sharply isolated from the surrounding mesen-
chyme. The first three ganglia are spindle-shaped, round in
transverse section, placed diagonally in the body with the proxi-
mal end slightly anterior, and end distally in the enlargement
formed by their fusion with their epibranchial placodes.
The posterior extremities of the last two are similarly attached,
but these two ganglia are fused in their proximal portions.
From the point of attachment of each ganglion to its placode
there is a large cellular mass which extends caudad from the
main body of the ganglion. This mass presumably will be

GANGLIA AND NERVES OF SQUALUS 49

incorporated into the main body of the ganglion, as in the vis-
ceral ganglion of VII. There are at this stage five of the true
gill slits open, but there is posterior to the last gill slit an epi-
branchial ganglion. This is interpreted as a vestigial fifth
branchial ganglion of X.

32. FIRST TRUNCUS BRANCHIALIS X

There are three rami arising from the distal and ventral end
of the first ganglion branchialis vagi. The first nerve is the
ramus pharyngeus (fig. 1, R.Ph.X,) which runs mesially and
ventrally to the roof of the pharynx. The second ramus is the
ramus pretrematicus (figs. 1 and 8, R.Pr.X,) which is sensory
and comes into contact with the anterior border of the gill slit.
Both these rami arise from the ganglion where it is still in con-
tact with the epibranchial placode and will doubtless contain
gustatory fibers.

The posttrematic nerve (figs. 1 and 9, R.Po.X,) is large and
arises from the extreme distal end of the ganglion. It pursues a
course ventral and slightly caudal in the gill bar. The first
two rami (fig. 1, Mo.1 and Mo.2) given off are motor, the third
(fig. 1, S.1) sensory, and the fourth and fifth or terminal are
again motor (fig. 1, Mo.3 and Mo.4). No posttrematic sensory
ramus similar to that on IX could be detected.

33. SECOND TRUNCUS BRANCHIALIS X

The second branchial trunk is quite like the first, possessing
a ramus pharyngeus (figs. 1, R.Ph.X2) and ramus pretrematicus
(fig. 1, R.Pr.X.), both sensory and arising from that portion
of the visceral ganglion fused to the epibranchial placode. The
posttrematic nerve (figs. 1 and 10, R.Po.X,) arises from the dis-
tal and ventral end of the ganglion and runs ventrally from this
point. The first twig given off is motor, the second sensory,
while the third and terminal twig is motor. All sensory twigs
from the first and second pretrematic nerves turn laterally
toward the ectoderm, while the motor twigs turn mesially to
the primordium of the branchial musculature.

50 F, L. LANDACRE
34. THIRD TRUNCUS,BRANCHIALIS X

The third branchialis ramus repeats the pattern of the sec-
ond, having a ramus pharyngeus (fig. 1, R.Ph.X;) and ramus
pretrematicus (figs. 1, 11, R.Pr.X;) with the same relation to
the epibranchial ganglion. The posttrematic ramus (figs. 1, 12,
R.PoX;) seems to lack sensory fibers; at least none could be
detected. The terminal ramus of the third posttrematic nerve
curves forward to end on the primordium of the branchial mus-
culature, and, in fact, the whole ramus forms a gentle curve
cephalad. The rami of the fourth branchial nerve are small,
particularly the ramus pharyngeus and the ramus pretrematicus.

35. FOURTH TRUNCUS BRANCHIALIS X

The first division of the fourth branchial nerve arises as two
minute twigs in the position occupied by the ramus pharyngeus
and ramus pretrematicus on the more anterior branchial gan-
glia. They (fig. 1, R.Ph.X,and R.Pr.X,) are identified as these
nerves although their minute size prevents their being followed
to their terminations. The ramus posttrematicus (figs. 1, 13,
R.PoX:) is much easier to follow and pursues a course behind
the last gill slit ventrally, then curves slightly forward to pass
to the heart, where it can be identified last near the wall of the
pericardium. It is identified provisionally as the ramus Car-
diacus X.

36. THE GANGLION VISCERALE X;

The fifth branchial ganglion (figs. 1, 12, 13, G.V.X;) extends
caudad from the fourth as a large mass of cells fully as large as
any of the preceding branchial ganglia. At its posterior end
it is fused with an indertation of the ectoderm, as are the more
anterior branchial ganglia at their attachment to the placodes
(fig. 1, G.P.X;). The attachment is small and there is no cor-
responding pharyngeal evagination. The large bundle of fibers
that has accompanied all the remaining branchial ganglia lying
on their ventral or mesial surfaces, disappears in this ganglion.

GANGLIA AND NERVES OF SQUALUS 51

37. RAMUS VISCERALIS X

There is in the specimen plotted no ramus visceralis arising
from the posterior end of the ganglion visceralis X; such as
Neal (14, plate 7, fig. 36) plots in a 25 mm. embryo. Neither
is there in my 25 mm. embryo any well defined ramus visceralis
or vagus nerve, although the posterior end of this ganglion is
ragged and seems to give rise to a very immature nerve trunk.
This is rather surprising in view of the condition of the first
two spinal nerves and the anterior sympathetic ganglia and
rami communicantes, all of which are well formed in the 22 mm. .
embryo. There are several minute twigs arising from the ven-
tral and mesial surfaces of this ganglion but their destination
could not be determined. Their general course is mesial but
they are quite short.

In a 30 mm. embryo, however, the ramus visceralis X (fig. 1,
R.Vis. X) is well formed and runs directly ventrad and caudad
and has the usual distribution of the vagus nerve.

SUMMARY AND DISCUSSION

Squalus acanthias possesses at the stage of 22 mm. eighteen
separate cerebral ganglia. Of these eighteen ganglia the gan-
glia profundus, Gasserian, lateralis VII dorsalis, and acusticus,
are isolated so that the nerves arising from them are pure and
readily identified. The remaining ganglia are in contact with
and sometimes fused with other ganglia so that, while they can
be identified, they are not separate as are those mentioned
above. The nerves arising from these ganglia which are in
contact are mixed with motor fibers only except in the follow-
ing cases. The nervus terminalis is combined with the olfac-
tory, the ramus auricularis X seems to be fused with the ramus
lateralis X,, and the hyomandibularis contains both lateralis
and visceral sensory fibers. The following table gives schemati-
cally the ganglia and rami as identified.

The nervus terminalis is placed provisionally under the general
cutaneous component where it is classified by Johnston (’06, p.
106). Brookover (’10), however, presents strong evidence for

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 1

52

F. L. LANDACRE

TABLE I

Showing the ganglia and rami of a 22 mm. embryo of Squalus acanthias

COMPONENT GANGLIA LATERAL LINE NERVES NERVES
No ganglion......... Olfactory
‘Rernainvalliisiapeerieere Terminalis?
Prokundtsseeree ase: R. Oph. Prof.
R. Oph. Sup. V
(Gasseriane oso" oe R. Max. V
j R. Mand. V
R. Oph. Sup. VII
hate Villidorse. se eee By Gerae. iil
ve Otamy aul
Lat. VIL vents...04.- \ R. Hyom, Vil
Re Ralsavalil
Geniculate.......... a Prett. VII
R. Hyom. VII
ACUSUICUSS-Eeeen eee Acusticus .
Watery 6 ie ccd R. Supt. IX
: 18, 12m, ID
ae or Viscer- i Deee tine
BASE Gk: sa BR ePospehie
IUPILATGe ss 4 ee R. Auricularis
IDEN Ket! Giegeoosen soni! Leto tsb atts
WatenalXG-ceec eel Meola tem Nel
ibyaemall SGocgesse sooo! Jats. bakes UC, 2
R. Phar. Xy
WViscenralexa.. eee B Prett. Xy
1RY. Postt. XG
R. Phar. Xe
WiASCeraliXG: 2... 2058 R Prett. Xe
R. Postt. X»
R. Phar. X3
Wiscerall XG. 26 24: i Prett. X3
R. Postt. X3
(R. Phar. X4
; i Rie enetoaeses
Waiscerall Xie pete: ‘5 Poet
\ or R. Car
WViscerdllents-. ee R. Visceralis

GENERAL CUTANEOUS

VISCERAL NERVES

classifying this nerve as a sympathetic nerve rather than as a
general cutaneous nerve.

The roots of these ganglia, in sharp contrast with the nerves,
are all complex and enter the brain, aside from the nervus ter-

GANGLIA AND NERVES OF SQUALUS 53

minalis, in three chief divisions: the Gasserian and the profun-
dus root complex, the VII—VIII root complex, and the [IX—-X
root complex.

The most striking features of the embryonic ganglia of Squalus
in comparison with Ameiurus and Lepidosteus (Landacre, ’10
and 712) are, first, the presence of three distinct lateral line
ganglia on X and, second, the immature condition of the gen-
eral cutaneous ganglia on X and the absence of a separate ramus
auricularis, this nerve being fused with the ramus lateralis X.1.
The presence on IX of a general cutaneous ganglion in several
vertebrate types including apparently man, and, particularly,
the presence of general cutaneous ganglia and fibers in both
VII and IX in Petromyzon (Johnston, ’05) would lead one to_
expect them in Squalus. A careful search failed to demon-
strate them. However, since the general cutaneous compo-
nent on X matures very late and the jugular ganglion is always
small and ill defined, a study of older material may show cu-
taneous ganglia and fibers in both VII and IX.

The special visceral or gustatory cells on VII, IX and X
cannot in a 22 mm. embryo be distinguished sharply from the
general visceral or branchial cells, although all of the bran-
chial ganglia on X are still in contact with their respective epi-
branchial placodes. In a 20 mm. embryo (Reed, ’16) the proc-
ess of contribution of cells by the epibranchial placodes and
their metamorphosis into ganglion cells can be observed.

The terminal buds or gustatory organs seem to be late in
appearance. They are present in the 30 mm. embryo but
material fixed in vom Rath is not particularly favorable for their
identification and their number and position are not described
in this paper. Taste buds could not be identified with certainty
in the embryo plotted.

The branchial ganglia of Squalus are well isolated in compari-
son with Ameiurus and Lepidosteus at similar stages. All of the
visceral ganglia on IX and X and most of the lateral line gan-
glia are in finger-like processes extending ventrally above their
respective gill slits. In Ameiurus and Lepidosteus and particu-
larly in Rana there is a large mass of cells from which the suc-

54 F, L. LANDACRE

cessive branchial ganglia extend ventrally. In Squalus this
mass of cells is replaced by the fibrous roots of IX and X and
contains no cells except those of the jugular ganglion which are
situated here up to the 25 mm. stage.

The very small size of the r. oph. sup. V in comparison with
the large r. oph. prof. is worthy of note. The disappearance
of the profundus nerve in higher forms is certainly not fore-
shadowed in Squalus. If we compare with these conditions the
condition in Amphibia (Coghill, ’01, 02, ’06), where the oph-
thalmic nerve is treated as an ophthalmicus profundus, it raises
an interesting question concerning the relationship of these
forms with the higher vertebrates which have apparently lost
both the ophthalmicus profundus ganglion and nerve. How-
ever, the ophthalmicus profundus ganglion is said to be present
in the cat between the stages of 10 and 21 somites but its fate
is not described (Shulte and Tilney, 15).

The usual conception of the sharks as generalized verte-
brates is borne out by the condition of the ganglia and nerves
with the possible exception of the general cutaneous component.

GANGLIA AND NERVES OF SQUALUS 55

LITERATURE CITED

Brooxover, Cuas. 1908 Pinkus’ nerve in Amia and Lepidosteus. Science,
N.S., vol. 27, p. 913.
1910 The olfactory nerve and nervus terminalis and the preoptic
sympathetic system in Amia. Jour. Comp. Neur., vol. 20.
Brookover, Cuas. AND Jackson, T. S. 1911 The olfactory nerve and the
nervus terminalis in Ameiurus. Jour. Comp. Neur., vol. 21, no. 3.

Cocuitt, G. E. 1901 The rami of the fifth nerve in Amphibia. Jour. Comp.
Neur., vol. 11, no. 1.
1902 The cranial nerves of Amblystoma tigrinum. Jour. Comp.
Neur., vol. 12, no. 3.
1906 The cranial nerves of Triton taeniatus. Jour. Comp. Neur.,
vol. 16, no. 4.
1916 Correlated anatomical and physiological studies of the growth
of the nervous system of Amphibia. II. The afferent system of
Amblystoma. Jour. Comp. Neur., vol. 26, no. 3.

Herrick, C. Jupson 1899 The cranial and first spinal nerves of Menidia. A
contribution upon the nerve components of bony fishes. Jour. Comp.
Neur., vol. 9, nos. 3 and 4.

Jounston, J. B. 1905 The cranial nerves of Petromyzon. Morph. Jahr., Bd.
34, Heft 2.
1906 The nervous system of the vertebrates. P. Blakiston’s Son
and Co., Phila.

Lanpacre, F. L. 1907 On the place of origin and method of distribution of
taste buds in Ameiurus. Jour. Comp. Neur., vol. 17.
1910 The origin of the cranial ganglia in Ameiurus. Jour. Comp.
Neur., vol. 20.
1912 The epibranchial placodes of Lepidosteus osseus and their rela-
tion to the cerebral ganglia. Jour. Comp. Neur., vol. 22, no. 1.
1914 Embryonic cerebral ganglia and the doctrine of nerve compo-
nents. Folia Neurobiologica, Band. 8, Nr. 6.

Lanpacre, F. L. anp McLetian, Marie 1912 The cerebral ganglia of the
embryo of Rana pipiens. Jour. Comp. Neur., vol. 22.

Lanpacre, F. L. anp Conamr, A. C. 1913 The origin of the lateral line pri-
mordia in Lepidosteus osseus. Jour. Comp. Neur., vol. 23, no. 6.

Locy, Wiitttam A. 1899 New facts regarding the development of the olfactory
nerve. Anat. Anz., Band 16, Nr. 12.
1905 On a newly recognized nerve connected with the forebrain of
Selachians. Anat. Anz., Band 26, Nr. 2 and 3, p. 33-63; Nr. 4 and 5,
epi 123.

MirropuHanow, P. 1893 Etude embryogénique sur les Sélaciens. Arch.
Zool. Exp., Sér. 3, tome 1.

Neat, H. V. 1898 The segmentation of the nervous system in Squalus acan-
thias. A contribution to the morphology of the vertebrate head.
Bull. Mus. Com. Zool. at Harvard, vol. 21, no. 7.
1914 The morphology of the eye-muscle nerves. Jour. Morph., vol.

_ 25, no. 1; reprinted in Tuft’s College Studies, Sci. series, vol. 3, no. 4.

56 F. L. LANDACRE

Norris, H. W. 1913 The cranial nerves of Siren lacertina. Jour. Morph.
vol. 24, no. 2.

ReEep, Cartos J. 1916 The epibranchial placodes of Squalus acanthias. The
Ohio Journal of Science, vol. 16, no. 8.

Scammon, Ricnarp E. 1911 Normal plates of the development of Squalus
acanthias. In Keibel’s Normentafeln, Zwélftes Heft.

Suuutr, H. von W. anv TiLney, FrepEeRiIcK. 1915 Development of the neu-
raxis in the domestic cat to the stage of 21 somites. Annals of the
N. Y. Acad. of Sci., vol. 24, pp. 319-346.

GANGLIA AND NERVES OF SQUALUS 57

ABBREVIATIONS

At., Atrium

Au.Ves., Auditory vesicle

B., Base of brain

B.V., Blood vessel

B.A., Bulbus arteriosus

Dien., Diencephalon

D.End., Ductus endolymphaticus

Epiph., Epiphysis

G.Au., Auditory ganglion

G.Gass., Gasserian ganglion

G.Gen., Geniculate ganglion

G.J.X., Jugular ganglion of X

Gl. 1-6, Gill slits

G.L.VII.D., Dorsal lateral line gan-
glion of VII

G.L.VII.V., Ventral lateral line gan-
glion of VIT

G.L.IX, Lateral line ganglion of IX

G.L.X,, First lateral line ganglion of X

G.L.X»2, Second lateral line ganglion
of X

G.L.X3, Third lateral line ganglion of
x

G.P.IX Epibranchial ganglion of IX

G.P.X,-X;, Epibranchial ganglia of X

G.Pro., Profundus ganglion

G.V.IX, Visceral ganglion of IX

G.V.X,-X;, Visceral or branchial gan-
glia of X

G.V.X,to X;+ Pl., Visceral ganglia of
X plus the placodal ganglia on X

G.Sp. 1-3, The first three spinal ganglia

Hyp., Hypophysis

H.C., Head cavity

L. 1-6, Lateral line primordia

M., Muscle primordia

Mo. 1-6, Motor rami of cerebral nerves

No., Notochord

N. ITJ-IV-VI, Nerves III, IV and VI

N.Au., Auditory nerve

N.Olf., Olfactory nerve

N.Ter., Nervus terminalis

Par., Paraphysis

Ph., Pharynx

Pro. 1, 2, 3, Twigs of the profundus
nerve

R.Aur., Ramus auricularis

R.Com., Ramus communicans

R.B.VIT, Ramus buccalis VIL

R.Hyo.VII, Ramus hyomandibularis
VII

R:.L.X.1, First lateral line nerve of X

R.L.X.2, Second lateral line nerve of X

R.Md.V, Ramus mandibularis V

R.Mz.V, Ramus maxillaris V

R.O., Ramus oticus

R.O.S.V, Ramus ophthalmicus super-
ficialis V

R.O.S.VIT, Ramus ophthalmicus su-
perficialis VII

R.Pal.VII, Ramus palatinus VII

R.PhIX, Ramus pharyngeus [X

R.Ph.X,-X;, Pharyngeal rami of X

R.PotIX, Ramus posttrematicus [X

R.Po.X,-X;, Posttrematic rami of X

R.Pr.IX, Ramus pretrematicus [X

R.Pr.X,-X;, Pretrematic rami of X

R.St.IX, Ramus supratemporalis [IX

R.St.X, Ramus supratemporalis X

Rt.Aud., Root of auditory ganglion

Rt.Gass., Root of Gasserian ganglion

Rt.Gen., Root of geniculate ganglion

Rt.L.VII.D., Root of dorsal lateral
line ganglion of VII

Rt.L.VIT.V., Root of ventral lateral
line ganglion of VII

Rt.L.IX+X, Lateralis root of IX and X

Rt.Vis.[X, Visceralis root of [IX

Rt.X-2-3-4-6, Visceral sensory and
motor roots of the second, third,
fourth and fifth branchial ganglia
of X

Rt.X-3-4-6, Visceral sensory and mo-
tor roots of the third, fourth and
fifth branchial ganglia of X

Rt.4-5, Visceral sensory and motor
roots of the fourth and fifth bran-
chial ganglia of X

PLATE 1

EXPLANATION OF FIGURES

1 A reconstruction of the cerebral ganglia and early nerves of a 22 mm.
embryo of Squalus acanthias. The embryo was fixed in vom Rath’s fluid and
mounted unstained. The length of all specimens was determined after fixation.
The plot was made from the left side of the specimen at a magnification of 50
and reduced to 40 for publication and gives true proportions in the anterior-
posterior and dorso-ventral diameters. The sections were 10 microns thick.

The ramus visceralis X has been added to the plot from a 30 mm. specimen.

The epibranchial ganglia on IX and X, which are presumably special visceral
or gustatory, are indicated by vertical lines, as are the general visceral ganglia,
since the exact limits of the cells contributed by the epibranchial placodes could
not be determined. The posterior cone-shaped projections on IX and X are in
each case in contact with the ectoderm.

The nervus terminalis in the specimen plotted contained two peripheral rami,
while only one was found on other specimens.

The r. lateralis X. 1 is double on the side plotted but single on the opposite
side of the same specimen.

The motor twigs are indicated at their separation from the chief ramus only,
the whole course of the motor fibers not being shown in order to simplify the
plot.

GANGLIA AND NERVES OF SQUALUS
F, L. LANDACRE

| | Ol
700 680 660 640

G.Sp.3 G.Sp.2 G.Sp.1

Loy haga
620 600 580

Lateralis ganglia

Cutaneous ganglia

Visceral ganglia

Brain wall

+

60

Le alee
120 100 80

PLATE 2
EXPLANATION OF FIGURES

2 to 13 Camera drawings of transverse sections of the same specimen plotted
in figure 1. The drawings are made at a magnification of 38 and reduced to 25
for publication. The sections were 10 microns thick for all figures. The section
numbers are to be identified on figure 1.

2 Camera drawing of section 249, which lies just anterior to the root of the
profundus ganglion and near the middle of that ganglion.

3 Taken from section 290 at the level of the root of the Gasserian ganglion.

4 Taken from section 321 at the level of the root of the dorsal lateralis gan-
glion of VIT.

5 Taken from section 360 at the level cf the origin of r. palatinus VIT from the
geniculate ganglion.

GANGLIA AND NERVES OF SQUALUS PLATE 2
F,. L. LANDACRE

L.
R. B. VII

R. Mx. V

63

PLATE 3

EXPLANATION OF FIGURES

6 Taken from section 455, is at the level of the origin of the r. supra-
temporalis IX from the lateralis IX ganglion. It also passes through the an-
terior end of the ganglion petrosum, or visceral IX.

7 Taken from section 482, is at the level of the origin of the r. supra-
temporalis X from the ganglion laterale X; and also passes through the point
of origin of the r. posttrematicus IX from the ganglion viscerale IX which is
attached to the epibranchial placode at this point.

8 Taken from section 526, is at the level of the origin of the r. pretre-
maticus X,; from the ganglion viscerale X; and passes through the placodal at-
tachment of that ganglion.

9 Taken from section 546, lies just posterior to the point of origin of r.
lateralis X-1, which arises from the ganglion laterale X2, and of the nerve iden-
tified as r. auricularis X. This section passes through the anterior end of g.
laterale Xs.

64

GANGLIA AND NERVES OF SQUALUS PLATE 3
F. L. LANDACRE

G. V. IX + Pl.
Ry Pos LX

65

PLATE 4

EXPLANATION OF FIGURES

10 Taken from section 569, is at the level of the origin of the r. post-
trematicus X». It also passes through the middle of the ganglion laterale X;
and through the extreme anterior end of the ganglion viscerale X3.

11 Taken from section 596, is at the level of the origin of the r. pretre-
maticus X3; from the ganglion viscerale X;. This is just posterior to the point
of fusion of this ganglion with its epibranchial placode. This section passes
through the posterior end of the ganglion laterale X3.

12 Taken from section 628, passes through the fusion of ganglion viscer-
ale X, with the ectoderm which is contributing cells to the ganglion at this
point.

This section also passes through the anterior end of the ganglion viscerale
X;. The division between ganglia visceralia X, and X; is shown better in this
section than in the reconstruction in figure 1.

13 Taken from section 647, passes through the posterior end of the ganglion
viscerale X; but anterior to its attachment to its epibranchial placode.

66

GANGLIA AND NERVES OF SQUALUS
F, L, LANDACRE

67

PLATE 4

Fig. 11

Oo’ | : 3
be eam ART F
feats 7 Nt ¢ P

NUCLEAR SIZE IN THE NERVE CELLS OF THE BEE
DURING THE LIFE CYCLE

W. M. SMALLWOOD anv RUTH L. PHILLIPS
(From the Zoological Laboratory of Syracuse University, C. W. Hargitt, Director.)
ONE FIGURE

The following study of nuclear size in the nerve cells of the
antennal lobe of the bee was undertaken for the purpose of
learning what are the normal conditions and what, if any, changes
they undergo during the life cycle.

Bees afford exceptionally good material for such work because
all members of a given swarm are of identical parentage; all
spend an inactive larval existence, and the life cycle of individu-
als varies according to type and season. Drones live through
the summer, queens may live for seven years, and the workers,
with which we are concerned in this paper, have a life cycle
varying from about six weeks in the summer to about six months
for the insects hatched from an autumn brood.

Hodge? (’92) published his observations on daily fatigue in the
bee, the sparrow and the cat. In this work he chose the cells
of the antennal lobes because they are easily located. We have
limited our study to the cells of this region for the same reason.
It is usually considered that excessive stimuli in the form of an
immense amount of normal daily work, electrical stimulation, or
surgical shock result in a decrease of nuclear size among the
nerve cells. That such assumptions are commonly held, the
work of Crile? and Hodge shows.

Conklin’ (12) has shown that there is a normal relation be-
tween the size of a cell and its nucleus, and Kocher‘ (’16) has

1 Journal of Morphology, vol. 7, 1892, p. 153.

2 Journal of the American Medical Association, vol. 57, no. 23, 1911, p. 1812.
3 Journal of Experimental Zodélogy, vol. 12, 1912, p. 1.

4 Journal of Comparative Neurology, vol. 26, no. 3, 1916.

69

70 W. M. SMALLWOOD AND RUTH L. PHILLIPS

questioned the results obtained by Hodge and Crile. Our work
was begun in 1910-11, but the opportunity for completing it
did not present itself until this summer. We have re-examined
our earlier work and supplemented it with additional material
collected and prepared in the same way as that obtained
previously.

This material consists of the following stages covering the life
cycle of the honey bee.

1. Recently hatched larvae.
. Half-grown larvae.
. Fully-grown larvae.
. Early pupae.
Mid-pupae.
. Late pupae.
. Newly hatched adults.
. Young adults taken at 6.30 a.m.
. Young adults taken at 6.30 p.m.

10. Old adults taken at 6.30 a.m.

11. Old adults taken at 6.30 p.m.

12. Adults taken at close of the winter season.

Several different fixatives were tried, but the only ones found
successful were osmic sublimate, 1 per cent osmic acid, 1 per:
cent glacial acetic, and sublimate to saturation, Carnoy’s and
OhlImacher’s fluids. Only one individual, that one of stage (8),
included in our study was fixed with osmic sublimate.

No attempt was made to dissect out the brains of the larvae,
which were embedded entire. The brains of pupae and adults
were excised. Sections were cut from four to seven micra thick
in paraffin of 54°, and stained in iron haematoxylin with Bordeaux
red as a counter stain.

The Zeiss and Leitz eyepiece micrometers were used, readings
being computed in micra. We tried to use the planimeter in
our work this summer, but found it impracticable in measuring
such small nuclei.

There are according to Kenyon,® four general regions in the
brain of the bee; the dorso-cerebron, the ventro-cerebron, and

COM D TP wd

No)

® Journal of Comparative Neurology, vol. 6, 1896.

NUCLEAR SIZE OF NERVE CELLS 71

the deuto-cerebron or antennal lobes. These latter arise from
the ventro-anterior side of the dorso-cerebron by two stalks of
fibrillar substance. Each stalk expands into a convoluted spheri-
cal mass of fibers from which the nerves of the antennae arise.
This fibrillar core is surrounded by nerve cells. In the adult
these cells are of three types as far as nuclear size is concerned,
which conform to the types described by Kenyon. These are,
multipolar giant cells, large and small ganglion cells. In the
larva and pupa we find large neuroblasts which give rise to the
cells of the last two types by mitosis and finally themselves
transform into the giant cells of the adult.

It is manifestly impossible to measure all the nuclei in any .
ganglion in such a study as this. We must be content to choose
and select with as much care as possible, such cells as appear to
belong in the same general group and from a study of their
measurements attempt to gain some insight into the problems
which concern the whole mass of cells. Such cells in each class
were chosen as appeared to be fair representatives of the respec-
tive groups. It is probable that others in going over the same
material would select and measure other cells and so arrive at
average measurements somewhat different from those given in
our tables. Our experience leads us to believe, however, that
the general form of the curves derived from a study of the data
would not be materially altered.

Usually we have found no difficulty in making a decision as to
the group in which any particular cell belongs. There have been
a few instances, however, where the mere matter of size seemed
to be insufficient to control the matter of classification. In such
cases we have taken into consideration the general appearance
of the cells, both as to nucleus and cytoplasm, before placing the
cell in one or another group. In the case of the giant cells care
was taken to choose those in which the plane of section passed
approximately through the center of the nucleus.

Each nucleus was measured in its longest and shortest. diame-
ter and the average of these taken as the mean diameter. The
results of these measurements are summed up in the following
table which gives the average nuclear diameter for the three

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, No. 1

PHILLIPS

M. SMALLWOOD AND RUTH L.

Wie

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NUCLEAR SIZE OF NERVE CELLS 13

types of cells in each stage, the number of cells measured and
the range of variation for each. The results are shown graphi-
cally in the curves which follow the table.

A study of these results together with the plotted curves in-
dicates a number of fundamental conditions. In all of the
stages in each of the three groups of cells measured, there is a
wide range of variation in the size of the nucleus. This cannot
be charged to the normal swelling of the nucleus just before
mitosis, for the same variation is present in the cells of the bee
that had lived through the winter and in the queens studied.
That variation is an ever-present condition in all living things
is a truism, but when we attempt to indicate which organ or
tissue 1s responsible for the variation most of the observations
have been simply a record of the organic fact of variation. This
study claims that the cells and their parts such as the nuclei
are the variable factors that are responsible for the variation in
the tissue or organ. Any explanation of the cause of such vari-
ations has to recognize the part played by cells. It seems to the
writers that this natural’‘and normal variation plays an important
part in explaining such conclusions as Crile comes to in regard
to the effects of shock. Before we can accept his conclusions,
we must determine what is the normal range of variation for the
group of. cells that he studied. It would have been a relatively
easy problem to indicate a definite tendency beginning with young
adults and passing to the winter bee by simply taking some of
the large cells in the young adult with nuclear diameter of 12
micra and comparing them with those that measure 7.9 micra.
This would give a definite shrinkage with age; but when the
average of some forty cells is taken, the total is 9.26 micra for
the young adult, and 9.45 for the winter bee. We interpret the
difference to be due to the normal variation present in these
cells and do not regard the larger average for the winter bee in
nerve cells of type I as a measure of the extent of change that
as come with fatigue or age.

The second inference to be drawn from these measurements
is the independent sequence of growth changes in these three
types of nerve cells. There is a more or less rhythmic variation

74 W. M. SMALLWOOD AND RUTH L. PHILLIPS

in the series of measurements made when the plotted curves are
viewed as a whole. Take the large multipolar cells which start
in with an average diameter of 9.05 micra; then increase to
9.89 during the mid-larva period, to be followed by a marked
decline to the mid-pupa period. This is followed by an increase
which is almost the same as the newly hatched adult and the
old adult taken at 6.30 a.m. The average nuclear diameter of
the winter bee is larger than the recently hatched larvae,
late larvae, early pupae and young adults taken early in the
morning. A similar study of the variations in nerve cells of
Type II indicates a different series of growth sequences. Here

the largest nuclear diameter is during the early pupa stage with
no marked variation in the average until the old adults are
reached. The averages for these three types of cells seems to
us to indicate that there is a definite series of growth sequences
that follow through the life cycle in the worker bee and that
they are not dependent on each other.

Beginning with the old adults taken at 6.30 a.m., there is a
noticeable decrease in nuclear size in cells of Types I and III in
the two following stages studied (11 and 12 of table) that is sim-
ilar to Hodge’s results. But there is a more marked decrease
in nuclear size in Type I from mid-larva to early pupa. <A simi-
lar change is indicated in the cells of Type II. If the change

NUCLEAR SIZE OF NERVE CELLS 75

in nuclear size in old adults is due to fatigue or old age or both,
how are we to explain the larval and pupal change? The larvae
of the bee are inactive so that they are more like the pupa than
is usual in insects. Metabolism is very active during the larval
period but this can hardly result in fatigue to nuclei in the brain.
One would expect that the changes that take place during the
metamorphosis in the pupa would drain heavily on the energy
of the insect, and yet the same rhythmic nuclear changes in size
continue through this period. So far as activity is concerned
these stages give us the two extremes, the larvae and pupae
at rest, the adults of summer extremely busy and the win-
ter workers relatively inactive. If there is a definite nuclear
change with work, the stages selected should give us some in-
dication of it. In place of definite nuclear change with age, we
find a constant variation which tends to be rhythmic.

CONCLUSIONS

1. In the honey bee worker there is a definite variation in the
nuclear size of the nerve cells studied.

2. Changes in nuclear size dependent on the life cycle are un-
like in cells of different type.

3. The changes in nuclear size can not be explained as due to
the effect of old age or fatigue.

w ry

1

A REVISION OF THE PERCENTAGE OF WATER IN
THE BRAIN AND IN THE SPINAL CORD
OF THE ALBINO RAT

HENRY H. DONALDSON

From the Wistar Institute of Anatomy and Biology

ONE CHART |:

In four studies previously published (Donaldson ’10, 11, 11a
and ’11 b), the percentage of water in the central nervous system
of the albino rat has’ been considered. A revision of this topic
was thought necessary, however, for several reasons. In the
first place, I wished to record the observed values on which had
been based an extensive table giving the percentage of water in
the brain and spinal cord according to age, each entry in this
table giving also the weight of the brain, or spinal cord to be ex-
pected. This table 74, from The Rat (Donaldson ’15)—is given
in full as an appendix to the present paper. Further, I wished
to state the conditions to be observed in the use of ‘his table.
In the second place, a statement previously made, required cor-
rection :—It has been stated (Donaldson ’10) that at a given age
the percentage of water in the brain or spinal cord is not sig-
nificantly modified by the absolute weight of the organ. This
conclusion is incorrect, for more careful study shows that weight
is a factor in modifying the percentage of water. The evidence
for such a conclusion will appear in the course of this paper.

It is the main purpose of the present study, then, to show how
the percentages of water, which appear in the appended table 74,
have been obtained, and, incidentally, to show how new data
must be treated when they are to be corrected for weight. In
considering the main points, the brain and the spinal cord will

77

78 HENRY H. DONALDSON

be treated separately, and for each there will be given in special
tables the observed values for the percentage of water, followed
by the values corrected for weight, and finally by the formula
(or table) values. Using the uncorrected values, the measures
of variability in this character will also be given.

THE BRAIN
The data and their treatment

The data used for table 74 (Donaldson 715) have been gradu-
ally accumulated during the past eight years, and were collected
by random sampling, mainly from the rat colony at The Wistar
Institute. The animals used represent, therefore, the general
rat population of the colony living, at successive periods, under
somewhat varying dietary conditions. In many cases, the rats
were small for their age. Although a number of litters are in-
cluded in the series, the majority of the animals were not closely
related to each other.

The records forming this series run from birth to 365 days of
age, and may be grouped as follows:

572 male brains—comprised in 61 age groups
375 female brains—comprised in 61 age groups

About one-half of the records here used were collected for an
earlier study (Donaldson 710), and the remainder have been
gathered since that time.

The method of removing and preparing the brain and the spinal
cord has been described earlier (Donaldson 710). Most of the
removals were made by my colleague, Dr. Hatai, and the pro-
cedure has been uniform. ‘The drying was done in a large water
bath at the temperature of 92°-97°C. We have no reason to
think that the observational errors have been important under
these conditions.

The accompanying chart 1 shows, for the males only, the course
of the percentage of water from birth to 365 days, in both the
brain and the spinal cord. The continuous graphs are based
on the formulas (pp. 104-105) and the separate entries give the
observed values—corrected for weight—for the percentage of

PERCENTAGE OF WATER IN BRAIN AND CORD 79

are
Tt

coat

fi aoaee

b

ae
Ba
AH

seeeeeeeet ee

ei
eT

See Cse04e8)

Chart 1 Showing the percentage of water on age in the central nervous sys-
tem of the albino rat. The upper graph gives the values for the water in the
brain as determined by the formulas (Hatai—in ‘The Rat,’ Donaldson, 1915).
The lower graph gives the corresponding values for the spinal cord, determined
in the same way. The small black dots indicate for the brain the corrected
(observed) values for the several age groups, and these corrected values form
the data on which the formulas have been based. The small black triangles
have a like significance in relation to the spinal cord.

water in the several age groups, as these appear in table 1, for the
brain, and table 2, for the spinal cord.

If we take the mean of the deviations of all of the corrected
(observed) values for the several age groups from the corre-
sponding formula values for the percentage of water, as given in
table 1, and shown in chart 1 (males only), we obtain the
following:

Mean of deviations—Males +0.19 per cent
Mean of deviations—Females +0.18 per cent

Thus it appears that for both sexes the observed values,
when corrected for brain weight, differ on the average approxi-
mately +0.2 per cent from the corresponding formula values.
Consequently new observations on random samples, which fall,
after correction, within +0.2 per cent of the formula values

80 HENRY H. DONALDSON

TABLE 1

Giving for the percentage of water in the brain the data used for the making of table
74 (Donaldson ’15), which is appended to this article. The records are entered
by age groups, male and female records being given separately. For any age
group of either sex the table gives the age to a day—or within a range of five days,
followed by the number of cases—and then by the percentage of water. This datum
appears first, as observed, second, as corrected for the brain weight and third, as
given in table 74, where the values have been computed by formulas (Hatai), these
formulas, in iurn, being based on the corrected values as here entered. When the
age is given within a range of five days the interval 5-10 is taken as 8, and 0-5 as
8; 7.e., 25—380 = 28 days, 30—35 = 33 days

MALES FEMALES
AGE IN
DAYS Noo ef Percentage of water No. of Percentage of water
Caserta <a cana en |e OSes
Observed | Corrected | In table Observed | Corrected In table
0 20 87.84 88.00 2 89.00 88.00
1 5 87.31 87.95 3 87.62 87.95
5 3 87.43 87.79 4 87.82 87.79
6 3 87.67 87.70 + 87.95 87.70
9 5 87.57 87.05 2 86.70 87.05
10 21 86.79 86.72 z 86.35 86.72
11 5 86.49 86.26
14 2 84.45 84.97
15 4 84.23 84.58
17 2 83.41 83.82
19 i) 83.28 83.12
20 13 82.68 82.91 82.80 3 82.11 82.37 82.82
21 9 82.69 82.87 82.49 3 82.42 82.50 82.51
22 5 82.52 82.67 82.19
25-30 25 80.58 80.80 80.72 5 80.63 80.60 80.74
30-35 9 79.97 80.16 79.91 3 80.09 80.34 79 .94
35-40 Dif 79.45 79.55 79.46 3 79.72 79.61 79.49
40-45 11 79.23 79.20 79 32 4 79.54.) 79.37 79.35
45-50 23 2 79.17 79 .22 20 78.93 78.79-| 79.25
50-55 Zi 79.30 79.23 79.14 22 79.34 79 .22 79.18
55-60 24 79.05 79.02 79.05 8 79.16 79.13 79.09
60-65 3 79.19 79.04 79.01
65-70 2 78.71 78.59 78.90 -f 79.04 78.73 78.94
70-75 9 78.85 78.68 78.84 7 79.06 79.00 78.88
75-80 12 79.11 78.90 78.77 12 78.97 78.71 78.82
80-85 10 78.61 78.52 78.72 9 78.66 78.60 78.77
85-90 8 78.66 78 33 78 .67 5 79.08 78.67 78.72
90-95 4 78.44 78.11 78.62 4 78.80 78.70 78 .67
95-100} 14 78.94 78.63 78.57 17 78.69 78.51 78 .62
100-105

or

78.44 78.47 78.48 78.03 78.53

w
~I
=
>
oo

105+110

PERCENTAGE OF WATER IN BRAIN AND CORD 81

TABLE 1—Continued

MALES FEMALES

Percentage of water Percentage of water

|| No. of
Cases! |aaa eee I Gases
Observed | Corrected | In table Observed | Corrected In table

110-115] 24 78.59 78.53 78.44
115-120 tl 78.53 78.43 78.40 6 78.58 78.37 78.45
120-125 | 10 78 .49 78.53 78.36 3 78.24 78.08 78.42
125-130] 10 78.30 78.16 78.33 13 78.39 78.21 78.39

130-135 4 78.28 78.12 78.35
135-140 7 78.68 78.59 78 32
140-145 | 27 78.48 78.35 78.23 13 78.57 78.39 78.29
145-150 | 22 78.48 78.24 78.20 10 78.45 78.21 78.26
150-155 8 78.25 78.09 78.18

155-160 | 22 78 .34 78 .23 78.15 14 78 .23 78 .09 78.21
160-165 | 11 78.40 78.34 78.13 c 78 .43 78 .32 78.19
165-170 9 78.22 78 .09 78.12 10 78.24 78.15 78.18
170-175 2 78.78 78 .44 78.12 f 78 .63 78.46 78.18
175-180 5 78.29 78 .02 78.11 14 78.28 78.13 78.7
180-185 7 78.29 78.13 78.11

185-190 4 78.19 78.18 78.11 2 78.58 78.56 (SAF
190-195 5 78.14 78.01 Ufsvea

195-200 9 78.14 78.08 78.10 6 78.14 78.12 78.17
200-205 7 78 .24 78.10 78.10 8 78 34 78.16 78.16
205-210 8 78.11 te ot 78.10 t 78.09 78 .02 78.16
210-215 8 78.34 78.26 78.09 3 78.40 78.30 78.16
215-220 3 78.06 CEO 78.08
220-225 2 78.28 78.04 78.07 12 78 .23 TOE 78.14
225-230 8 78.31 78.28 78.06 4 78.35 78.16 78.13
230-235 8 78.16 78.20 78.05 2 78.70 78.39 78.12
235-240 | . 4 78.29 78.59 78.04 8 78 .26 78.20 78.11
240-245 9 78.36 78.25 78.10
245-250 6 78.29 78.21 78.02 2 78.16 78.09 78.09
250-255 t 78.03 77.85 78.00

255-260 2 78.08 77.81 46299 6 78.30 78.25 78.06
260-265 2 78.05 77.75 77.98 2 78.14 77.99 78.05
265-270 2 78.45 78.13 77.96 5 77 .86 77.84 78.03
270-275 6 77.85 77 47 77.94

275-280

280-285

285-290

290-295 3 77.88 78.15 77.95
295-300 4 78.09 77.90 77.85 a 78.31 78.58 77.93
300-305

305-310 4 77.42 77.54 77.89

82 HENRY H. DONALDSON

TABLE 1—Concluded

MALES FEMALES

Percentage of water Percentage of water

a Cc ———. —]— KK —
Observed | Corrected | In table Observed | Corrected | In table

310-315 2 78.04 78 .06 Chee)

315-320 4 77.59 77.53 77.84
320-325 4 78 .24 77.99 77.74 5 77.92 77.81 17.82
325-330

330-335 2 77.95 77.90 CRE (H
330-340 of 78.09 77.89 77.74
340-345 3 reel 77.62 rian
345-350

300-355

300-360 5 77.83 77.62 77.62
360-365 6 77.64 77.49 77.59

may be considered as in agreement. Where one is making com-
parisons within a litter or within a homogeneous series, less
deviation is to be expected and agreement may be limited to
values that fall within +0.1 per cent of the standard which is
used. Where data from test animals are contrasted with those
from controls of the same litter deviations of 0.05 per cent, if
constant or nearly so, may be regarded as significant.

Thus far nothing has been said of the way in which the factors
for correction were obtained or how they have been applied.
These questions will now be considered.

Sources of variations in the percentage of water

If identical in other respects, two brains of the same age should
have the same water content. Two brains are, however, never
found to be exactly alike even in the terms of our rather crude
measurements, and the differences, which we can at present
appreciate, fall into two classes, those which are gross, and those
which depend on histological structures.

1. Variations due to gross differences. ‘There are at least two
possible causes of variation in the water content dependent on
gross characters.

PERCENTAGE OF WATER IN BRAIN AND CORD 83

a. The amount of fluid in the ventricles. This is, as a rule,
negligible in brains more than 25 days old; but in younger
brains and especially during the first 10 days, when the ven-
tricles are relatively large, it may be a modifying factor of
importance.

b. Variations in the relative weights of the several parts of the
brain. If the brain is divided into the stem, the cerebellum,
the cerebral hemispheres, and the olfactory bulbs, it is found that
the most variable part of the brain is that formed by the olfac-
tory bulbs. At times these may differ from one another by 50
per cent, in two brains of nearly the same total weight, ranging
therefore from 4 per cent to 2 per cent of the weight of the
entire brain.

The water content of the mature bulbs is high, 82 per cent.
If, as an example, we take the water content of the entire brain
as 78 per cent, a reduction of the relative weights of the bulbs
from 4 per cent to 2 per cent would cause a loss of 0.1 per cent
in the water content of the entire brain, thus reducing it to 77.9
per cent.

Variations in the density of the meninges or in the quantities
of blood do not appear to have any significant influence.

2. Variations in the water content of the brain due to histologi-
cal differences. At the same age large rats have absolutely
heavier, and small rats absolutely lighter brains. As there is
reason to think that in a given mammalian species, the Norway
rat for instance, the number of neurons composing the brain
is approximately constant, the difference in the size of the
entire brain must therefore mean a difference in the size of its
constituent neurons and not a change in their number. How-
ever, under the usual conditions of growth, shortly after birth
myelin begins to appear on the axons. It has been shown that
myelin is the constituent mainly responsible for the progressive
loss of water from the brain (Donaldson, 716), and although its
formation is closely correlated with age, it must be considered
probable that slight fluctuations in the relative amount of
myelin may occur. These fluctuations would produce in turn
small changes in the percentage of water observed.

84 HENRY H. DONALDSON

Inspection of the data at hand suggests that size differences
of the brain may depend either on a mere magnification or re-
duction of the neurons in a strictly proportional manner or on a
disproportional growth caused by a relative excess of white sub-
stance, in the heavier brain and vice-versa.

As will be pointed out further on, the least variability in the
water content of the brain is found within the same litter, and
it seems probable that here the differences in the brain size which
occur are mainly due to a strictly proportional growth of the
neurons. On the other hand, it appears that the brain which is
large at a given age has commonly anticipated some of the
growth changes whch belong to a later period, and this means
that the relative abundance of the myelinated axons has been
increased—a change necessarily accompanied by a lowering of
the water content. This, the more common relation found be-
tween two unrelated rat brains of the same sex at like age,
where the larger brain usually has the smaller percentage of
water, is considered therefore to be due to the relative excess
of myelinated fiber substance in the larger brain. As will be
seen, this statement constitutes a reversal of the opmion which
I formerly held (Donaldson, 710).

If this corrected opinion is accepted, the next step is to de-
termine the factors to be used for reducing any observation on
the percentage of water.

On the relation of the percentage of water to the absolute brain
weight

According to our hypothesis the relatively small brain is
likely to be retarded in development, i.e., to be a trifle behind
the stage characteristic for its age and so to have a less pro-
portion of myelin and therefore a higher percentage of water,
while on the other hand the relatively large brain is likely to be
precocious and to show as a consequence a lower percentage of
water.

The test of this assumption. was therefore made by averaging
for the first and last thirds or halves of each age group, ar-

PERCENTAGE OF WATER IN BRAIN AND CORD 89

ranged, according to increasing brain weight, the observed per-
centages of water. The corresponding weights for the relatively
light and for the relatively heavy brains were also averaged.
If it turned out that on the average the higher percentage of
- water went with the light brain lot, and the lower percentage
with the heavy brain lot, the determination was considered
‘accordant.’ The opposite relation was designated as ‘reverse.’
As it seems probable that the myelin is the principal cause of
the differences for which correction should be made, it is not
advisable to introduce corrections for brains from rats less than
20 days of age, since previous to that age myelination is quite
incomplete. On looking at table 1, it will be found that begin-
ning with the 20 day records there are observations for the
percentage of water on a number of age groups, which com-
prise five or more individuals. Of such groups we have used 38
from the male, and 32 from the female records. Each of these
groups has been treated as indicated in the sample table which
follows. The details of this entire operation are here given.

The data for each individual were arranged according to in-
creasing body weight. After the body weight of each rat was
entered the observed brain weight, and after this the brain
weight to be expected for the body weight (not the age) was also
entered, using table 68 (Donaldson 715) for the expected brain
weight values. If the observed brain weight is found to be
greater than that to be expected from the body weight then the
brain is large, and vice-versa.

Using the brain weight values taken from table 68 as the
standards, the percentage value of each difference was deter-
mined, and this was entered opposite the brain weight to which
it applied. These percentage differences were next arranged in
regular order from the most minus to the most plus, and of the
series thus formed either the first third or first half Gn this in-
stance the first three) of the cases was used for comparison with
the ‘ast third or half.

The three minus cases give an average deficiency in brain
weight of 6.2 per cent and show 78.57 per cent of water ob-

86 HENRY H. DONALDSON

Sample table, to illustrate the procedure for obtaining a correction factor for the
percentage of water as modified by the brain weight. Albinoralt. Male. Brain.
Age growp 210-215 days.

DIFFERENCE IN PER CENT OF

OBSERVED TABLE 74 DIFFERENCE, GRAMS TABLE VALUE
VALUES
BRAIN tt it ais?
aa Brain Weight | W=!GHT = ce = ae
grams grams grams
160 iL gala 1.807 0.096 DES
171 ike aU 1.824 0.117 6.5
216 1.982 1.886 0.096 HO)
224 1.765 1.895 0.130 6.9
251 1.911 1.925 0.014 0.7
273 Pale 1.947 0.250 12.8
276 2.215 1.948 0.267 13SEC
Ave. 1.890
ARRANGEMENT OF PERCENTAGE DIFFERENCES IN CORRESPONDING PERCENTAGES OF WATER
BRAIN WEIGHT FROM MINUS TO PLUS OBSERVED
~ - ~ +
£ 6.9 77.9
ra | 6:5 78.7
[5.3 79.1
Ave. 6.2 Ave. 78.57
13).0) 78.1
rene 12.8 78.0
: 130, 78.4
Ave. 10.5 Ave. 78.17

served. Similarly the three plus cases give an excess of brain
weight of 10.5 per cent and show 78.17 per cent of water ob-
served. The relatively heavier brain group has, therefore, the
less percentage of water and the relation is ‘accordant.’

If we use the mean of the table values for brain weight—
1.890—as the standard and calculate the absolute difference
for the brain weights, between the minus group (= —6.2 per
cent) and the plus group (= +10.5 per cent) we find this to be
315 mgm. This difference 315 mgm. corresponds to a difference
in the percentage of water of (78.57 per cent —78 17) =0.40

PERCENTAGE OF WATER IN BRAIN .AND CORD 87

per cent so that a difference of 1 mgm. of brain in this group
corresponds to a difference of 0.0012 in the percentage of water.

Factors for correcting the percentage of water according to
brain weight

The result of treating the data in this manner was to show
that of 38 male groups 64 per cent, and of 32 female groups 66
per cent were accordant. In the case of each age group further
calculations were made. Taking as the standard the average
of all the tabular brain weight values entered, the absolute dif-
ference between the average weights of the light and the heavy
brains was computed and expressed in milligrams. The differ-
ence between the mean percentage of water for the light and
for the heavy groups was also found. Then, by dividing this
difference in the percentage of water by the difference in weight,
expressed in milligrams, the difference for 1 mgm. was found.
The number thus found I designate the ‘correction factor.’
The preceding paragraphs give an example of the foregoing
procedure.

Of course, such a factor was obtained for each age group in
each sex and was found to be accordant, as stated, in 64 per
cent of the male, and 66 per cent female groups, but reverse in
the remaining groups. In the case of each sex the sum of the
reverse factors was deducted from the sum of the accordant
factors and the remainder divided by the number of accordant
cases. This gave the correction factor for each sex.

The results are as follows:

Correction Factors

Male brain: 0.0013 per cent water for a difference of 0.001 gram
Female brain: 0.0012 per cent water for a difference of 0.001 gram

The correction factor selected for both sexes was 0.0013 and
this has been used in correcting the observed percentages of
water as given in table 1.

The object of this treatment of the crude data was to reduce
the deviations in the percentage of water which depended on

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 1

88 HENRY H. DONALDSON

differences in the absolute size (weight) of the brain Therefore
by getting the difference in milligrams between the observed
brain weight and the brain weight for body weight, as given in
table 74, and multiplying this by 0.0013, a correction was ob-
tained which could be applied to the crude values for the per-
centage of water, which appear in table 1.

From this table there have been omitted, however, both the
body weights and the observed brain weights for the several
age groups, so that the final results there given cannot be con-
trolled except by reference to the original records which are on
file at The Wistar Institute.

Application of the correction factor in the case of new data

To obtain the corrected value for the percentage of water in the
brain in the case of a new observation, the necessary data are
the body length, the body weight, the observed brain weight, the
percentage of water in the brain, and the age and sex of the rat.

It is necessary also to have access to reference tables which
give the body weight normal to the body length, and also the
brain weight and percentage of water (for that brain weight)
normal to the age.

With these data it is possible in a given case first to deter-
mine what correction should be made in the observed percentage
of water in order to make that value comparable with the per-
centage of water to be expected when the brain weight was
normal to the body length. This may be illustrated by an
example taken from a recent investigation. The data for the
rat selected are as follows:

Body weight, 133.5 grams

Body length, 179 mm.

Age, 173 days—female

Brain weight, 1.581 grams

Percentage of water in brain, 78.61 per cent

If we turn to table 68 in ‘The Rat’ (Donaldson, 715), it ap-
pears that for a female rat 179 mm. long a body weight of 144.4
grams is to be expected. Therefore this rat is under weight.

PERCENTAGE OF WATER IN BRAIN AND CORD 89

Moreover a brain of 1.750 grams should be found with the
above body length, but the observed brain weight was only
1.681 grams. It is therefore deficient by 0.069 grams. The
observed percentage of water in the brain was 78.61 per cent.
The percentage of water to be expected for a female having a
body weight of 144.4 grams was 78.62 per cent (table 74, here
appended). As the observed brain weight was 69 mgms. too
low and the correction factor is 0.0013 per milligram, the total
correction amounts to 0.09 which is to be subtracted from the
observed value 78.61 per cent, thus giving 78.52 per cent as the
corrected value for the percentage of water.

This result may be interpreted as follows: The growth of the
brain was retarded in this animal so that although the animal was
173 days old, it had nearly but not quite the water content of a
younger rat, the age of which was normal to the body weight.

In view of the fact that we have a series of computed values
for the percentage of water found in brains of the standard size
at various ages, it is possible in this case to determine the prob-
able percentage of water in the brain under examination, if it
had reached the size characteristic for its age—173 days.

At this age the brain, according to table 74, should weigh
1.835 grams and have 78.18 per cent of water. This tabular
brain weight is 154 mgm. above the observed weight 1.681
grams, with a water content of 78.61 per cent. The difference
154 mgm. multiplied by the correction factor 0.0013 gives a cor-
rection of 0.2 which is to be subtracted from the water content
observed, 78.61 per cent, giving as the corrected value 78.41
per cent against the tabular value of 78.18 per cent. This
shows a deviation from the tabular value of about 0.23 per cent
or an amount just outside the range of +0.2 for random sam-
pling. This somewhat elaborate process seems necessary to re-
duce the crude data to a form in which they may be compared
with each other.

Percentage of water according to sex

When the values given in table 74—here appended—are ex-
amined, we note that from the age of 60 days on the body

90 HENRY H. DONALDSON

weights and brain weights of the female are regularly less than
those for the male. It we determine for a series of cases the
difference in the percentage of water between the male and
female brains of like age, we find that a difference of 1 mgm. in
brain weight corresponds to a water difference of about 0.001
per cent, thus giving an amount which is a trifle below the cor-
rection factor for the brain within each sex. It seems prob-
able that the perikarya of the neurons in the male are relatively
somewhat larger than in the female, and this would account
for the slightly lower value found by this method of comparison.

Measures of variability in the percentage of water

The material represented by the data in table 1 makes it
possible for the first time to determine the variability of the
percentage of water in brains of like age, not only when the
brains are taken by random sampling, but also when they be-
long to a single litter. The measures of variability determined
for each sex were the standard deviation (c) and the coefficient
of variability (C).

For the determination of the standard deviation o we used
the formula

| MEGS)

n
and for the probable error of the standard deviation

oO

EL, = + 0.6745 Wa
n

For the coefficients of variability, C, the formula

= 7 x 100
and for the probable error of the coefficient of variability
E, = + 0.6745 a
; V 2n

(Davenport 04).

PERCENTAGE OF WATER IN BRAIN AND CORD 91

The uncorrected values for the percentage of water were used
in this series, as well as in all of the other series examined. The
differences between the results based on the corrected, and
those based on the uncorrected values are however negl gible.

In the case of the males, as will be seen from table 1, we have
from birth to 163 days of age, 19 groups containing 10 or more
entries and averaging 18 observations in a group.

From the treatment of this material we obtained the following:

Standard Deviation—Males

Range in 19 groups _—_0.21+0.028 to 0.50+0.080.
Average of 19 groups 0.320.035

Coefficient of variabiliiy—Males

Range in 19 groups 0.26+0.034 to 0.640.090
Average of 19 groups 0.40+0.047

In the case of the females we have 11 groups averaging 14
individuals in a group and ranging from 48 to 223 days of age.
From the treatment of this material we obtained the following:

Standard deviation—Females

Range in1l groups 0.22+0.030 to 0.45+0.016
Average of 11 groups 0.310.040

Coefficient of variability—Females

Rangein 11 groups 0.280.038 to 0.57+0.077
Average of 11 groups 0.40+0.050

It appears from the foregoing that the variability in the per-
centage of water is nearly alike in the two sexes, and that it is
remarkable small (¢ = 0.31 and C = 0.40 per cent), thus sup-
porting the conclusion that normally the water content in the
brain is highly constant when taken in relat’on to age. A num-
ber of age groups, used for the preceding determinations, contain
records that belong to one or to several litters. It seemed
probable that the variability would be less within a given litter
than in the mixed population, or in a group composed of al] the
members of the several litters. Among the 19 male groups,
just examined, 9 contained from one to four litters each com-
posed of three or more individuals. In all there were 21 litters

92 HENRY H. DONALDSON

available for examination. The average variability in the 9
groups from which the 21 litters are taken was

o = 0.26 + 0.029 and C = 0.32 = 0.035, ;

while the average of the variabilities of the 21 litters, within
these 9 groups, was

o = 0.14 + 0.033 and C = 0.17 = 0.041.

Thus the variability of the male litters is only about one-half
that of the age groups in which they are found.

Among the 11 female age groups, there were 7 which con-
tained 11 litters of sufficient size for study. Here we find
much the same relations as appeared among the males. The
average variability of the 7 female groups from which the 11
litters were taken was as follows:

o = 0.25 +0.032 and C = 0.32 +0.041

while the average of the variabilities of the 11 female litters

was:
o = 0.13 +0.30 and C = 0.17 +0.043

Again the litter variability is about half that of the groups from
which the litters were taken.

It appears from the foregoing that the variability of the per-
centage of water in brains belonging to the same age group is
small—and that it is about the same for both sexes—but that
within a given litter it tends to be much less than in the age
group formed by a combination of the litters

THE SPINAL CORD

Although the number of records for the spinal cord is a trifle
less than the number for the brain, yet all the spinal cords which
were used are from rats that also furnished brains for the brain
series. What has been said already (p. 78) in connection with
the brain, concerning the material and the general character of
the data, applies therefore to the spinal cord series also. In

PERCENTAGE OF WATER IN BRAIN AND CORD 93

discussing the data we shall follow the same order of presenta-
tion as was followed for the brain. The records for the spinal
cord run from birth to 365 days of age and may be grouped as
follows:

569 male spinal cords comprised in 61 age groups

363 female spinal cords comprised in 56 age groups
Thus in the case of the females there are five age groups less for
the spinal cord than for the brain.

For the graph representing the course of the loss of water in
the cord and the relation of the corrected (observed) male values
to those computed, the reader is referred to chart 1, p. 79. If
we take the mean of the deviations of all of the corrected values
for the percentage of water from the corresponding formula
values for the several age groups as given in table 2, and shown
in chart 1 (males only), we obtain the following:

Mean of deviations — males =+0.61 per cent
Mean of deviations — females +0.55 per cent

Thus it appears that the corrected observed values for the
water in the spinal cord deviate on the average approximately
+ (.6 per cent from the corresponding formula values. This
deviation is about three times that found for the brain. As a
consequence new observations on random samples which after
correction fall within + 0.6 per cent of the formula values may
be considered as in agreement. Where one is dealing with very
uniform material less deviation is to be expected and agreement
may be limited to values that fall within + 0.3 per cent of the
standard which is used. Where data from test animals are con-
trasted with those from controls of the same litter deviations of
0.1 of a per cent, if constant or nearly so, may be regarded as
significant.

Sources of variation in the percentage of water—spinai cord

The gross differences already noted as modifying the per-
centage of water in the brain do not apply to the cord, because
of the dissimilarity in its architecture; but so far as the differ-

94 HENRY H. DONALDSON

TABLE 2

Giving daia for the percentage of water in the spinal cord used for the making of
table 74 (Donaidson 715), which is appended to this article. The records are
entered by age groups, male and female records being given separately. For any
age group of either sex the table gives the age to a day, or within a range of fwe
days, followed by the number of cases—and then by the percentage of water. This
datum appears first, as observed, second, as corrected for the spinal cord weight,
and third, as given in table 74, where the values have been computed by formulas
(Hata), these formulas, in turn, being based on the corrected values as here en-
tered. When the age is given within a range of five days the interval 5-10 is taken
as 8, and 0-6 as 3, %.e., 25 — 80 = 28 days, 30 — 35 — 33 days.

MALES " FEMALES
ee, Novant Percentage of water No. of Percentage of water
Cases Cases) |---27272727YN-

Observed | Corrected | In table Observed | Corrected In table

0 20 86.85 86.75 2 84.80 86.75

1 5) 84.71 86.42 3 84.83 86.42

5 3 84.98 85.07 4 85.40 85.07

6 3 85.51 84.73 4 86.31 84.73

9 5 85.46 83.73 2 83.50 83.73
10 21 82.95 82.33 83.40
11 5 84.14 83.97 82.98
14 2 81.15 80.78 81.77
15 4 80.61 80.26 81.39

19 5 79.70 79.57 79.90
20 13 79.39 78.81 79.55 3 78.59 78.24 79.47

21 9 79.84 79.51 79.21 3 78.74 78.39 79 .02

22 5 79.58 79.43 78.87
25-30 25 75.96 76.88 77.00 5 76.02 75.78 76.76
30-35 9 75.37 76.21 75.64 3 74.00 74.04 75.40
35-40 27 73.98 74.36 74.46 3 74.93 74.43 74.26
40-45 11 73.83 73.13 73.74 4 74.08 73.44 73.60
45-50 23 73.95 73.33 73.17 20 73.60 72.72 73.12
50-55 27 73.21 73.03 72.69 22 73.96 73.08 72.69
55-60 24 73.02 72.86 72.27 8 73.49 73.21 72.27
60-65 2 72.69 71.92 TLE
65-70 2 72.35 69 .90 71.60 4 73.50 71.73 71.61
70-75 9 72.61 (plsrar 71.32 7 72.21 71.65 71.36
75-80 12 73.17 71.27 71.09 12 73.20 71.31 71.15
80-85 10 72.36 71.30 70.89 9 72.43 71.56 70.96
85-90 8 72.63 69.63 70.71 5 73.52 71.05 70.80
90-95 4 72.46 69.06 70.56 4 72.80 71.16 70.67
€5-100| 14 73.00 69.70 70.43 14 73.02 71.66 70.55
100-105
105-110 5 70.72 70.56 70.23 3 70.16 69.04 70.38

AGE IN

PERCENTAGE OF WATER IN BRAIN AND CORD

TABLE 2—Continued

MALES

95

FEMALES

Percentage of water

Percentage of water

BESS No. of No. of
Cases Cases

- Observed | Corrected | In table Observed | Corrected | In table
110-115] 24 71.68 70.71 70.15
115-120 Uf 70.88 70.59 70.09 6 G23 70.50 70.27
120-125; 10 ales (al 70.68 70.05 2 a4. 70.61 70.24
125-130} 11 71.36 69.40 70.02 13 71.68 70.15 70.22
130-135 4 Tale 68.96 70.22
135-140
140-145 | 27 71.62 70.30 70.00 20 CAP 70.64 70.22
145-150] 22 72,.20 69.95 70.00 10 72.26 70.39 70.22
150-155 8 70.99 69.35 70.00
155-160] 22 71.32 69.95 70.00 14 71.28 69.62 70.22
160-165 | 10 TAL Bil 70.27 70.00 7 aleo5 70.73 70.22
165-170 9 71.16 70.12 70.00 10 lve ZA: 70.10 70.22
170-175 2 42.32 70.70 70.00 4 72.01 70.70 70.22
175-180 5 71.19 68.63 69.99 14 71.35 70.26 70.22
180-185 7 71.14 69.65 69.99
185-190 4 leaky) 70.77 69.99 2 lsat 70.26 70.22
190-195 5 70.96 70.01 69.98
195-200 8 (A225 70.49 69.97 6 71.25 76.39 LO)s2ik
200-205 7 ALOT 70.30 69.96 8 71.39 70.31 70.20
205-210 8 70.48 69.72 69.95 4 70.59 69.56 70.19
210-215 8 11.33 70.63 69.93 3 70.76 69.76 70.18
215-220 3 70.34 69.12 69.92
220-225 2 71.35 70.45 69.90 12 Giese 70.21 70.15
225-230 8 71.45 70.60 69.88 4 72.20 70.70 70.14
230-235 8 70.06 70.42 69.87 2 71.09 69.54 70.12
235-240 2 70.67 71.30 69.85 8 71.138 70.04 70.11
240-245 9 71.49 70.36 70.09
245-250 6 71.31 70.10 69.80 2 70.63 69.81 70.06
250-255 +f 71.46 69.26 69.75
255-260 2 70.92 69.08 69.75 6 71.68 71.07 70.01
260-265 2 70.07 69.45 69.73 2 Gla 69.47 69.99
265-270 2 71.50 69.99 69.71 5 70.89 69.67 69.97
270-275 6 71.23 68.93 69.68
275-280
280-285
285-290
290-295 3 70.56 70.72 69.85
295-360 4 wales; 69.49 69.55 4 71.50 69.89 69.83
300-3805
305-310
310-315 2 70.04 70.00 69.46

96 HENRY H. DONALDSON

TABLE 2—Concluded

MALES FEMALES
ay res Noee Percentage of water NaWGE Percentage of water
Cases Cases

Observed | Corrected | In table Observed | Corrected | In table
320-325 4 71.02 69.36 69.40 5 70.83 70.14 69.70
325-330
330-335 2 71.18 71.05 69.64
335-340 i 70.67 69.68 69.61
340-345
345-350
350-355
355-360 5 70.12 68.96 69.48
360-365 3 71.01 69.93 69.45

ences depend on histological composition, the sources of variation
for the spinal cord are similar to those for the brain (p. 83).

On the other hand, we have the condition of adaptation by
enlargement emphasized in the cord, and represented there par-
ticularly by the passive lengthening, whereby the cord adapts
itself to the varying lengths of the vertebral canal: an adapta-
tion which seems to be accomplished mainly by changes in the
quantity of the white substance.

Factors for the correction of the percentage of water according to
the spinal cord weight

Theoretically there can be little question that the conditions
represented by the relative weight (i.e., relative to the body
weight) act as in the case of the brain to produce a high per-
centage of water in the cord which is relatively small, and vice
versa. But the cord data cannot be used in the same way as
we used the data for the brain, because the absolute weight of
the cord is the dominating factor, owing to the fact that the
increase in the weight of the spinal cord is so largely due to the
addition of myelinated fibers. ‘To obtain correction factors for
the cord it has been necessary therefore, to deal with the data
from the standpoint of absolute weight. Our assumption is
that at the same age the absolutely heavier spinal cord will

PERCENTAGE OF WATER IN BRAIN AND CORD 97
have the smaller percentage of water, and vice versa. ‘There
were tested 50 male and 37 female age groups. Each of these
groups has been treated as follows: The data were arranged ac-
cording to the increasing spinal cord weights and after each
cord weight the percentage of water found in it was set down.
Then the averages of the spinal cord weights and of the cor-
responding percentages of water for the first third or half of the
groups were compared with respective averages for the last
third or half.

Where the lighter cord was associated with the greater per-
centage of water, the data were considered as ‘accordant,’ but
where the opposite relation was obtained as ‘reverse.’ When
treated in this way it was found that of 50 male age groups, 84
per cent, and of 37 female age groups, 70 per cent, were accord-
ant. Thus in the cord a heavier weight was associated with a
less percentage of water somewhat more frequently than in the
case of the brain (p. 87). To obtain the correction factors the
difference between the averages for the percentage of water was
divided by the number of milligrams by which the correspond-
ing average cord weights differed, and the value for one milli-
gram of cord weight was thus obtained. This gave the cor-
rection factor for a single age group. The correction factors to
be applied at the different phases of growth were determined
arbitrarily by taking the average of the accordant correction
factor values in the several age groups within each phase. ‘Two
such phases were recognized, as given in table 3.

By the use of the factors thus obtained the corrected percent-
ages of water in table 2 were determined. ‘To obtain these the
observed weight of the cord in each age group was subtracted
from the cord weights characteristic for that age and this dif-
ference in milligrams multiplied by the appropriate correction
factor. The observed percentage of water was then corrected
by the amount of this product. —

It is to be noted that in the case of the cord, in which the
myelination process begins during the first or second day after
birth, corrections can be applied as early as the tenth day of
life.

98 HENRY H. DONALDSON

TABLE 3

Albino rat. Change in the percentage of water for each 0.001 gram of spinal cord
weight as obtained from the comparison of the light and heavy cords in the same
age group

CORRECTION FACTORS

PHASE AGE IN DAYS
Males Females
1 10-58 0.010 0.008
2 58-365 0.009 0.007

As noted in the case of the brain, neither the body weight nor
the observed cord weight for the several age groups are given.
These data, however, have been placed on file at The Wistar
Institute.

Application of the correction factors in the case of new data

To obtain the corrected value for the percentage of water in
the case of a new observation on the spinal cord, the same data
are required as in the case of the brain (p. 88). The details
are presented in following paragraphs:

Body length is a more reliable guide than body weight. If
we continue the illustration of procedure with the same case
as that which was used for the brain (see p. 88) we have as
data: body length 179 mm., age 173 days, female, cord weight
0.458 gram, percentage of water 72.00 per cent.

If we compare the observed value with that in table 74 for
this age—173 days—it appears that the cord weight expected
was 0.580 gram, or 0.122 gram in excess of the observed
weight.. Using 0.007 as the correction factor for 1 mgm., the
total correction amounts to 0.854 to be subtracted from 72 per
cent, the observed water content, thus giving the final percent-
age as 71.15 per cent. ‘Table 74 gives 70.22 per cent for this
age, so that when tested in this manner the corrected value is
about 1 per cent too high. We conclude in this instance, as we
did previously, in the case of the brain, that the growth changes
in the spinal cord of this rat had been somewhat retarded.

PERCENTAGE OF WATER IN BRAIN AND CORD 99

When the factors for correction in the case of the spinal cord
are compared with the single factor for the brain, it is at once
evident that those for the cord are much larger.

Although it is not possible to explain this difference in detail
or with precision, nevertheless the fact that there should be a
difference and one of about the amount found, can be shown
readily. In the first place it must be remembered that the
correction factors, both for the cord and the brain, have been
computed for an absolute weight—0.001 gram. The cord,
however, weighs on an average only one-fourth as much as the
brain. The relative value of 0.001 gram in the case of the cord
is therefore four times that for the brain, and consequently the
equivalent factor for correction would be some four times as
large as that for the brain.

Further, a study of the formation of the lipoids (Koch, W.
and Koch, M. L. 713) shows that the myelination process in
the cord is accompanied by the formation of about twice as
much lipoid substance as in the brain, and because the lipoid
formation is a rough indicator of the formation of myelin
sheaths—and these in turn mean a less percentage of water—
it follows that the change of 0.001 gram of absolute weight in
the cord involving as it does a larger change in the lipoids will
for this reason have a greater effect in terms of the percentage
of water than does the corresponding change in the case of the
brain. This would again increase the correction factor for the
spinal cord. Thus, although we cannot justify the factors for
the cord in detail, the foregoing considerations indicate that
values are to be expected similar to those which have been ac-
cepted and used. Further, since the weight of the cord is so
largely a matter of white substance the fact that for the female
cord—which is typically lighter than that of the male at the
like age—the correction factor is smaller, is in accord with the
general relation of the white substance as here described.

100 HENRY H. DONALDSON

Percentage of water according to sex

After 60 days of age, a comparison can be made, by the use
of the data in table 74, of the weights and water content of the
spinal cords in the males and females at like ages. Such a com-
parison shows the correction factor between the sexes to be
about 0.008 per cent of water per milligram. This lies roughly
between the correction factor values for the two sexes, as pre-
viously determined.

Measures of variability in the percentage of water—spinal cord

As in the case of the brain, it has been possible to obtain for
the spinal cord, in a number of age groups of both sexes, the
measures of variability as represented by the standard devia- -
tion and the coefficient of variability. The formulas used
have been given on p. 90. In the case of the males, as will be
seen from table 2, we have from birth to 163 days of age 19
groups containing 10 or more entries, and averaging 18 obser-
vations per group.

Using the uncorrected values we obtained the following:

Standard deviation

Range in 19 groups’ = 0.74+0.07 to 1.86+0.26
Average of 19 groups 1.06+0.12

Coefficient of variability

Range in 19 groups _0.96+0.09 to 2.64+0.37
Average of 19 groups 1.46+0.17

In the case of the females we have 11 groups, averaging 14
individuals in a group, and ranging from 48-223 days of age.
We obtained the following:

Standard deviation
Range in 11 groups __0.60+0.08 to 1.13+0.12
Average of 11 groups 0.81+0.10
Coefficient of variability

Range in 11 groups 0.85+0.11 to 1.56+0.17
Average of 11 groups 1.13+0.14

PERCENTAGE OF WATER IN BRAIN AND CORD 101

It is evident from the foregoing that the variability in the per-
centage of water is somewhat greater in the case of the males
than in the case of the females. When the corresponding mean
values for the variability of the brain are compared with those
for that of the cord we find that the values for the male cord
are about 3.4 times that for the brain and the values for the
female cord about 2.6 times.

Among the 19 male groups examined there were 9 which con-
tained from 1 to 4 litters, composed of three or more individ-
uals. In all there were 21 litters.

The average variability in the 9 age groups in which the 21
litters occurred was

¢ = 0.80 + 0.091 C=h0s == 05120

while the average of the variabilities of the 21 litters taken
from these 9 age groups was

Ge — sor = 10.04 C— 0.46 = 0.11

Thus among the males the variability within the litters was
only about one half of that found for the age groups. It seems
not improbable that myelin formation, which is so much more
active in the cord than in the brain, may also be relatively
more variable in the cord and thus contribute to the higher
variability of this organ in general, and of the cord of the male,
in particular.

Among the females there were 7 age groups which contained
from 1 to 3 litters composed of three or more individuals. In
all there were 12 litters. The average variability of the 7 age
groups in which the 12 litters occurred was

¢ = 0.72 + 0.094 Creve aks
While the average of the variabilities of the 12 litters was
¢ = 0.31 = 0.08 C042 —= 0:01

Again the litter variability is less than one half of that for the
groups from which the litters were taken.

102 HENRY H. DONALDSON

SUMMARY

Evidence has been adduced for the view that both the rela-
tive and the absolute weight of the brain and the absolute weight
of the spinal cord, at a given age, are factors tending to modify
the percentage of water present, in the sense that the heavier
brain or cord usually shows the smaller percentage of water.

A presentation has been made also of the data which were
used as a basis for the formulas by which the percentages of
water in table 74—here appended—have been determined, and
of the manner in which the observed values for the percentages
of water in the brain and in the spinal cord have been corrected
for the weights of the respective organs. Factors for correction
have also been given for reducing the observed values for the
percentage of water in the brain or cord to a form in which they
may be fairly compared with one another or with the values in
table 74, when it is desired to use such a table for reference.
The factors for correction are given on p. 87 for the brain, and
in table 3 on p. 98 for the spinal cord.

It is understood, of course, that when an investigator has
homogeneous data, a comparison of these data with one an-
other can be perfectly well made without cross reference to a
table such as that here given. On the other hand, where the
series of data are from different researches or from different
strains of rats they should be referred to such a table before they
are compared with each other. The measures of variability
have also been found for the percentage of water both in the
brain and in the spinal cord, and it has been pointed out that in
both organs the variability is small, but that the variability for
the cord is about three times that for the brain. Further, it
appears that the variability within litters is only about half
that found in the age groups to which these litters belong, a
relation similar to that already found for the body weight by
Jackson (713) and by King (’15). The measures of variability
are given on pages 91-92 for the brain and on pages 100-101
for the spinal cord.

In the appendix are reprinted the formulas for the determina-
tion of the percentage of water in the brain and in the spinal

PERCENTAGE OF WATER IN BRAIN AND CORD 103

cord, as well as table 74 (Donaldson 715) giving the percent-
age of water in the brain and spinal cord for the first 365 days
of life.

LITERATURE CITED

DaveEnrort, C. B. 1904 Statistical methods with special reference to bio-
logical variation. 2d ed., 1904, John Wiley and Sons, New York.

Donatpson, H. H. 1910 On the percentage of water in the brain and in the
spinal cord of the albino rat. Jour. Comp. Neur., vol. 20, no. 2, pp.
119-144.

1911 On the influence of exercise on the weight of the central nerv-
ous system of the albino rat. Jour. Comp. Neur., vol. 21, no. 2, pp.
129-1387.

191la The effect of underfeeding on the percentage of water, on the
ether-aleohol extract, and on medullation in the central nervous
system of the albino rat. Jour. Comp. Neur., vol. 21, no. 2, pp. 139-
145.

1911b An interpretation of some differences in the percentage of water
found in the central nervous system of the albino rat and due to con-
ditions other than age. Jour. Comp. Neur., vol. 21, no. 2, pp. 161-
176.

1915 The Rat. Reference tables and data for the albino rat (Mus
norvegicus albinus) and the Norway rat (Mus norvegicus). Memoirs
of The Wistar Institute of Anatomy and Biology, no. 6, pp. 1-278.
1916 A preliminary determination of the part played by myelin in
reducing the water content of the mammalian nervous system (albino
rat). Jour. Comp. Neur., vol. 26, no. 4, pp. 443-451.

Harar, 8. 1915 In ‘The Rat.’ (Ed. by Donaldson.) Memoirs of The Wistar
Institute of Anatomy and Biology, no. 6, pp. 1-278.

Jackson, C. M. 1913 Postnatal growth and variability of the body and of
the various organs in the albino rat. Am. Jour. Anat., vol. 15, pp.
1-68.

Kane, Heten D. 1915 On the weight of the albino rat at birth and the factors
that influence it. Anat. Rec., vol. 9, pp. 213-231.

Kocu, W. anp Kocn, M. L. 1913 Contributions to the chemical differentia-
tion of the central nervoussystem. III. The chemical differentiation
of the brain of the albino rat during growth. J. Biol. Chemistry, vol.
15, pp. 423-448.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 1

104 HENRY H. DONALDSON
APPENDIX

Formulas for the percentage of water in the central nervous
system (Hatai, in The Rat, Donaldson 715, p. 170-172).

PERCENTAGE OF WATER IN BRAIN

The formulas do not apply to rats under 10 days of age.
The data for the first 10 days are from direct observations.
Percentage of water in brain—(male) =
92.122 —0.614 Age +0.00739 Age? (Phase 1) (40)

[10 <Age <40]

= 82.756 —2.103 log Age (Phase 2) (41)
[40 <Age <160]

= 77.671 +0.00537 Age —0.000016 Age? (Phase 3) (42)
[160 <Age <365]

To transform any determination for the male into that for
the female, the value for the male at a given age (see formulas
(40) (41) (42) ) is modified by a plus correction (Hatai).

Correction (plus) =0.0555 log (age +3) —0.0606 (42a)
[10 <Age <365]

The foregoing (40)—(42a) replace the formulas given in the
paper by Donaldson (’10).

Formulas (40) (41) (42) (42a) were used for table 74.

PERCENTAGE OF WATER IN SPINAL CoRD

The formulas do not apply under 10 days of age. The data
for the first 10 days are from direct observations.
Percentage of walter in spinal cord—male =
87.976 —0.494 Age +0.00364 Age? (Phase 1) (43)

[10 <Age <40]

= 100.8 +0.0548 Age —17.7 log Age (Phase 2) (44)
[40 < Age <150]

= 62.186 —0.0121 Age +4.434 log Age (Phase 3) (45)
[150 <Age <365|

To obtain from the values for the male at different ages the
corresponding value for the female, several corrections are re-
quired and these differ according to age.

PERCENTAGE OF WATER IN BRAIN AND CORD 105

From 10 to 50 days the following correction formula (45a) is
used:
Correction (minus) =0.0006 Age? —0.0386 Age +0.3 (45a):
The values thus obtained are subtracted from the computed
values for the males at the corresponding ages.
From 50 to 65 days no correction is made.
From 65 days to 135 days, correction is made according to
the formula (45b)
Correction (plus) =0.823 log (Age +1) —0.000542 (Age +1) — 1.4616
(45b)
From 135 and 165 days the correction is uniform thus:
Correction (plus) =0.22 (45e)
From 165 to 365 days correction is made by the following —
formula:
Correction (plus) =0.22 +0.0005 (Age — 165) (45d)
The foregoing (43)—(45d) replace the formulas given in the
paper by Donaldson, ’10.
Formulas (43)—(45d) were used for table 74.

Table 74, which follows, is reprinted from The
Rat (Donaldson 715). It gives, for the albino rat,
the brain weight and the percentage of water in
the brain and in the spinal cord for each sex,
on age.

106 HENRY H. DONALDSON

TABLE 74

Giving the percentage of water inthe brain and in the spinal cord for each sex, on age

———___~

MALES FEMALES
AGE 3

Ls] : Per cent Per cent - |Per cent ft
DAYS Body Brain or ody Brain Cord |cent of
weight | woight [°h rain | weight ("cord || weight | weight | brain” | weight | water
B 4.7 0.217 88.00 0.038 86.75 4.6 0.213 88.00 0.083 86.75
1 5.5 0.290 87.95 0.0388 86.42 5.4 0.269 87.95 0.037 86.42
2 5.9 0.333 87.90 0.041 86.08 5.8 0.323 87.90 0.041 86.08
3 6.4 0.395 87.85 0.046 85.74 6.3 0.373 87.85 0.045 85.74
4 6.9 0.442 87.83 0.050 85.41 6.8 0.421 87.83 0.050 85.41
5 7.6 0.509 87.79 0.056 85.07 425 0%492 187579) 02056) 85207
6 8.5 0.581 87.70 0.064 84.73 8.4 0.564 87.70 0.064 84.73
7 9.5 0.657 87.50 0.072 84.40 9.4 0.645 87.50 0.073 84.40
8 10.5 0.708 87.30 0.081 84.06 10.4 0.697 87.30 0.082 84.06
9 11.8 0.840 87.05 0.091 83.73 11.6" 0.811. 187.05: 0.091 S83e76
10 13.5 0.947 86.72 0.104 83.40 13.0 0.909 86.72 0.102 83.40
11 13.9 0.969 86.26 0.106 82.98 13.7 0.940 86.26 0.107 82.96
12 14.4 0.991 85.82 0.110 82.57 14.4 0.979 85.82 0.112 82.52
13 14.9 1.011 85.89 0.114 82.17 15.1 1.003 85.40 0.117 82.10
14 15e5) 120387 784297 (OMS VSG 15.8 1.031 84.98 0.122 81.68
15 16.1 1.057 84.58 0.122 81.39 16.5 1.048 84.59 0.127 81.28
16 16.7 1.077 84.19 0.126 81.00 17.3 1.079 84.20 0.133 80.88
WA 17.3 1.095 83.82 0.131 80.63 18.1 1.099 83.82 0.188 80.49
18 18.0 1.112 83.46 0.185 80.26 18.9 1.118 838.47 0.142 80.11
19 See lelolesoel2 OMS Oe ORo0 19.8 1.140 83.18 0.148 79.73
20 19.5 1.150 82:80 0.144 79.55 20.7 1.159 82.82 0.154 79.47
21 20.3 1.169 82.49 0.149 79.21 26) elie S21 Onto0mecoeo2
22 21.1 1.184 82.19 0.154 78.87 22.5 1.195 82.21 O:165 WS367
23 22.0 1.202 81.91 0.159 78.54 23.4 1.208 81.93 0.170 78.33
24 22.9 1.219 81.64 0.165 78.22 24.4 1.226 81.66 0.176 78.00
25 D379n Ae 2a0 Sle39 OF169) 77.90 25.4 1.241% cial MOL182 een
26 DAROMMEDD 2 Olli Onion ailaroo 96°55 (e2bt estat OLS] Saeou
27 25.9 1.266 80.98 0.179 77.29 27.5 1.269 80.95 0.193 77.06
28 270) e282) 80872) 0-186) 77200 28.6 1.282 80.74 0.198 76.76
29 OS Mee2oie o0-oo | OFL9S 16a 29.7 1.297 80.55 0.204 76.47
30 29.2 1.311 80.385 0.198 76.438 30/9 1/310 80.37 02210) 76nL9
31 30.4 1.324 80.19 0.204 76.16 32.0. 1.822 80.21 0.216) 75.92
32 31.6 1.3388 80.04 0.210 75.90 33.2 1.334 80.07 0.221. 75.66
33 3208) Wool (omole OL 215) vono4 34.4 1.346 79.94 0.227 75.40
34 BA 17365.7 909) AOL 221 7539 35.7 1.858 79.82 0.2838 75.16
35 35.4. 1.375 9 79269) 102227, 75-1 37.0 1.369 79.72 0.239 74.92
36 36.8 1.389 79.60 0.233 74.91 38.3 1.380 79.63 0.245 74.69

PERCENTAGE OF WATER IN BRAIN AND CORD 107

TABLE 74—Continued

MALES

. Per cent
mars | Body | Brain lef water
gms. gms.

37 38.1 1.899 79.52
38 39.6 1.411 79.46
39 41.0 1.4238 79.42
40 42.5 1.434 79.39
41 44.1 1.446 79.36
42 45.7 1.457 79.34
43 47.3 1.468 79.32
44 48.9 1.478 79.30
45 50.6 1.488 79.28
46 52.3 1.498 79.26
47 54.1 1.507 79.24
48 55.9 1.518 79.22
49 Hind bevel 7h Pal
50 D980 eos ai9R19
51 Cle owe o4G 79d,
52 6324 e555) 79215
53 65.4 1.563 79.14
54 Ge Ameleola) COR L2
55 6955) 125815 79210
56 71.6 1.589 79.08
57 Woh Asse PO A0e
58 75.9 1.606 79.05
59 78.1 1.614 79.04
60 80.3 1.622 79.02
61 82.5 1.629 79.00
62 84.9 1.637 78.99
63 87.2 1.644 78.97
64 89.6 1.652 78.96
65 92.0 1.659 78.94
66. 94.5 1.666 78.93
67 OPOM MAG OMSL O2
68 99.5 1.681 78.90
69 102.1 1.688 78.89
70 104.7 1.695 78.88
(A Oe ome le OZmeiSron
4 IO) Wee). AS Ss
(By WPA IL ecAlss. “ree!
TE SSE oy 22) SE 82

FEMALES

Cord Beco Body Brain pee cent Cord Ene
eee ea | geen temic | Wome |e
0.239 74.68 39.6 1.391 79.55 0.250 74.47
0.245 74.46 40.9 1.400 79.49. 0.255 74.26
0.251 74.25 42.3 1.411 79.45 0.261 74.06
0.257 74.04 43.7 1.422 79.42 0.267 73.86
0.264 73.95 45.1 1.482 79.389 0.272 73.78
0.269 73.87 46.6 1.441 79.387 0.278 73.72
0.276 73.74 48.1 1.451 79.35 0.284 73.60
OR281 a 7on62 49.6 1.460 79.33 0.289 73.50
0.288 73.50 51.1 1.468 79.31 0.294 73.39
0.293 73.39 52.7 1.478 79.29 0.300 73.30
0.299 73.28 04.3 1.487 79.27 0.306 73.21
0.305 73.17 55.9 1.495 79.25 0.311 73.12
OFS 73)07 57.5 1.5038 79.24 0.316 72.05
0.317 72.97 59.2) -1.512> 79.23 0.322 72'.97
0.323 72.88 60.9 1.520 79.21 0.3827 72.88
0.329 72.79 62.6 1.528 79.19 0.3382 72.79
0.334 72.69 64.3 1.535 79.18 0.3388 72.69
0.340 72.60 66.1 1.548 79.16 0.348 72.60
0.346 72.51 67.9 1.551 79.14 0.348 72.51
0.352 72.43 69.7 1.558 79.12 0.8538 72.48
0.358 72.35 (126 12565" 79.11 02359) 72235
0.363 72.27 73.4 1.573 79.09 0.364 72.27
0.369 72.19 75.3 1.580 79.08 0.3870 72.19
0.375 72.11 tied) Meb8e W906 0-375) 72.11
0.380 72.04 79.2 1.594 79.04 0.380 72.04
0.386 71.97 81.2 1.601 79.02 0.385 71.97
0.391 71.91 8322) 12607) 79201 08389) 71291
0.397 71.84 85.2 1.614 78.99 0.394 71.84
OF402 aleid 87.3 1.621 (78.98 0-399 71.77
0.407 71.71 89.4 1.627 78.97 0.404 71.72
0.413 71.65 91.5 1.633 78.96 0.409 71.66
0.418 71.60 93.6 1.639 78.94 0.414 71.61
0.424 71.54 95.8 1.645 78.93 0.419 71.54
0.429 71.48 98.0 1.651 78.92 0.424 71.50
0.434 71.43 100.2) 1.657 -78.91 0.429 71:45
0.489 71.38 102.4 1.663 78.89 0.433 71.41
0.445 71.32 ~ 104.7 1.669 78.88 0.488 71.36
0.450 71.27 107.0 1.675 78.86 0.442 71.32

108

HENRY H. DONALDSON

TABLE 74—Continued

MALES

Body
weight

gms.

118.
121.
124.
126.

129
132

134.
136.
138.
140.
142.

143

145.

147
148

150.

152.

153
155
156

158.
160.
16k.
162.

164

165.

167.
168.
Lie

171
172
174

175.

176.6

eed

179.

180.
181.

nnmnoor

nAonmantonruan

Oowororh OWN

wontwoaw

3

HOD

a >

Brain

weight

ee i

=

ee

a ee ee

eee

gms.

029
735
741
746
752
758

762
765
.769
072
776
eS)
782
785
788
791

794
Oe.
799
.802
805
.807
.810
.812
815
817

.819
821
824
.826
.828
.830
.832
.833
.835
837

839
841

FEMALES

Per cent Per cent - |Per cent
obwater! wetant [LMa4e"| weight | weight |T3te
18.81 (OF 455) Wile 22 109.3 1.681 78.85
78.80 0.460 71.18 111.6 1.687 78.84
78.79 0.465 71.13 114.0 1.692 78.83
78.77 0.470 71.09 116.4 1.698 78.82
78.76 0.475 71.04 118.8 1.703 78.81
78.75 04800 71000) 1211.3) 11709" 78.80
78.74 0,488 70.96 12276 eZ S eo
78.73 0.486 70.92 124.0 1.715 78.7
78.72 0.488 70.89 12D AS Leon’
78.71 0.491 70.85 126.8 1.720 78.76
78.70 0.494 70.81 128.1 12723) 18S
78.69 0.497 70.78 129.5 1.726 78.74
78.68 0.499 70.74 130.8 1.728 78.73
IK WEA WeAl 132 AME T3leGs ere
78.66 0.504 70.67 138324) Iles Sac
78.65 0.507 70.64 184.6 1.786 78.70
78.64 0.509 70.61 135.8 1.738 78.69
78.63 0.511 70.58 137.1 1.740 78.68
78.62 0.514 70.56 138.3 1.748 78.67
78.61 0.516 70-53 139.4 1.745 78.66
78.60 0.518 70.50 140.6 1.747 78.65
78.59 0.520 70.48 141.8 1.749 78.64
78.58 0.522 70.45 142.9 1.751 78.638
78.57 0.525 70.438 144.0) 1.752 78.62
78.56 0.527 70.40 145.1 1.754 78.61
78.55 0.529 70.38 146.2 1.756 78.60
78.54 0.531 70.36 i fy Aes uel Wn 32 ks EAL)
78.53 0.533 70.34 148.3 1.760 78.58
78.53 0.534 70.32 149.4 1.762 78.58
78.52 0.5386 70.30 150.4 1.764 78.57
78.51. 0.538 70.28 151.4 1.766 78.56
78.50 0.540 70.26 152.4 1.768 78.55
78.49 0.541 70.25 1538.4 1.770 78.54
78.48 0.548 70.23 154.4 1.772 -78.53
78.47 0.544 70.22 155.3 1.774 78.52
78.46 0.546 70.20 156.3% Lib) 78750
78.45 0.547 70.19 WV (ag?) Narehay 7s f510)
78.44 0.549 70.17 158.2 1.778 78.49

Cord

weight

gms.

0.447
0.451
0.456
0.460
0.465
0.469

0.471
0.474
0.476
0.479
0.481
0.483
0.485
0.488
0.490
0.492

0.494
0.496
0.497
0.499
0.501
0.503
0.505
0.506
0.508
0.510

0.512

0.514

0.515
0.517
0.519
0.520
0.522
0.523
0.525
0.526

0.527
0.528

| Per

cent of
water
cord

71.27
71.23
VL AS
71.15
gL
71.07

71.03
71.00
70.96
70.93
70.89
70.86
70.83
70.80
70.77
70.74

70.72
70.69
70.67
70.64
70.62
70.60
70.58
70.55
70.53
70.51

70.49
70.47
70.46
70.44
70.42
60.41
70.40
70.38
70.37
70.36

70.35
70.34

PERCENTAGE OF WATER IN BRAIN AND CORD 109

Body
weight
gms.

182
184.
185
186.
187
188.
189.
190

OWTIMKM EW O

192.
193
194
195
196.
ee
198
199
200
201

WDwwwwnNnnerese

202
203
204.
205.
206.
206.
207.
208.
209.
210.

NAOntnwnooorenn

211.
212.
213.
213.
214.
215.
216.
217.

bo
—
NI
co

218.7

eBewonwroonht w

Brain

weight

gms.

ee i ee Se sO

er ee ee

842
844
846
848
849
851
852
854

899
857
808
.860
861
862
863
.865
.866
867

868
.870
Park
873
874
875
876
STT.
878
879

.880
881

.882

883
884

885
886
887
887
.888

MALES

Per cent
of water
brain

78 .44
78 .43
78 .42
78.41
78 .40
78 .40
78 .39
78 .38

78.37

78 .24
78 .23
78 .23
78 .22
78 .22
78.21
78.21
78 .20
78.20
78.19

TABLE 74—Continued

Per cent

weit [Eg] weet
0.550 70.15 159.1
0.552 70.14 160.0
OF553) 7013 160.9
0.555 70.12 161.8
0.556 70.11 162.6
0.558 70.09 163.5
0.559 70.08 164.3
0.561 70.07 165.2
0.562 70.06 166.0
0.563 70.06 166.8
0.564 70.05 167.6
0.565 70.05 168 .4
0.566 70.04 169 .2
0.567 70.03 170.0
0.569 70.03 170.7
0.570 70.02 ib7(Taeeay
0.572 70.02 We
0.573 70.01 173.0

FEMALES

Per
cent of
water
cord

Per cent
of water

brair

Cord
weight
gms.

Brain
weight
gms.

779 78.49 °0.5380. 70.32
781 78.48 0.5381 70.31
.782 78.47 0.532 70.30
783 78.46 0.533 70.29
.785 78.46 0
786 78.45 0.536 70.27
.788 78.45 0
789 78.44 0

a a aaron

.790 78.43
791 78.43
.793 78.42
794 78.42
795 78.41
196 78.4

798 78

RN) Ther.
.801 78
.802 78

pe ye a

.803 78.36 0.549 70.22
.804 78.36 0.550 70.22
.804 78.35 0.551 70.22
.805 78.35 0.552 70.22
.806 78.34 0.553 70.22
.807 78.33 0.554 70.22
.808 78.32 0.555 70.
.809 78.32 0.555 7
.810 78.31 0.556 70.
Olt; 78230) O:55% 70.

a
bo
bo

to by wy
wwpy

.812 78.30
.813 78.29
.813 78.29
.814 78.28
.815 78.2
.816 78.2
201% 18:
ZO Alor
.818 78.
soils) fe).

On
>
o
pa |
iw)

~]

er a
a

SS eae) Soy Sy Sy
. ae os

>

—

=I

oO

bo

bo

Dw Ww bw

Or

HENRY H.

MALES

Per cent

TABLE 74—Continued

Per cent

mre bees t Ee ae ht os eet eae pe a ee
gms gms. gms gms.
151 219.5. 1.889 78.19 0.591 70.00 186.7
152 220.2 1.890 78.18 0.592 70.00 187.2
153 221.0 1.891 78.18 0.592 70.00 187.8
154 221.7 1.892 78.17 0.593 70.00 188 .4
155 222.5 1.893 78.17 0.594 70.00 188.9
156 223.2 1.894 78.16 0.595 70.09 189.5
157 223.9 1.895 78.16 0.586 70.00 190.0
158 224.7 1.896 78.15 0.596 70.00 190.6
150) 225noie LCoS mOn aoe lO L00) 191.1
160 226.0 1.898 78.14 0.598 70.00 191.6
161 226.7 1.899 78.14 0.599 70.00 192.1
162 227.4 1.900 78:13 0.600 - 70.00 192.6
163 228.1 1.901 78.13 0.600 70.00 193.2
164 228.8 1.902 78.12 0.601 70.00 193.6
165 229.4 1.902 78.12 0.602 70.00 194.2
166 230.1 1.903 78.12 0.603 70.00 194.6
167 230.7 1.903 78.12 0.603 70.00 195.1
168 231.4 1.904 78.12 0.604 70.00 195.6
169 232.0'1.904 78.12 0.604 70.00 196.1
170 232.6 1.905 78.12 0.605 70.00 196.5
tiie 623320 12906) 7812 02605: 70200 197.0
172 233.9 1.906 78.12 0.606 70.00 197.5
178° 2345 1-907 78.12 0-606" 70200 197.9
WAL 8235-0 12907 78el2) 0607 70200 198 .4
175 235.7 1.908 78.12 0.608 70.00 198.8
176 236.3 1.909 78.12 0.608 70.00 199.3
177 =. 236.9 1.909 78.12 0.609 70.00 199.7
178 237.4 1.910 78.11 0.609 69.99 200.1
179 238.0 1.910 78.11 0.610 69.99 200 .6
180 238.6 1.911 78.11 0.610 69.99 201.0
181 239.1 1.912 78.11 0.611 69.99 201.4
182 239.7 1.912 78.11 0.612 69.99 201.8
183) 24052) A213 78a (0.612 69299 202 .2
184 240.8 1.913 78.11 0.618 69.99 202.6
185 241.3 1.914 78.11 0.613 69.99 203 .0
186 241.8 1.915- 78.11 0.814 69.99 203 . 4
187 242.3 1.915 78.11 0.614 69.99 203.8
188 24%.9 1.916 78.11 0.615 69.99 204 .2

DONALDSON

FEMALES

Brain
weight
gms.

.820
821
821
822
823
824
825
.825
826
827

pear a a a — a — Gt — a — at

.828
.829
.829
830
831
832
.832
833
.833
.834

a ee a a

834
835
835
836
837
837
.838
.838
.839
839

re ee

.840
841
841
.842
842
843
843
844

ee ee ee ee ee

Per cent
of water

brain

78 .25
78.24
78 .24
78 .23
78 .23
78 .22
78 .22
78.21
78.21

Cord

weight

gms.

0

oo

Soy fey ee)

Soro te Soe SSJ(oQey ey Se (=|) =)

Sis ens ererey(S)

.065
0.
0.
0.
0.

566
567
568
568
569
570
571
571
572

73
574

.ot4
O75
.076
576
O17
.Ol7

578
578

O19
O79

Per

cent of

water
cord

70.22
70.22
70.22
70.22
70.22
70.22
70.22
70.22
70.22
40.22

70.22
70.22
70.22
70.22
70.22
70.22
70.22
70.2

70.22
70.22

70.22
70.22
70.22
70.22
70.22
70.22
70.22
70.22
70.22
70.22

~1
oS

No bh bo
bo bw bo

lo bo

PERCENTAGE OF WATER IN BRAIN AND CORD

111

TABLE 74—Continued

AGB
IN
DAYS

189
190

191
192
193
194
195
196
197
198
199
200

201
202
203
204
205

206 .

207
208
209
210

211
212
213
214
215
216
217
218
219
220

221
222
223
224
225
226

bo

rs

a
Onmwawnoro fk

bo
on
S
ome NwWoh OSS

bo

or

ns
ORrFNTWOONN >

Brain
weight
gms.

1.916
LOIy

mo
918
.918
.919
.919
.920
.920
921
921

.922

pee ee

.922
.923
.923
. 924
.924
.925
.925
.926
.926
927

— Ot

927
.928
928
929
929
.929
.930
.930
.930
931

— ee ot

931
931
.932
.932
.932
1.933

= = —

MALES

Per cent
of water

brain

78 .09
78 .09
78.09
78.09
78.09
78 .09
78.09
78 .08
78.08
78.08

78.08
78.08
78 .07
78 .07
78.07
78 .07

FEMALES

Per cent - |Per cent Per
weight | ° lay oa eae went etamater Ww ae ne ; a es
ms gms. gms. gms. cord
0.615 69.99 204.6 1.844 78.17 0.588 70.22
0.616 69.99 204.9 1.845 78.17 0.588 70.22
0.616 69.99 ZO5RS ME S4oN Sle OkOSSs O22
0.617 69.99 205.7 1.846 78.17 0.589 70.22
0.617 69.98 206.0 1.846 78.17 0.589 70.22
0.618 69.98 206.4 1.847 78.17 0.589 70.22
0.618 69.98 206.7 1.847 78.17 0.590 70.21
0.618 69.98 ZO SA eon lt OK oFOn iO m2
0.619 69.97 ZU ARIE SS Sond 0.591.) fOP2I
0.619 69.97 207.8 1.848 ‘78.17 0.591 70.21
0.620 69.97 208.1 1.849 78.17 0.591 70.21
0.620 69.97 208.4 1.849 78.17 0.592 70.20
0.620 69.96 208.8 1.849 78.17 0.592 70.20
0.621 69.96 PRY) LW tse PAS LU) TAD AD
0.621 69.96 209.4 1.850 78.16 0.5938 70.2
0.622 69.96 ZOORSMIE S51 ariel 6) Ono ome Oe2
0.622 69.95 Zl OR ie Shilly 7S al 6m Oko9a 0e20
0.622 69.95 210.4 1.851 78.16 0.594 70.19
0.623 69.95 DNOR Me S52) 968. 16 025945 019
0.623 69.95 211.0 1.852 78.16 0.594 70.19
0.624 69.94 PALS 18a 7G Oss 70) 108)
0.624 69.94 FALL. Isis 7G OLE 7O.Iy)
0.624 69.94 PAUL) eS} Asia) WSS 70a
0.625 69.94 212.2 1.854 78.16 0.596 70.18
0.625 69.938 212.5 1.854 78.16 0.596 70.18
0.626 69.938 ZI2ES e855 18-16 ON59T 70E18
0.626 69.93 ZIM SODNe Sn OM ORO a (Os
0.626 69.93 213.4 1.855 78.16 0.597 70.18
0.627 69.92 ZA ler SOOM On OMmOL OT me On ll
0.627 69.92 PASSO ILS) Asai) (OAs ss 7(eal/
0.627 69.92 QIAP 2 e856 oeloenOLo98) Ons
0.628 69.91 214.4 1.857 78.15 0.598 70.16
0.628 69.91 214.7 1.857 78.15 0.598 70.16
0.628 69.90 ZISROMM ESS Selon OOO a ORLG
0.629 69.90 215.2 1.858 78.14 0.599 70.15
0.629 69.90 215.5 1.858 78.14 0.599 70.15
0.629 69.89 215.8 1.858 78.14 0.599 70.15
0.630 69.89 216.0 1.859 78.14 0.600 70.14

112

Body
weight

gms.

259.
259
259
260.

260.
260.
261.
261.
261.
262.
262
262.
263.
263

263.
263.
264
264.
264
265.
265.
265
265.
266.

266.
266
266.
267.
267 .
267.
267.
268.

268 .¢
268 .!

268 .
269.
269.
269.

Nw oan

WoOomMDRHOANOD

HK ORMWOAMNOD

NMWwOMDAWH DOOD w

HENRY H. DONALDSON

TABLE 74—Continued

MALES FEMALES
Bosin [Der cent] con Per cert] Body | Brain [BES] Cora foont of
weight |°hrain | WEIGH | cord || WeMgE® | weight |°hruin'| weight | wate
1.933 78.07 0.630 69.89 216.2 1.859 78.14 0.600 70.14
1.938 78.06 0.630 69.88 216.5 1.859 78.138 0.600 70.14
1.933 78.06 0.6380 69.88 216.7 +1 .859' 78.13 OF 600" 7014
1.934 78.06 0.631 69.88 27 301 S60! (78213) Or60 lee 0RlS
1.9384 78.06 0.631 69.87 2 eo TE SHOT 7S: 13 ORG OOS
1.9384 78.06 0.631 69.87 Zio) 860) °78).13;) OL601 70ers
12935087805 JORGS2GOEST PARC AUER Tash. OO 170) 1
1.935 78.05 0.6382 69.86 2729) MES6l 78-12) OL 602 ne Omie
1.985 78.05 0.632 69.86 ZS VAL S61 78212 OF 602 70mi2
1.936 78.05 0.633 69.85 218-8 “12862 778.12 10602 70RnT
1.986 78.05 0.633 69.85 218.6 1.862 (78.12 (07602) 70
1.936 78.04 0.633 69.85 218.8 1.862 78.11 0.603 70.11
1.937 78.04 0.634 69.84 219.0 1.868 78.11 0.603 70.10
1.937. 78.04 0.634 69.84 2192) 863) 782 Ll ORCS a0 RO
1.9387 78.04 0.6384 69.84 219.4 1.868 78.11 0.603 - 70.10
1.9388 78.03 0.634 69.838 219.6 1.868 78.10 0.608 70.09
1.988 78.03 0.685 69.83 219.8 1.868 78.10 0.604 70.09
1.988 78.03 0.685 69.82 220.0 1.864 78.10 0.604 70.08
1.988 78.03 0.685 69.82 220.3 1.864 78.10 0.604 70.08
1.9389 78.02 0.6385 69.81 220.4 1.864 78.09 0.604 70.07
1.939 78.02 0.636 69.81 220.6 1.864 78.09 0.604 70.07
1.9389 78.02 0.636 69.80 220.8 1.864 78.09 0.605 70.06
1.940 78.01 0.636 69.80 221.0 1.864 78.08 0.605 70.06
1.940 78.01 0.6386 69.79 221 2 1.865 78.08 (026055) 70805
1.940 78.01, 0.637 69.79 221.4 1.865 78.08 0.605 70.05
1.940 78.01 0.637 69.78 221.6 1.865 78.08 0:605 70.04
1.941 78.00 0.637 69.78 221.7 1.865 78.07 0.606 70.04
1.941 78.00 0.637 69.77 221.9 1.865 78.07 0.606 70.08
1.941 78.00 0.638 69.77 222.1 1.865 78.07 0.606 70.03
1.941 78.00 0.688 69.76 222.3 1.866 78.07 0.606 70.02
1.942 77.99 0.6388 69.76 222.4 1.866 78.06 0.606 70.02
1.942 77.99 0.6388 69.75 222.6 1.866 78.06 0.607 70.01
1.942 77.99 0.639 69.75 222.8 1.866 78.06 0.607 70.01
1.943 77.98 0.639 69.74 223.0 1.866 78.05 0.607 70.00
1.948 .77.98 0.639 69.74 223.1 1.866 78.05 0.607 70.00
1.943 77.98 0.6389 69.73 223.3 1.867 78.05 0.607 69.99
1.9438 77.98 0.640 69.73 223.4 1.867 78.05 0.608 69.99
1.944 77.97 0.640 69.72 223.6 1.867 78.04 0.608 69.98

PERCENTAGE OF WATER IN BRAIN AND CORD 113

oP)

Nw dD Ww
SSS ST on
ooo oo ©
NON © OD

bo

TABLE 74—Continued

MALES d FEMALES
Brain [Percent Cora [Per eent!| Body | Bein [Pet cert] Cord feont of
weight |main| WeiDt |°cord || weight | woight [cin | weight | water
1.944 77.97 0.640 69.72 223.7 1.867 78.04 0.608 69.98
1.944 77.97 0.640 69.72 223.9 1.867 78.04 0.608 69.98
1.944 77.96 0.640 69.71 224.0 1.867 78.03 0.608 69.97
1.944 77.96 0.640 69.71 224.2 1.867 78.03 0.608 69.97
1.945 77.96 0.640 69.70 224.3 1.867 78.03 0.608 69.96
1.945 77.95 0.641 69.70 224.5 1.868 78.02 0.609 69.96
1.945 77.95 0.641 69.69 224.6 1.868 78.02 0.609 69.95
1.945 77.94 0.641 69.69 224.8 1.868 78.02 0.609 69.95
1.945 77.94 0.641 69.68 224.9 1.868 78.01 0.609 69.94
1.945 77.94 0.641 69.68 225.0 1.868 78.01 0.609 69.94
1.946 77.93 0.641 69.67 225.1 1.868 78.01 0.609 69.94
1.946 77.93 0.641 69.67 225.3 1.868 78.00 0.609 69.93
1.946 77.93 0.641 69.66 225.4 1.868 78.00 0.609 69.93
1.946 77.92 0.642. 69.66 225.5 1.869 78.00 0.610 69.92
1.946 77.92 0.642 69.65 225.7 1.869 78.00 0.610 69.92
1.946 77.92 0.642 69.65 225.8 1.869 77.99 0.610 69.91
1.947 77.91 0.642 69.64 225.9 1.869 77.99 0.610 69.91
1.947 77.91 0.642 69.64 226.0 1.869 77.99 0.610 69.91
1.947 77.91 0.642 69.63 226.1 1.869 77.98 0.610 69.90
1.947 77.90 0.642 69.63 226.2 1.869 77.98 0.610 69.90
1.947 77.90 0.642 69.62 226.4 1.869 77.98 0.610 69.89
1.947 77.89 0.643 69.62 226.5 1.870 77.97 0.611 69.89
1.948 77.89 0.643 69.61 226.6 1.870 77.97 0.611 69.88
1.948 77.89 0.643 69.61 226.7 1.870 77.97 0.611 69.88
1.948 77.88 0.643 69.60 226.8 1.870 77.96 0.611 69.87
1.948 77.88 0.643 69.60 226.9 1.870 77.96 0.611 69.87
1.948 77.88 0.648 69.59 227.0 1.870 77.96 0.611 69.86
1.948 77.87 0.648 69.59 227.1 .1.870 77.95 0.611 69.86
1.948 77.87 0.6438 69.58 224-2) 1870) 77395) (OF611 69.85
1.948 77.86 0.643 69.58 227.3 1.870 77.94 0.611 69.85
1.948 77.86 0.644 69.57 227.4 1.870 77.94 0.611 69.84
1.948 77.86 0.644 69.56 227.5 1.870 77.94 0.611 69.84
1.949 77.85 0.644 69.56 227.6 1.871 77.93 0.612 69.83
1.949 77.85 0.644 69.55 22M RSs ait 9S) 10,612 69E83
1.949 77.84 0.644 69.55 22780 Vesdl ms92), 0.612) s69Es2
1.949 77.84 0.644 69.54 227.9 1:87) 77.92. 0.612) 69-82
1.949 77.84 0.644 69.53 228-0" TES Te i792) OLGI2 OOrSl
1.949 77.83 0.644 69.53 228.0) 187L 77-91, OF612) GSest

114 HENRY H. DONALDSON

TABL 74—Continued

MALES FEMALES

AGE é
vs Body Brain Per SOEs d Per cent Body Brain Per cent Cord eeneteE

ReeNe MSM prmin | TSE |Teora || meant | Malet paca ae lees
303 275.9 1.949 77.83 0.645 69.52 223-1 VEST WOU ORG G9es0
304 276.1 1.949 77.82 0.645 69.52 228.2 1.871 77.90 0.612 69.80
305 276.2 1.949 77.82 0.645 69.51 228.3 1.871 77.90 0.612 69.79
306 276.3 1.949 77.82 0.645 69.50 228.3 187i 77290 7 OnGI2 69 R79
307 276.4 1.949 77.81 0.645 69.50 228.4 1.871 77.89 0.612 69.78
308 276.5 1.949 77.81 0.645 69.49 Z2SeON MeSil ilas9) (ORGl2mOOmiS
309 276.6 1.950 77.80 0.645 69.49 228.6 1.872 77.88 0.613 69.77
310 276.7 1.950 77.80 0.645 69.48 223) MeSi2) Wiha so) VOROlomOo aia
311 276.9 1.950 77.80 0.646 69.47 228 NESi2) VWie88, OLGlsn ole76
312 «277.0 1.950 77.79 0.646 69.47 228.8 1.872 77.87 0.613 69.76
313 277.0 1.950 77.79 0.646 69.46 228.8 1.872 77.87. 0.618 69.75
314 277.1 1:950 77.78 0.646 69.46 228.9 1.872 77.86 0.6138 69.75
315 9277-2) 1.950 77.78 0-646 69745 229.0 1.872 77.86 0.613 69.74
316) 238 15950) “Wiad (O2G46) 469r44" = 229.07 1872 7285, OL Olonmoo mie
317 277.5 -1.950 77.77 0.646 69.44 2297, Le Si2) Tie 8a. Oko Sa Como
318 277.5 1.950 77.76 0.646 69.43 22971 1.872 77.84 0.618 69.72
319 277.6 1.950 77.76 0.646 69.43 229.2 1.872 77.84 0.613 69°72
320 277.7 1.950 77.75 0.646 69.42 229.3 1.872 77.83 0.6138 69:71
321 277.8 1.950 77.75 0.646 69.41 229.3 W872) 77-83 “O2613 ooe7Tt
SID) Le9oll ide OFG47, SCOR 229.4 1.873 77.82 0.614 69.70
323 278.0 1.951 77.74 0.647 69.40 229.4 1.873 77.82 0.614 69.70.
324 - 278.0 1.951 77.73 0.647 69.40 229.5 1.873 77.81 0.614 69.69
BoD eon TOSI Mien ONGATeI69R39 229.5 1.873 77.81 0.614 69.68
S20R iors 951 Mite 2004s 169738 229.6 1.873 77.80 0.614 69.68
327 278.3 1.951 77.72 0.647 69.38 229.6 1.873 77.80 0.614 69.67
328 278.4 1.951 77.71 0.647 69.37 229.7 1.873 77.79 0.614 69.67
329 278.4 1.951 77.71 0.647 69.37 229.7 1.873 77.79 0.614 69.66
330 278.5 1.951 77.70 0.647 69.36 229.8 1.873 77.78 0.614 69.66
Sol eee On ool Wii 0) JORGATaGO R35 229.8 1.873 77.78 0.614 69.65
332 278.6 1.951 77.69 0.647 69.35 229.8 1.873 77.77 0.614 69.64
333 278.7 1.951 77.69 0.647 69.34 229.9 1.873 77.77 0.614 69.64
334 278.7 1.952 77.68 0.648 69.34 229.9 1.874 77.76 0.615 69.63
335 278.8 1.952 77.68 0.648 69.33 229.9 1.874 77.76 0.615 69.63
336 6278.9 1.952 77.67 0.648 69.32 230.0 1.874 77.75 0.615 69.62
337 278.9 1.952 77.67 0.648 69.32 230.0 1.874 77.75 0.615. 69.62
338 279.0 1.952 77.66 0.648 69.31 230.0 1.874 77.74 0.615 69.61
339 279.0 1.952 77.66 0.648 69.31 230.1 1.874 77.74 0.615 69.61
340 279.1 1.952 77.65 0.648 69.30 230.1 1.874 77.73 0.615 69.60

PERCENTAGE OF WATER IN BRAIN AND CORD

TABLE 74—Concluded

AGE
IN
DAYS Body Brain
weight | weight
gms gms.
341 279:2 1.952
342 «27922 1.952
S40, 2/983) 1.952
344 279.3 1.952
345 «279.3 1.952
O40) ee (9RA 952
347 9279.4 1.953
348 279.5 1.953
349 9279.5 1.953
350 279.6 1.9538
351 279.6 1.953
oo2, 2/96 1.953
353 ©40279.7 + 1.9538
Dotee29eG 1953
355 279.7 1.953
356 279.8 1.9538
357 «279.8 1.953
358 279.8 1.953
359 279.8 1.954
360 279.8 1.954
361 279.8 1.954
362 279.9 1.954

954
.954
954

w
for)
(JX)
bo
~]
oO
oOo
See ee

MALES

Per cent
of water

brain

77.64
77 .64
77 .63
77.63
77.62
77.61
77.61
77 .60
77 .60
77.59

67 .58
77.58
Ghat
77.57
77.56
77.55
77.55
77 .54
77 .54
77.53

77.52
77.52
77.51
77.51
77.50

FEMALES

Per cent
of water

cord

69
69
69
69
69
69
69
69

69.
69.
69.
69.
69.
69.
69.
69.
69.
69.

69.
69.
69.
69.
69.

29
29
28
20
27
.26
25
25
69.
69.

Per cent
of water
brain

Brain
weight
gms.

874 7
874 7
874 7

ane 00:
874 77.
874 77.69
874 77.69
874 77.68
.874 77.68
.874 77.67

ert eel oe

.874 77.66
.874 77.

.875 77.65
.875 77.65
.875 77.64
.875 77.63
875 77.63
875 77.62
.875 77.62
875 77.61

el ee

-815 77.60
.875 77.60
.875 77.59
875 77.59
1.875 77.58

—_ ft et

Cord

weight

gms.

0.615
0.615
0.615
0.615
0.615
0.615
0.615
0.615
0.615
0.615

0.615
0.615
0.616
0.616
0.616
0.616
0.616
0.616
0.616
0.616

0.616
0.616
0.616
0.616
0.616

115

Per

cent

of

water
cord

69.
69
69
69
69
69
69
69
69
69.

69
69
69
69
69
69
69
69
69.
69

69
69
69.
69
69.

59

9
.08
mY |
OT
06
.06
00
54

54

53
02
.52
ol
.00
.50
49
48

48

AT

AT
46

45

45

44

Le
bal
ean
i]

3
4
al

_ ni , r
i i vane ; F

* tone

1

SOME EXPERIMENTS ON THE NATURE AND FUNC-
TION OF REISSNER’S FIBER

GEORGE E. NICHOLLS
Beit Memorial Fellow

Zoological Department, King’s College, London
THIRTY-FIVE FIGURES
CONTENTS

ea linbrocduetyonmsy src eee RENO oir ois cok ie ores eR ete te os 119
A. A review of the suggestions which have been made concerning
the nature and function of Reissner’s fiber and the sub-com-

TESTS OH RTH UO BTN Cie Ot On a ee 5 > co eR Oe eee ae 119
B. Earlier attempts to determine the function of Reissner’s fiber
by. expernimentalbmethods.: ..; . ister sces cs oan Blane oes 125
C. An account of the present state of our knowledge of Reissner’s
LT OVE Noha Big RG es ONES RS ok Uc eae ae Pe 128
bi he scone of the present investigation. «conc... c.:-s6seds bass ome = 133
PbeViaternalvand Methods. 5... i0h 8 ce. vo sae ee Me ieee Ona cede ees 136
iY “Observations upon the living animals: . 2220432. 00.. eee ces dee ee 145
V. A summary of the record of the experiments and an account of the
MctecistUponyWeIssner SrHlO Or... 5.2.2 acs eae tata nel nee as Ciccks 149
VI. The relation between the condition of Reissner’s fiber and the reac-
POET ACY OVS(ey EV ot0 Nee REA Ais OI SERRE SB SRE oe >. RRP anaes a PR Reg ee Ae 166
NM DS CHAS TOMm ree nt Piney remorse tnt corsga eet Moves exe rs- oeu ach CO hes BS inate vas 175
1. The function and mode of action of the Reissner’s fiber ap-
FOPN EG DES parece trie eh bs oh ann te Rent MRA AeA oie ag ka, Nk nena nn Raa 175
2. The spiral winding of the fiber and the occurrence of ‘snarls’... 180
3. The duration of the reaction and the problem of regeneration... 183
ISD Pan SCLIN ATS AS ny ese eke eo ye Send 2 teas Wee Tee ns Satta a at 188
Pe MMIMIGE TNLINGE CELLO Gs crane sad oe the ctre os kscheete ORE aK Ree ER os 190

It is probable that concerning no part of the vertebrate ner-
vous system have there been held views more widely divergent
than those which have been entertained concerning Reissner’s
fiber.

In 1907, when I took up the study of this structure, Sargent’s
‘optic reflex’ theory had met with very general acceptance. At
an early stage in my work, however, I obiained proof that the

117

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2
FEBRUARY, 1917

118 GEORGE E. NICHOLLS -

fiber, although undoubtedly a preformed structure, was certainly
not a nerve fiber and, therefore, could not have the function
ascribed to it by Sargent. In 1909, I published a statement to
that effect and in the following summer, following my discovery
of the practical accessibility of the fiber in the tail region, I
carried out some experiments upon elasmobranchs, in an en-
deavour to ascertain the function of the fiber.

The results of these experiments, which were performed upon
less than a dozen dogfish and rays, were hardly sufficient to give
a conclusive answer to the question of the function of the fiber
but were, nevertheless, extremely suggestive. An account of
these experiments was published, therefore, in a short prelimi-
nary paper which did not, however, appear until 1912. In
the meanwhile, a much more extensive series of experiments
had been carried out but there had been no opportunity to ex-
amine this material microscopically before the paper in question
was published.

The completion of the investigation has been very considerably
delayed, for these further experiments were scarcely completed
when I left England to take up an appointment in India. I had
purposed however, to carry on, there, the work of preparing the
necessary serial sections. The material is, unfortunately, ex-
ceedingly refractory, so that under the best of circumstances the
preparation. of ‘serial sections demands much time and patience.
In India, there were added difficulties, due to the climate, and
the preparation of the sections went on very slowly, it being
possible to attempt this work only during the cold weather. An
attempt to get some of the material sectionized in England was
unsuccessful, essential portions of some of the specimens being
ruined in the attempt to prepare the sections and the remaining
tails were returned to me as being too refractory to yield satis-
factory sections by ordinary methods. In the end I was com-
pelled to postpone the preparation of my remaining material for
microscopic study until my return to England. Only recently
has this part of the task been completed.

In the interval, I have published a paper (12 a) dealing with
the subject of Reissner’s fiber and its relation to the central

THE FUNCTION OF REISSNER’S FIBER 119

nervous system. Accordingly, there will be need, at this time,
only for a brief review of the various suggestions which have
been put forward as to the nature and function of the fiber and
a short account of the present state of our knowledge of the fiber
and its connections, the reader being referred to the above men-
tioned work for further details.

I gladly avail myself of this opportunity to express my nee
to Professor Dendy for valuable advice and criticism throughout
the progress of the work: also, to the Government Grant Commit-
tee of the Royal Society, for Grants in Aid; to the British Asso-
ciation for the Advancement of Science and the Senate of the
University of London for placing at my disposal their tables at
the Plymouth Marine Laboratory, and to Dr. Allen, Director of
the Laboratory, for the facilities afforded me in the prosecution
of the research.

I. INTRODUCTION

A. A review of the suggestions which have been made concerning
the nature and function of Reissner’s fiber and the
sub-commissural organ

1. Reissner (’60), by whom the fiber which now bears his name
was discovered, believed that this ‘Centralfaden’ was simply a
nerve fiber and to him, therefore, it was remarkable principally
on account of its peculiar situation. He found it, as is well
known, lying freely as an axial thread in the central canal of the
spinal cord of the lamprey. Since the diameter of the fiber in
this animal (in which alone he had observed it) is, approxi-
mately, that of a moderately coarse nerve fiber, it is scarcely
surprising that, its unusual situation notwithstanding, Reissner
came to this conclusion. Kutschin (’63) who confirmed Reiss-
ner’s discovery, accepted that author’s view of its nature.
Neither of these observers was able to trace the fiber into the
brain ventricles and they believed it to be confined to the cen-
tral canal of the spinal cord.

2. That a nerve fiber should occur in such a situation seemed
to Stieda (’68, ’73) altogether improbable and he decided that
Reissner’s fiber (‘jenen riithselhaften Strang’) must be an arti-

120 GEORGE E. NICHOLLS

fact. He suggested that the alleged fiber was produced by the
coagulation of the cerebro-spinal fluid under the action of the
fixing reagent, pointing out that there was no evidence of its
being related to any nerve cell.

For thirty years this view passed almost unquestioned, Viault
(76), Rohon (77), Sanders (’78, 794) and Gadow (’91) all ac-
cepting it. More recently Kalberlah (’00), Streeter (03) and
Edinger (08) have expressed themselves in agreement with
Stieda’s view. That this view was so widely held is, doubtless,
the explanation of the fact that during this period there are found,
in the literature, so few references to the occurrence of the fiber.

3. Interest in this structure revived, however, when Stud-
ni¢ka (’99) reasserted the preformed nature of the fiber. This
author suggested that it was to be regarded as an epithelial se-
cretion, comparable to that which has produced the crystalline
style of the lamellibranch gut. He believed that it is produced
by the cells lining the central canal of the spinal cord and that
it is capable of growing forward, to end freely in the brain ven-
tricles but he made no suggestion as to its function. Kolmer
(05) appears to be the only author who has endorsed this view
and Studniéka has, himself, since abandoned it (713).

4. It is a very surprising fact that the extraordinary and quite
conspicuous development of the epithelium beneath the posterior
commissure, should have remained for so long unnoticed. A
brief mention of it, indeed, appears to have been made by Fulli-
quet (86) but not until 1892 was it figured (very diagrammati-
cally) by Edinger (’92) who conjectured that it might be a
glandular body producing some secretion to be discharged into
the cerebro-spinal fluid. Its histology was first carefully de-
scribed by Studni¢ka (’00) who gave figures of its finer anatomy
in dogfish and lamprey but did not, apparently, realize its con-
nection with Reissner’s fiber.

5. A little later the sub-commissural organ of the Ammocoete
was described and figured by Dendy (’02) who noted the exist-
ence of close-set cilia clothing its ventricular surface and sug-
gested that, in conjunction with certain folds of the choroid plexus

THE FUNCTION OF REISSNER’S FIBER 121

of the midbrain, it served to establish currents which promoted
the circulation of the encephalic fluid.

6. In the meanwhile Sargent had also asserted the preformed
nature of Reissner’s fiber but had denied that Studni¢ka was cor-
rect in interpreting it as a secretion. In Sargent’s view the fiber
was a nervous structure.

In several subsequent papers (’01, ’03, ’04) Sargent endeay-
ored to establish this view stating that Reissner’s fiber consists
of ‘‘numerous axis cylinders closely applied to each other and
surrounded by a single thin medullary sheath of myelin.” These
axis cylinders were supposed to be derived in part from the nu-
merous large cells of the ‘Dachkern’ and from alleged multi-
polar cells in the habenular ganglion as well as from other mul-
tipolar cells said to be situated actually within the lumen of the
central canal, towards the hinder end of the spinal cord. In
teleosts, in which group Sargent overlooked the remnants of the
‘Dachkern,’ he claimed that the alleged midbrain constituent
“axons” of Reissner’s fiber were derived from the myriad
cells of the torus longitudinalis.

Reissner’s fiber was, therefore, according to this author, built
up of two sets of axons running in opposite directions and a
comparison was made between this structure and the giant fibers
of Amphioxus and Annelida. Concerning the destination of the
forwardly running axons there is nothing stated, but those which
were said to arise in the brain were regarded as motor axons
having a very great length, each being supposed to stretch from
the midbrain roof direct to one of the trunk muscles. Sargent
stated that he had seen such fibers leaving the main Reissner’s
fiber in the region of the spinal cord and that these passed out
directly to the musculature, probably by way of the ventral
spinal roots. In the midbrain roof the related nerve cells were
described as in direct connection with the proximal ending of the
retinal neurons so that there was said to be interposed but a
single nerve element between the sensory (retinal) nerve cell
and the muscle-fiber in the trunk. Sargent suggested that, by
this means, the delay in the transmission of motor stimuli along

12, GEORGE E. NICHOLLS

the ordinary (tecto-spinal) conduction paths through a number
of neurons could be lessened in cases of urgency.

Houser (’01) claimed that he had been able to confirm Sar-
gent’s observations, while numerous observers seem to have ac-
cepted Sargent’s theory concerning the function of the fiber.

That, notwithstanding many weighty objections, this theory
met with such general acceptance is doubtless to be attributed
very largely to the fact that Sargent claimed (04) that his ob-
servations had been fully confirmed by actual experiments upon
living animals (vide infra).

7. Although Sargent (03) was the first to describe the con-
nection between Reissner’s fiber and the sub-commissural organ
(his ‘ependymal groove’) he attributed comparatively little
importance to this latter structure, asserting that it served
merely as a support and anchorage for Reissner’s fiber. In this
view he has been followed recently by Tretjakoff (13).

K6lliker (02) recording the occurrence of Reissner’s fiber
in the blind Proteus and other Amphibia, admitted that he had
become convinced of the preformed nature of the fiber. He
appears, however, to have been unable to choose between the
conflicting views advanced by Studnicka, Sargent and Kalberlah.

8. The work of Ayers upon ‘Ventricular Fibers in Myxinoids’
is of interest in that it contains the first suggestion that Reiss-
ner’s fibers might be composed of numerous united delicate
fibrillae springing from ependymal epithelial cells. Whether,
however, he considers these fibrillae as of the same nature as
the ependymal fibers which serve as supporting structures within
the central nervous system, or not, Ayers does not make clear,
and his work unfortunately contains a number of erroneous
statements. He does not, indeed, refer to the fiber by name and
appears to have been wholly unaware of previous work upon the
subject.

Thus, in Bdellostoma, he figures numerous more or less parallel
ventricular fibers which, while they may perhaps represent
several lengths of a much folded and snarled fiber, may equally
well represent some artifact. It certainly is not the normal
condition in this animal. Moreover, it would appear that

THE FUNCTION OF REISSNER’S FIBER 123

Ayers never saw Reissner’s fiber in the lamprey, since his de-
scription of the ‘ventricular fibers’ in that animal as ‘‘a fine-
meshed network of fibrils which . . . . in life practically
fills the ventricular cavity”’ certainly can not apply to Reissner’s
fibers. It is extremely probable, therefore, that Ayers failed
to distinguish clearly between coagulum and the fibrillae of
Reissner’s fiber. His conclusion that the fiber was certainly
“an organ of relation bringing all parts of the ventricular cavity
into. intimate connection”? (my italics) is likewise mistaken, for
Ayers did not correctly identify the brain cavities in this animal,
in which of the iter little remains but the sub-commissural canal.
Accordingly he failed to recognize the distinction which exists
between the tract of modified epithelium which constitutes the
sub-commissural organ and the flattened epithelium which
lines other parts of the ventricular cavity. Concerning the
function of the fiber he conjectured that it might be ‘‘ connected
with the control of the ventricular lymph supply by vaso-motor
control.”

9. Horsley (08) describing the occurrence of Reissner’s fiber
in certain apes stated that, in these forms at least, the fiber
had not the structure of a tract of nerve fibers nor, when cut,
did it exhibit Wallerian degeneration. While not denying the
accuracy of Sargent’s statements in so far as they relate to this
structure in the lower vertebrates, Horsley expressed the opinion
that, in its resiliency, the fiber resembled a chitinous or skeletal
structure and suggested that, in the higher vertebrates, it had
become nothing more, perhaps, than a residual structure.

10. In 1909 I gave an account (’09) of the behavior of the
fiber in recoil and stated that, in my opinion, the fiber was cer-
tainly non-nervous. At the same time Dendy (’09) put for-
ward an entirely novel suggestion concerning the function of
the fiber. His suggestion was that the fiber itself was a strand
of connective tissue which played a merely mechanical part,
variations in its tension being produced by the flexure of the
body and every such variation might be supposed to result in a
stimulus being transmitted to the cells of the sub-commissural
organ This latter structure was interpreted as a sensory

124 GEORGE E. NICHOLLS

organ, controlling automatically the flexure of the body. He
concluded by expressing the hope that some way would be found
of overcoming the apparently insuperable obstacles which stood
in the way of satisfactory experiments upon the fiber by which
alone could the hypothesis be tested.

11. A study of the development of Reissner’s fiber in Cyclo-
stomes (and Amphibia) led me to the conclusion (712, ’12a, 713)
that this structure was formed by the coalescence of cilia-like
processes springing from cells which, while largely collected
upon the sub-commissural organ, are not limited to that organ,
other cells occurring scattered in the ependymal lining of the
central canal contributing to the fiber. In my opinion, the
fiber is to be regarded as a thread of protoplasm. This view
is supported by the staining reactions of the fiber, while its
high refractivity, its power of regeneration and the rapidity
with which it apparently disintegrates after death are facts
easily explicable upon this hypothesis. Further, its mode of
contraction is paralleled, only, so far as I am aware, in the
searcely modified protoplasm which forms the stalk of certain
Protozoa.

This view that the fiber is, in fact, a protoplasmic thread
has since been accepted by Dendy (’12), Studnicka (713) and
by Tretjakoff (13). The latter author, however, appears to
have misread Sargent’s papers, for he attributes this view to
that investigator, saying (’13, p. 110) “Sargent zeigte namlich,
dass der Faden noch in embryonalen oder larvalen Leben als
ein Bundel von feinen, cilienahnlichen Fortsatzen der Zellen
der Sub-kommissuralen Grube ensteht.”’

12. Tretjakoff (’13), however, while accepting this view of
the nature and function of the fiber suggests that we are mis-
taken (‘“‘ich glaube deswegen, dass in diesem Punkt die ‘Theorie
von Dendy und Nicholls falsch ist’”’) in attributing any sensory
function to the sub-commissural organ. He believes that the
sensory cells connected with Reissner’s fiber are found only in
the epithelium which lines the central canal and holds, with
Sargent, that the sub-commissural organ serves merely for the
support or anchorage of the fiber. These sensory cells are

THE FUNCTION OF REISSNER’S FIBER 125

described by Tretjakoff as projecting into the lumen of the
central canal where each is said to end in a small knobbed proc-
ess, which Tretjakoff compares to the bellpush of an electric
bell. He supposes that the stimulation of these cells is effected
by the pressure of the fiber upon these processes whenever the
body is flexed.

That Tretjakoff’s investigations were made upon material
in which the fiber had been broken and had retracted is suggested
by his figures. Two only of these depict the central canal. In
one (fig. 20) the fiber (which is invariably very fine in the Ammo-
coete) is seen indistinct and vastly swollen. In the other (fig.
19) the fiber is absent and the lumen of the central canal is
occupied by nuclear bodies, the remains probably of epithelial
cells dislodged from the ependymal epithelium by the fiber in
its withdrawal. Under these circumstances it is not surprising
that Tretjakoff failed to find the delicate filaments which seem
to join the fiber at frequent intervals as I have described (12 a)
and the occurrence of which has been confirmed by Studincka
(435 py 585)

The little knobs (Tretjakoff’s bell-pushes) are almost certainly
the retracted remnants of the fibrillae of those cells which, in
my view contribute to the formation of the fiber and which,
torn free by the dislocation of the fiber, have shrunk back upon
the sensory process of the parent cell.

B. Earlier attempts to determine the function of Reissner’s fiber by
experimental methods

The first reference to experiment in connection with the ques-
tion of the function of Reissner’s fiber occurs in a preliminary
paper by Sargent (01). These experiments were subsequently
described in greater detail in 1904.

In these experiments an attempt was made to break the fiber
by a means of incision made through the choroid plexus of the
fourth ventricle of certain elasmobranchs. Such experiments,
involving, as they necessarily did, the risk of serious disturbance
to the central nervous system or even actual injury to the brain

126 GEORGE E. NICHOLLS

itself, were of little value, for it could not be established that
any of the reactions observed were the results simply of the
interruption of the ‘optic reflex short-circuit’ alleged to be pro-
vided by Reissner’s fiber.

I gather, moreover, that Sargent relied upon observations
made from dissections to determine whether or not the experi-
mental incision had really broken the fiber, which appears to me
as an altogether unsatisfactory method. Whether there was a
subsequent microscopical examination of the material is not
clear nor does Sargent state what precautions were taken to
prevent a disturbance of the fiber during the dissection. The
statement that ‘“‘the cord and medulla of each individual was
preserved for microscopical examination” suggests that a part
only of the nervous system was subsequently cut out. If this
were the case, it is practically certain that, whatever the result
of the experiment upon the fiber, it. would be found retracted
in the preserved material.

It is, therefore, a little difficult to ascertain the grounds for
his remark (’01, p. 450) that ‘“‘animals on which the equivalent
operation was performed without breaking the fiber are nearly
or quite normal.”’

Otber experiments were made by Sargent (’01) to determine
the effect of artificial extirpation of the eye upon the fiber but
the results obtained were never recorded. Several years later,
experiments were made upon Reissner’s fiber by Horsley (08).
In this case the subjects of the experiments were individuals of
two species of Macacus. Minute electrolytic lesions were made
in the spinal cord, at the level of the fifth cervical segment, in
order to break the fiber. No observations are recorded, how-
ever, upon the behavior of the living animals nor are details
given as to the duration of the experiments. Concerning the
appearance of the fiber under the microscope, Horsley remarked
that Wallerian degeneration was not observed in the broken
fiber.

I find, however, some little difficulty in interpreting the ap-
pearance of Reissner’s fiber in the sections figured by Horsley.

THE FUNCTION OF REISSNER’S FIBER 127

In his figure 10, Reissner’s fiber is seen in transverse section,
occupying quite an appreciable part of the lumen of the central
eanal. As it is traced backwards from this level (the first cervi-
cal segment) through the third cervical segment (fig. 9) towards
the point of lesion in the fifth cervical segment (fig. 8), it is seen
to constantly diminish in size. Behind the point of lesion this
diminution in size continues as will be seen in figures 11 and 12
but, more caudally, the diameter of the fiber is again seen to
increase (fig. 13), this latter figure representing a section through
the spinal cord in the lumbar region.

Now this is not at all what one would expect to find where
Reissner’s fiber had been broken experimentally. Usually it
would be found that on either side of the lesion there was a
stretch of canal devoid of fiber. Still further from the lesion
the severed ends of the fiber might be found swollen and perhaps
knotted if the material were killed and fixed soon after the lesion
had been made. ‘Tracing the fiber distally, in either direction,
from these knotted or swollen ends one would expect to find that
the fiber diminishes in diameter until the normal size is reached.
If, however, the killing of the material were postponed for a
considerable time after the experimental operation the swollen
end and the spiral twisting would have disappeared and the
fiber-would have straightened out backwards, extending practi-
cally to the point of lesion, nearly normal except that it might
not have regained its taut condition. The piece lying posterior
to the lesion might have retracted wholly backwards to the
end of the cord. If the material were not killed until several
weeks after the operation it is probable that regeneration would
have largely re-established the normal condition throughout.

The condition figured by Horsley, in which the fiber is most
swollen anteriorly, regularly diminishes in diameter towards
and past the lesion (and probably becomes normal in the thoracic
region) but shows a renewed swelling very far back, suggests
that the condition of the fiber may have had nothing to do with
the actual experiment. I should judge that sufficient time had
elapsed after the experiment to permit of regeneration, and the

128 GEORGE E. NICHOLLS

actual condition of the fiber figured was due to its rupture in
removing the central nervous system from the body for pres-
ervation. The appearances are those which would be observed
if the fiber were broken accidentally in the hindbrain and in the
region of the filum terminale by section of the nervous system
in those regions, or by handling during dissection.

From the figures it would appear that there may have been a
somewhat considerable local disturbance of the central nervous
system in consequence of the experiment. While this might
have obscured, to some extent, the reaction consequent upon the
breaking of the fiber (especially as a point’very far forward was
selected for the operation) it is nevertheless much to be regretted
that nothing is recorded as to the behavior of the living animals
as the result of the experiments.

C. An account of the present state of our knowledge of Reissner’s
fiber

Reissner’s fiber is an extremely delicate. protoplasmic thread,
having, in general, a diameter of more than 1y and less than 3un.
It possesses a high refractivity and, in the normal tense con-
dition, appears to be absolutely structureless.

It is normally present in the central nervous system of practi-
cally all vertebrates and may be seen, most readily, in longi-
tudinal (sagittal) sections of the spinal cord. It is necessary,
however, that the nervous system shall have been preserved
entire and immediately after the death of the animal; even then,
carelessness in handling during the dissection may cause the
fiber to snap, or it may chance that the fiber was broken prior
to the death of the animal. Generally, however, if the central
nervous system has escaped damage, Reissner’s fiber will be
found everywhere in the central canal stretched taut and lying
centrally in the canal. It maintains a uniform thickness and
shows no trace of spiral winding. At frequent intervals it
appears to be connected with the ependymal epithelial cells by
delicate cilia-like protoplasmic filaments (figs. 29, 30).

THE FUNCTION OF REISSNER’S FIBER 129

In this undisturbed state the fiber issues (in the lower verte-
brates at least) from the posterior end of the central canal through
a terminal opening (the ‘terminal neural pore’) into the peri-
neural space, where it ends in an elongated conical expansion
(the ‘terminal plug’). There is, in these forms, a widening of
the lumen of the central canal at its posterior end to form a
chamber (the ‘terminal sinus’) which is only incompletely
enclosed by the walls of the filum terminale, this being, in this
region, reduced to a simple epithelial tube. The posterior wall
of the terminal sinus is formed by the meningeal sheath into
which the terminal plug is inserted (text-figs. 1, 3).

——— = =

Mane ge boaecesmees --
aM TNT Niel! ae a

Halfuittbey Uy sitot Za SSH woh Vivant
PaWR0-509 ede Gernieea.e.

Text-fig. 1 Slightly diagrammatic median sagittal section through the end
of the filum terminale to show the normal (undisturbed) arrangement of the
sinus terminalis and the insertion of Reissner’s fiber. c.c., central canal of the
spinal cord (and terminal filament); f.t., filum terminale; mn., meninges, form-
ing the hinder wall of the sinus terminalis; R.f., Reissner’s fiber; s.t., sinus ter-
minalis; ¢.p., terminal plug.

Traced forward, the normal fiber is found to pass from the
central canal of the spinal cord into the fourth ventricle. It
maintains its position in the middle line and appears, in this
part of its course, to lie absolutely freely at the level of the middle
of the height of the ventricle.

At the anterior part of the hindbrain, however, the fiber
stretches in contact with the lower surface of the cerebellum.
There is frequently, upon the lower surface of the rhombo-
mesencephalic fold, a narrow groove (the ‘isthmic canal’) which
may show traces of a paired character and which serves for the

130 GEORGE E. NICHOLLS

reception of the fiber. Emerging from the anterior end of this
groove, sometimes as a paired structure (12a, figs. 10, 11),
Reissner’s fiber stretches freely through the midbrain ventricle
to the neighborhood of the posterior commissure.

The ventricular surface of the posterior commissure is clothed
by a band of highly developed epithelium which is often folded
in both the longitudinal and the transverse planes. It is to this
remarkable tract of epithelium that the name ‘sub-commissural
organ’ has been given. Owing to its longitudinal folding it has
usually, in transverse sections, a horseshoe shape and partly
encloses a median dorsal groove (the ‘sub-commissural canal’).
Reissner’s fiber, if it has continued as an unpaired structure so
far forward, breaks up at the hinder end of the posterior com-
missure into two or more strands which subdivide within this
median groove into numerous delicate fibrillae which are con-
nected with the cells of the sub-commissural organ.

A study of the development of the fiber indicates that it
arises by the confluence of numerous filaments springing from
sub-commissural organ and that the composite thread so formed
extends backwards into the central canal of the spinal cord.
Within the central canal it probably receives numerous additional
components from scattered cells in the epithelium which lines
the central canal.

Perhaps the most remarkable characteristic of the fiber is its
extreme elasticity. In life it appears to exist under quite con-
siderable tension and to be somewhat prone to accidental break-
age. In that event, or following artificial section, the free ends
may recoil sharply to form tangled knots or ‘snarls.’ The re-
traction is accompanied by a marked increase in the diameter
of the fiber.

This elasticity usually disappears very rapidly during the
process of fixation and the preserved fiber may become distinctly
brittle (fig. 21). If, however, the fiber be severed before fix-
ation is completed a retraction will still take place, but much
more gradually, and it will then be found that the fiber has
become wound in a more or less open spiral. Even where the
recoil has been an abrupt one, resulting in the formation of the

THE FUNCTION OF REISSNER’S FIBER a

characteristic knot, a careful examination of this mass will,
almost invariably, reveal the fact that the retraction was ac-
complished by a spiral winding of the fiber.

Such a knot of retracted fiber has, indeed, the form of a con-
torted mass similar to that which may be produced in any thin
stretched elastic thread of which one end is held fast and the
other end twisted continuously in one direction. I have been
able to obtain practically all stages intermediate between such
complicated knots and the simplest spiral (text-fig. 2). Unlike

D.
a eee ac
| E. f
G. Meee
UT ORIRS — SASHECKERR,

Text-fig. 2 Stages in the twisting of Reissner’s fiber in its withdrawal from
the point of breakage. A, B, D from Scyllium eanicula (9); C, from Petro-
myzon fluviatilis; HZ, F, G, H, from Raia blanda (3).

the simple twisted elastic thread, however, the spiral winding
may appear interruptedly in Reissner’s fiber, spiral stretches
alternating with swollen but untwisted lengths. Moreover, the
twisting does not always make its appearance at the free end
but may arise at a greater or less distance from the point where
the fiber has been broken.

If, therefore, the spinal cord has been cut prior to fixation,
Reissner’s fiber may be found to have withdrawn for a relatively
considerable distance from the point of section and a great stretch
of the central canal may be found devoid of fiber. The extent
of such retraction apparently varies with the region in which the

NS 4 GEORGE E. NICHOLLS

fiber has been broken and depends, possibly, upon the size of the
central canal in that particular region for, in the case of a sud-
den recoil, the spiral winding may produce at or near the severed
end a mass of coiled fiber which apparently checks further re-
traction. With the retreating end of the fiber may be dragged
numerous epithelial cells and, around it, will collect a quantity
of coagulum (fig. 17) which may render it difficult to distinguish
exactly the condition of the knotted end.

On the side of the tangle remote from the point of section, the
fiber usually emerges as a coiled thread and thence passes gradu-
ally into a more open spiral. If the fiber has been cut at a
sufficient distance from its attachment, this open spiral may pass
into a swollen but straight stretch and ultimately be found to
pass almost or quite into the normal condition. Broken, how-
ever, near to one of its attachments, the fiber will almost cer-
tainly withdraw violently and completely to that attachment,
from which it may even tear itself free, dragging with it many
of the epithelial cells.

While this retraction which is so characteristic of Reissner’s
fiber is, as I have pointed out (’09), altogether unlike anything
known in a nerve, neither does it altogether resemble the recoil
of a simple (homogeneous) elastic thread. It is, therefore, of
especial interest that I have been able, recently, to detect in a
greatly swollen and retracted fiber what appears to be a fine
deeply staining central axis (fig. 17); the resemblance of the fiber
to the stalk of a Vorticella (with which I have already compared
it, 712 a, p. 25) is thereby greatly enhanced. ‘This appearance
is somewhat inconstant and never to be made out in the unrelaxed
condition.

I have been unable to decide whether the numerous delicate
fibrillae (fig. 29) seen in the central canal of the spinal cord are
in organic continuity with Reissner’s fiber or whether they are
merely unusually long cilia which have been cemented to the
fiber after death by the coagulated cerebro-spinal fluid. That
the former view is probably correct is indicated, I believe, by
the fact that in the cases in which some retraction of the fiber
has occurred it is very rare to find any of these fibrillae apparently

THE FUNCTION OF REISSNER’S FIBER 133

related to the swollen and retracted portion of the fiber. In-
stead, in the region in which there has been a dislocation of the
fiber, minute spherules of some highly refracting substance are
found plentifully, close to or in contact with the free surface of
the ependymal cells. That these are the contracted remains of
such connecting fibrillae, which were, indeed, component fila-
ments of Reissner’s fiber is therefore extremely probable. The
withdrawal of the fiber would inevitably snap such connecting
filaments in the region affected and these broken protoplasmic
strands would naturally shrink backwards towards the surface
of the parent cells.

The view that Reissner’s fiber is a thread of modified proto-
plasm, formed by the complete coalescence of numerous delicate
filaments (or hypertrophied cilia) is indicated by its origin and
is confirmed by its staining reactions. Moreover, it is an inter-
pretation which renders comprehensible its singular elastic recoil
notwithstanding its apparent structureless condition. Such spiral
retraction is met with only, so far as I. am aware, in the little
differentiated protoplasm of the Protozoa, among which gr ou
the fusion of cilia is also no uncommon feature.

II. THE SCOPE OF THE PRESENT INVESTIGATION

From what has been stated above it will be seen that, while
there has been a great variety in the suggestions made as to the
nature of Reissner’s fiber, there have been put forward but three
theories as to its function.

The disproof of Sargent’s statements as to the nature of the
fiber disposed, at the same time, of his ‘optic reflex theory.’

Ayer’s suggestion was based, as I have shown, almost entirely
upon an erroneous idea of the nature and normal condition of —
the fiber and its relation to the ventricles; in any case his view is
not one which could easily be tested experimentally.

There remained Dendy’s theory which might readily be put
to the test of experiment if a way could be devised of breaking
the fiber without damage to the central nervous system.

Such an operation became possible with my discovery (’10,
p. 527) of the actual condition of the hinder end of the filum

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

134 GEORGE E. NICHOLLS

terminale in the Ichthyopsida. Elsewhere completely enveloped
by the brain and spinal cord, Reissner’s fiber is peculiarly
accessible at the extremity of the tail, the more so that there is
practically an absence of nervous tissue in the hinder part of
the filum terminale. This structure is, indeed, little more than
a simple tube of columnar epithelium. At the actual hinder
end, Reissner’s fiber may be said to be protected only by the

Text-fig. 3 A sagittal section through the extremity of the tail of Raia blanda
(III—experiment 3) to show the position of the sinus terminalis. c.c., cen-
tral canal of the spinal cord (and terminal filament); f.t., filum terminale; mn.,
meninges, forming the hinder wall of the sinus terminalis; nch., notochord; R-f.,
Reissner’s fiber; s.t., sinus terminalis; t.p., terminal plug; v.c., vertebral column.

skin and the delicate meninges, between which there lies but a
film of connective tissue (text-fig. 3).

A cut made in the vicinity of the end of the terminal filament
would break the continuity of the fiber, therefore, but would be
quite unlikely to produce physiological results such as to mask or
interfere with the reactions resulting from the disorganization
of the mechanism of which Reissner’s fiber forms a part.

My experiments, then, were intended primarily as an attempt
to determine the function of Reissner’s fiber and its related

’ THE FUNCTION OF REISSNER’S FIBER 135

structures by means of observations made upon the living ani-
mals in which the continuity of the fiber had been intentionally
destroyed, but I had other objects, also, in view.

At the time when the experiments were undertaken practically
nothing was known concerning the mode of recoil of the fiber.
Sargent had stated that when cut before fixation the free ends of
the fiber retracted into a knotted mass or ‘snarl’ but he had not
observed that this snarl was spirally wound. I had myself
seen such snarls in several cases in material which had not been
specially preserved for the study of this structure and in which
the spinal cord had been cut previous to fixation (712 a, figs. 17,
18, 19). In most of such material the fiber was ill preserved
and, in the main, my own attention had been confined to a
determination of the normal anatomical relations of the fiber.
Accordingly, I had taken special precautions to thoroughly fix
and harden my material before severing the spinal cord. Never-
theless, I had come, in the previous year, upon a few examples
of this spirally wound condition which had been obtained unin-
tentionally by a premature cutting of the spinal cord (12 a,
figs. 12, 16). These accidents, however, had yielded no in-
formation concerning the behavior of the fiber cut in life. It was
naturally supposed that a breaking of the fiber in the living
animal would be followed by a sharp recoil of the severed ends
similar to that which was known to occur when the fiber was cut
in freshly killed material. It was desirable, however, to ascertain
if this were so.

It was anticipated, moreover, that the results of the experi-
ments would throw light upon the question of the natural limits
of this recoil. It must be remembered that beyond the mere fact
of the occurrence of a recoil nothing had been recorded, and it
was not even known whether the recoil started by the section
of the living fiber would continue until both free ends had re-
tracted to their respective points of attachment or whether, on
the contrary, there would be formed speedily, in the living animal,
a tangle (or tangles) which might (on reaching a size sufficient
to block the lumen of the canalis centralis) automatically check
further recoil in one or both directions.

136 GEORGE E. NICHOLLS

In the latter event the tangled end (or ends) might perhaps
afford a temporary hold and so prevent the fiber from being
put completely out of action.

And, finally, there was the problem of regeneration. It was
uncertain whether a tangle, if it were formed, would remain as a
permanent record of the breaking of the fiber, or, if it were a
transient feature, whether it would simply uncoil or whether the
whole fiber, or the tangled part of it, simply disappeared to be
replaced by a new growth.

Upon some of these points a certain amount of light was shed
by the results of the few preliminary experiments carried out in
1910 but upon others the information was too meager to supply
a decisive answer. Upon many of these points much additional
knowledge has been gained from the more extended investi-
gation carried out in the following year.

Ill. MATERIAL AND METHODS

The curiously exposed condition of the filum terminale in
fishes, coupled with the fact that in both elasmobranchs and
teleosts, Reissner’s fiber is particularly well developed, largely
influenced my choice of material. My final preference for elas-
mobranchs was determined by the idea that the absence of
bony tissue in the vertebral column would facilitate the prep-
aration of the inevitable large number of series of sections.

I planned, originally, to experiment principally upon the
common dogfish (Seyllium canicula) and to use rays only in the
event of dogfish of suitable size being unobtainable. Knowing
nothing certainly as to the probable extent of the recoil of the
fiber, I was anxious to make use of comparatively small speci-
mens, for it was possible that serial sections of the entire length
of the central nervous system of all of the specimens might
have to be prepared—a task of no little magnitude.

As it happened, only a couple of reasonably small dogfish were
obtained during my stay in Plymouth in July, 1910; relatively
small rays were, however, moderately plentiful, and for the most
part the preliminary experiments were performed upon these
animals.

THE FUNCTION OF REISSNER’S FIBER 137

Subsequent examination of this material under the microscope
indicated that in most cases it would be necessary to examine
only an inch or so of the spinal cord in front of the place where
the incision was made. This point was almost always within
a third of an inch of the extremity of the tail. The size of the
specimen thus appeared to be of no great importance but this
fact was only ascertained when the material had been prepared
for microscopic examination nearly a year subsequently to the
completion of these preliminary experiments.

Accordingly in the summer (August) of 1911 I was less care-
ful to restrict my experiments to specimens of small size. I was
thus enabled to obtain, more readily, the many specimens which
I required. In all, a dozen comparatively small dogfish, ranging
from 14 to 20 inches in length, were secured, and, upon these
were performed experiments varying in duration from a few
(three) hours in some cases to more than eighteen days in others.
_ Of the rays, three species were employed, but of one of these,
Raia microcellata, I had but a single specimen and, as in the
previous year, the greater number of the experiments were made
upon specimens of R. clavata and R. blanda. These included
rays which were barely 6 inches in length and which were, pre-
sumably, just escaped from the egg case, while others ranged up
to 16 inches. The duration of the experiments, in the case of
the rays varied from a few (ten) minutes to as much as thirteen
days.

The actual operation consisted in severing Reissner’s fiber
at a point quite near to the hinder end of the terminal filament
and was practically nothing but a simple prick which rarely
drew a drop of blood although, in some cases, the sections showed
that there had been some effusion of blood into the canalis
centralis.

Notwithstanding its trivial character, however, I was obliged
by the conditions under which the vivisection license was issued,
to perform the operation only upon anaesthetized specimens.

Some trial experiments with the anaesthetic indicated that
dogfish were curiously susceptible to chloroform and, despite

138 GEORGE E. NICHOLLS

my precautions, two of the subjects of the experimentis subse-
quently failed to recover from the anaesthetic.

Finally it was found that a short immersion of the specimen
in. sea-water in which had been shaken up a small quantity of a
mixture of chloroform and ether would induce a sufficient de-
eree of insensibility, and this method was adopted throughout
my series of experiments in 1911. Under this treatment, none
of the specimens died.

The operation was quite easily pertormed, the subject being
removed from the chloroform water and placed upon the table
with its tail turned upon the side. The necessary prick was
inflicted with the point of a very fine scalpel (which had pre-
viously been sterilized by passing through a gas flame) at a
point usually considerably less than a third of an inch in front
of the sinus terminalis. In the dogfish, therefore, the incision
perforated the caudal fin near its hinder border while in the
rays the cut was generally made behind the last dorsal fin (figs.
2, 8). The animal was at once returned to its tank, having
been out of water for, perhaps, thirty seconds. Recovery was
usually rapid and, as might be expected, there was no evidence
of shock.

None of the specimens died from the effect of the operation,
nor in the subsequent examination of the tissues in serial sec-
tions, was there found any indication that morbid or sepiic
conditions had been set up. Indeed, apart from certain pecu-
liarities of behavior about to be described, and which I attribute
to the breaking of Reissner’s fiber, the animals suffered no ap-
parent ill-effects.

Nevertheless, two or three specimens were lost during the
progress of the experiments from causes indirectly connected
with the experiments. In the second series of experiments a
number of photographs were taken, of normal specimens as
well as of the subjects of the experiments. I could find no record
of previous attempts to photograph living fish, and had accord-
ingly to make a number of trial exposures. At first, attempts
were made to obtain the photographs out of doors by daylight.
Numerous difficulties cropped up however, for none of the out-

THE FUNCTION OF REISSNER’S FIBER 139

side tanks were glass fronted, and the only available glass-
fronted tank, of a size to be readily transported, held but a
comparatively small quantity of water and there were no facili-
ties for connecting this tank with the aerating apparatus. A
prolonged sojourn of the fish in this tank was not possible so
that attempts to photograph under these conditions involved
disturbing the specimens, transferring them in a bucket to the
small tank and then waiting for them not only to settle down. but
to settle in a position in which it would be possible to photo-
graph them. One or two lucky snapshots were obtained but
the method was, in general, a failure.

An attempt to photograph the fish in their proper tanks in the
laboratory encountered other difficulties. Of these the chief
was connected with the light. With subdued daylight a com-
paratively long exposure was needed and it was found in practice
that the head region was always blurred by the respiratory
movements even if the fish did not elect to move bodily during
the process.

In the end flash-light photographs were taken. The camera
was fixed up opposite the tank in which was the specimen of
which a photograph was desired and by the hght of an incan-
descent gas lamp it was focussed upon a part of the tank a
little within the glass front. Above the camera was stretched
a piece of string upon which were placed a number of bent strips
of magnesium ribbon. Usually some twenty inches of the ribbon
were required, divided into four or more pieces. The gas lamp
was then extinguished, and the aerating tube and bulb removed
from the tank to do away with movement in the water. As
soon as the specimen settled in a suitable position the strips of
magnesium were lit, as nearly as possible, simultaneously.
The reflection from the glass front of the tank was considerable,
but in some of the later photographs this was diminished by
igniting other strips of magnesium suspended immediately
above the tank, care being taken to shield the lens from the
direct rays from this source of illumination. Most of the
photographs reproduced here were taken in this way.

140 GEORGE E. NICHOLLS

In all, experiments were performed upon sixty-seven elasmo-
branchs, of which twelve were dogfish and the remaining fifty-
five were rays. Two only, as already mentioned, died from the
effect of the anaesthetic, while two others died from suffocation
consequent upon my omission to replace the aerating tube in the
tank after the specimens had been photographed.

They were killed by being plunged into a mixture of spirit
and chloroform and, after a brief stay in this fluid, were evis-
cerated. In this way the blood vessels were practically drained,
which greatly facilitated the rapid dissection necessary to expose
brein and spinal cord, there being no troublesome effusion of
blood from cut vessels within the brain case. The partially
dissected specimens were immersed in a large vessel of fixing
fluid (Tellyesnicky’s bichromate-acetic mixture) and the further
dissection required to expose the greater part of the spinal cord
wes completed under the fluid. To dissect away the vertebral
column from the hinder part of the spinal cord and the filum
terminale is, however, a very delicate operation, which involves
considerable risk of damaging the nervous system. The exposure
of the spinal cord was, therefore, carried only to within a couple
of inches of the end of the tail. Behind this point I was con-
tent to strip away most of the skin and muscles, about half an
inch at the actual extremity being left quite untouched. In the
case of the dogfish the last inch (or even more) of the tail was
left intact.

The preparation of the series of sections proved unexpectedly
difficult. In general, a piece of the tail, about an inch in length,
was removed—this piece including the point of experimental
lesion—and prepared for sectioning.

My intention was to cut this terminal piece sagittally in order
that the point of experimental incision and a_ considerable
length of the filum terminale before and behind this point
might be seen in one and the same section. To avoid risk of
damage to the sinus terminalis it was found expedient to retain
undisturbed the skin upon the last half inch or so of the tail and
the terminal piece, therefore, contained the bases of numerous
spines embedded in the skin, and separated from the axis of

THE FUNCTION OF REISSNER’S FIBER 141

partly calcified cartilage by particularly tough connective tissue
with contained fin-rays. These several structures became
greatly indurated during the prolonged paraffin embedding
which was found to be necessary. Moreover, the various tis-
sues contracted unequally during this process with the result
that despite many precautions a very troublesome crumpling
was often produced.

This was most in evidence near the actual extremity of the
tail and thus affected, principally, the region behind the incision
so that, while it was usually easy to determine if the fiber had
retracted backwards from the lesion it was sometimes extremely
difficult to certainly recognize the contracted piece of fiber.
Especially was this the case when a considerable infiltration of
blood into the sinus terminalis had accompanied or followed the
recoil of the fiber.

In such sections, the filum terminale appears as a number of
isolated pieces, often cut quite obliquely and a diagrammatic
sagittal section through the sinus terminalis, such as that seen
in text-figure 3, was but rarely obtained.

Apart from this crumpling the tail usually becomes bent at
the place where the incision was made, so that the lengths of
filum terminale before and behind the incision rarely lay in the
same plane, notwithstanding that weights were used during the
process of embedding to keep the tissue as nearly flat as might
be. In front of the experimental incision the crumpling was less
noticeable, the vertebral axis being more rigid, and the muscular
and other soft tissues liable to contraction having been, for the
greater part, removed. Nevertheless, even here, a certain cur-
vature almost invariably occurred. Further, the greater hard-
ness of the cartilage in this region often caused the sections to
cut very unevenly. This irregularity could be largely avoided,
it was found, by cutting rather thick sections (not less than 30 1).
The lumen of the central canal, however, in the hinder part of
the spinal cord of the rays examined has a diameter which rarely
exceeds 30 4 and in such sections, therefore, the whole of the
central canal may be included within the thickness of a single
section or a relatively thick layer of overlying tissue may seri-

142 GEORGE E. NICHOLLS

ously obscure the lumen, and a structure so slight as is Reissner’s
fiber normally can scarcely be distinguished with certainty, if
viewed through the thickness of the epithelial wall of the filum
terminale. In the swollen or spirally twisted condition the fiber
becomes much more conspicuous, it is true, but even so there is
still considerable difficulty in making out details. In order,
therefore, to make reasonably sure of recognizing the fiber, the
sections ought not to have a thickness greater than 20 nu. In
such sections the fiber, if present, would be likely to appear as a
well defined thread in an open canal and even if the sections
should chance to include also an underlying or overlying layer of
epithelium, this would almost certainly be quite thin.

Accordingly, the attempt was made to cut the tails in sagittal
sections of 20 u in thickness, the resulting series consisting gen-
erally of comparatively thin sections alternating with others
considerably thicker, the latter often permitting the presence
(and extent) or the absence of the fiber to be ascertained, details
being filled in from the thinner sections.

As already observed, the almost invariable distortion ae ae
material led, very generally, to parts of the filum terminale being
cut very obliquely (fig. 26). Thus it happened sometimes, even
where all the sections of a series were thick, that parts of the
lumen of the central canal were exposed clearly to view. On the
other hand, even where moderately thin sections had been ob-
tained, there was occasionally some difficulty in deciding whether
or no Reissner’s fiber was present. In experimental material in
which the fiber has been broken, the relaxed fiber may frequently
be found lying closely against the surface of the cells which line
the central canal. This epithelium has a clear and highly re-
fractive internal border which stains, with borax carmine, a
delicate pink, precisely like a lightly stained Reissner’s fiber. A
very slight alteration of the focus of the microscope produces,
along the cut edge, the effect of a double line and gives rise to an
appearance which may readily be mistaken for the fiber lying in
juxtaposition, optically or actually, with the epithelial surface.
It has been found impossible, in some cases, to be absolutely
certain whether one is viewing the cut internal edge of this epi-

THE FUNCTION OF REISSNER’S FIBER 143

thelium, or Reissner’s fiber lying against it or beyond it. Usu-
ally, however, the relaxed fiber does not lie, everywhere, in a
perfectly straight line and, if one is actually dealing with the
fiber, a careful tracing of the central canal will almost always
show the fiber, sooner or later, turning centrally away from the
wall of the central canal (fig. 27) and standing for a longer or
shorter stretch as a distinct and free central thread.

There would be less difficulty, perhaps, in certainly recogniz-
ing the fiber if it invariably maintained its normal thickness (2
to 3 w in the rays) or swelled, as it may do after being cut, to as
much as 8 uw or 10 w in diameter. Not altogether infrequently,
however, the fiber appears extremely fine, of a thickness which I
estimate to be less than 0.3 u. Of such a diameter is the fiber in
early development in larval cyclostomes and amphibians, and I
can only conjecture that the occasional occurrence in these small
rays of this delicate fiber is an indication that there has taken
place a retraction of the fiber so extensive that repair has taken
on the character of a completely new growth which is at first
much thinner than in the adult state.

In yet other cases the fiber may be wanting in the region ex-
amined but there may be found, lying centrally, a shadowy
structure which seems to be a hollow cylinder (fig. 18) whose
diameter is considerably greater than that of a much swollen
fiber. Were it not that a swollen and displaced length of fiber
often lies nearby, I should have been disposed to regard this
structure as the product of the disintegration of the fiber. Pos-
sibly it represents a film of coagulated cerebro-spinal fluid which
has formed around a swollen and gradually withdrawing fiber.

The tissues were stained (in bulk) with borax carmine.
Double staining was soon. abandoned as it was found that parts
of thick sections were at times imperfectly fastened to the slide
and were liable to be lost in the staining or decolorizing fluids
and it was most important that no parts of Reissner’s fiber
should be lost in this way. In the few cases in which double
staining was resorted to, the second stian was invariably picro-
indigo-carmine.

144 GEORGE E. NICHOLLS

One other point must be mentioned here. In the ray the ac-
tual position of the terminal sinus varies slightly, it was found,
in different individuals. In the case of the specimen of Raia
blanda figured (text-fig. 3) this terminal chamber extended
downwards behind the extremity of the notochord, which is, I
believe, the strictly primitive condition. It occurs, however,
less frequently in this position than might be expected, and in
many cases it lies altogether dorsal to the notochord, not always
extending even to the posterior extremity of that structure.
Whether there has been some mutilation in these cases or whether
on the contrary there takes place, normally, a certain amount of
resorption of the tissue of the terminal filament, I can not
decide.

In some teleosts I have found what are, almost certainly,
stages in the disappearance of the postero-ventral (post-chordal)
part of the neural tube. I find, moreover, that the corrugation
of the hinder end of the filum terminale in small rays which I
have described (12, p. 423) as so strongly suggestive of neuro-
meric constriction, is likewise frequently met with in the vanish-
ing vestiges of the filum terminale in the region of the disappear-
ing tail in the recently metamorphosed anuran.

While these facts suggest that the variation in position of the
sinus terminalis of the ray may be due to some extent to the ab-
sorption of tissue in this region,! the possibility of mutilation
must not be ignored. The actual end of the tail of the ray is
soft and not protected by spines, and specimens which have suf-
fered quite considerable mutilation are by no means rare. The
terminal sinus, too, in those specimens in which it lies wholly
dorsal to the notochord (fig. 19) rarely shows that bulbous ex-
pansion which is seen in examples in which the sinus terminalis
has the postero-ventral position (fig. 20) but has quite a marked
resemblance, in shape, to the secondary terminal sinus which I

‘That an absorption of tissue in this region does occur in rays is suggested
by Beard’s statements (’96, p. 55, footnote 2), that the young (Raia radiata)
immediately prior to escape from the egg case are shorter by a centimeter or so
than embryos a month younger. Some of my own specimens which were six

inches or less in length must, almost certainly, have been quite newly escaped
and the process of resorption was possibly incompleted.

THE FUNCTION OF REISSNER’S FIBER 145

have found produced as the result of my experiments (12,
text-fig).

Be the reason for this variation in position what it may, it
has a certain importance in this investigation, for in one or two
cases where the sinus terminalis lay unexpectedly far forward,
the incision (which was made in the postero-ventral region of the
tail, being planned to break the fiber actually in the sinus ter-
minalis) missed the terminal filament altogether.

Of young dogfish, only recently emerged from the egg-case, I
have had no material but in the adult there appears to be little
variation in the position of the terminal sinus.

In several cases, both dogfish and rays, the cut was made in
the region of the terminal filament but just a trifle too far dor-
sally, and the sections show that, although the cut penetrated
the neural canal, the filum terminale and surrounding pia mater
escaped damage.

Such specimens in which the experimental incision failed to
break the fiber served well as control specimens. Other control
specimens were simply anaesthetized without undergoing the
usual operation. These latter on recovery behaved in perfectly
normal manner.

IV. OBSERVATIONS UPON THE LIVING ANIMAL
1. Upon normal material

The experiment carried out in 1910 had almost immediately
directed my attention to the fact that a frequent, if not an invari-
able, consequence of the operation was the assumption by the
subject of the experiment of a very distinct attitude while at
rest. Accordingly, during the time spent at Plymouth both in
1910 and 1911, while the experiments were going on in the
Jaboratory, very constant and careful attention was given to the
numerous normal specimens which were kept in confinement in
the adjoming aquarium. Control specimens, too, were kept
under observation in small tanks in the laboratory under condi-
tions precisely similar to those in which the subjects of the
experiments were maintained.

146 GEORGE E. NICHOLLS

It was found that the normal dogfish would, after a period of
activity, settle indifferently upon any part of the aquarium floor
apparently neither shunning nor choosing the well lighted parts
of the tank. Whether, however, they came torest upon the
floor of the tank or upon a rocky ledge in the aquarium, it was
observed that they almost invariably settled in some position
which gave room for the body to stretch out freely with the
tail extended horizontally in the line of the long axis of the
body. In such a position (figs. 1, 6) the wedge-shaped head lies
with its ventral surface lifted from the floor, but the long axis
of the brain has an approximately horizontal position. The
trunk, from the branchial region almost to the end of the pelvic
fins, lies slightly flattened ventrally against the supporting sur-
face. The pectoral fins are disposed nearly horizontally out-
wards and backwards. The anal fin is bent over, near its base,
sharply to one side so that the actual ventral surface of the ani-
mal, behind the pelvic region, is supported just clear of the
bottom. Behind the anal fin, however, in which region the trunk
tapers off into the tail, the ventral surface no longer touches the
bottom but is supported well clear of the tank floor. The
caudal fin rests upon the bottom so lightly that its flexible ven-
tral border is searcely bent. In this attitude, which was found
to be invariably assumed by fish confined in the small tanks, the
long axis of the central nervous system (which coincides with the
position of Reissner’s fiber) is maintained, practically, in the hori-
zontal plane. Only at its hinder end, in the heterocercal tail,
is this axis slightly upturned.

In the aquarium, in which an attempt is made to reproduce
more nearly the natural condition, the bottom is frequently
uneven. Whether, however, the fish settles upon the roughly
level floor or perches itself upon some jutting rocky shelf, it
will be found to maintain the posture described. Upon an
uneven supporting surface it will be seen that the body bridges
stiffly the gaps between inequalities of the surface and the tail
maintains its nearly horizontal position even if there be no
contactual surface beneath the caudal region. It is not unusual
to see a dogfish resting with the trunk supported upon a rocky

THE FUNCTION OF REISSNER’S FIBER 147

ledge and the tail projecting out stiffly. This is not to be attrib-
uted to a natural rigidity of the tail, for this region of the body
is peculiarly flexible, and it must be assumed that the posture is
maintained by muscular effort.

At times, however, dogfish will wedge themselves into crevices
between the rockwork in a nearly vertical position, but even then
they maintain a posture in which the long axis is approximately
straight.

As will appear, a quite different attitude is assumed by speci-
mens in which the fiber of Reissner has been accidentally or
otherwise injured. Indeed, specimens which have received in-
jury resulting in the breaking of the fiber can be easily recog-
nized by the attitude in which they rest.

The normal attitude of the rays is strictly comparable to that
just described for dogfish. These animals will, in confinement,
settle, apparently indifferently, either upon a horizontal or a
smooth vertical surface. While, however, the rays may often be
seen adhering to the smooth surface of the wall of the tank, or
the glass front of the tank, they appear unable to maintain
themselves for long in this position. In the larger tanks and
aquaria they seem to exhibit a preference for smooth and level
horizontal surfaces.

In either case the whole ventral surface (including that of the
head and flattened tail) is applied to the supporting surface
(fig. 7). The snout, it is true, may be very slightly lifted from
that surface (fig. 10). The flexible tail stretches backwards, its
long axis being a continuation of that of the trunk.

2. Upon experimental material

I propose, here, to give a general outline of the reaction ob-
served in the subjects of the experiments in order that the sig-
nificance of the various experiments, a detailed account of which
is given in the succeeding section, may be more readily appre-
ciated.

In the subject of the experiments, recovery from the anaes-
thetic occurred usually within a very few minutes and was fre-

148 GEORGE E. NICHOLLS

quently followed by a period of marked activity. In this case
the animal would dash about the tank, commonly blundering
heavily into the confining walls. This phase rarely endured for
long, but gave place to a quiescent stage in which the animal
apparently exhibited a preference for the darker part of its tank.
Settling down, it might remain inactive for comparatively long
periods, moving only when disturbed. In other cases the speci-
men, recovering from the anaesthetic, passed directly into this
lethargic condition. I imagine that this difference in behavior
was due to the varying degree in which the animal had been
affected by the anaesthetic, a slight degree of insensibility being
marked by the erratic activity when volition was recovered.

Be this as it may, the assumption of some posture of the body
unlike that which I have described above as normal, was fre-
quently manifested very soon after the quiescent stage was
reached. In some cases it appeared within ten minutes of the
operation. Both the head and tail would be gradually lifted
until the long axis of the body, from being a straight line would
become markedly curved (figs. 2-5). The tail was, in general,
sharply upturned from its base, while the trunk region was up-
lifted upon the pectoral fins from a region just behind the head.
In the rays, owing to the great development of the pectorals,
this appears to give rise to a transverse curvature of the anterior
part of the body, as seen from in front (figs. 9, 13, 14).

There may be also a distortion of the long axis in the hori-
zontal plane, the trunk and tail being bent several times from side
to side (in some of the dogfishes) or with a single sharp bend of
the hinder part to one side (rays and dogfish).

It is probable that a disturbance of the poise of the body
exists, ikewise, while the animal is in motion. It is, however,
very difficult to be sure of this. In some of the dogfish, certainly,
uniform undulation of the body in swimming seemed to be re-
placed by a less even movement which is perhaps best described
as a wriggling action.

These reactions did not always make an appearance quickly
after the operation. In some cases their advent was delayed for
days even, and in yet others, as will be seen from the detailed

THE FUNCTION OF REISSNER’S FIBER 149

record given below, they never appeared at all. The explanation
of these apparent exceptions must be deferred until after the
account of the microscopical examination of the experimental
material.

The duration of the reaction also varied considerably, persist-
ing in some cases for a few hours only, while in others it endured
for several days. In a few cases it appeared to be intermittent.

Vv. A SUMMARY OF THE RECORD OF THE EXPERIMENTS AND AN
ACCOUNT OF THE EFFECTS UPON REISSNER’S FIBER

A. Scyllium canicula

2. The experimental incision was made at noon on July 7, 1910.
The specimen quickly recovered and, although somewhat sluggish,
appeared to swim normally. At rest, its body was bent slightly but
otherwise the posture seemed normal. No change was observed
until July 11, when the ventral border of the caudal fin was seen to
be lifted slightly (about half an inch) from the tank floor. The ani-
mal became more sluggish and, if disturbed, soon returned to rest,
exhibiting an apparent preference for the darkest corner of its tank,
which rendered observation more difficult. The tail rested, more-
over, against a sloping part of the tank where wall and floor met.
It was impossible, therefore, to be sure whether the tail was really
slightly lifted by muscular effort or merely upraised on account of the
elevation of its support. The whole body, however, was seen to be
considerably curved. On July 13 and 14 the fish was more restless
and upon the 15th, when seen at rest, the long axis of the body was dis-
posed in a straight line (for the first time since July 8). During the
following day it was noticed that the body of the fish was once more
bent from side to side in long wavy curves, but with the tail, as be-
fore, supported upon the sloping part of the tank. Next day, however,
it was found resting well away from the back of the tank and the tail
was uplifted, a clear two inches, from the floor. By midday on the
18th this reaction was still more marked and the hinder part of the
trunk and tail were bent sharply to one side. Throughout the two
succeeding days this reaction was pronounced. On July 21 the fish
had reverted to an earlier posture, with the tail supported against
the sloping part of the floor, but by midday it was once again well
out in the tank with the tail held well off the floor. On the next day
the reaction was less marked though the body was still bent. During
July 23 the reaction was scarcely discernible and later in the day, when
it was decided to kill the specimen, the fish appeared normal. It
showed very marked activity in its attempts to avoid the net, swimming
with a wriggling movement (the head and forepart of the body being
twisted quickly from side to side). This action in swimming had been

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

150 GEORGE E. NICHOLLS

noticed on several previous occasions. This specimen, then, gave a
marked reaction lasting for 13 days. Duration of the experiment 16
days +.

The sections showed that, in front of the incision, a secondary sinus
terminalis had been produced into which Reissner’s fiber is seen to ex-
tend and, in contact with the hinder (meningeal) wall of which, it
flares. Apparently it has just become attached thereto (fig. 31).
Behind the incision the fiber has apparently entirely disappeared.

9. This specimen, for nearly a week after the operation (performed
on July 12), showed a curious restlessness, not once being observed at
rest until the morning (10 a.m.) of July 18. This activity was fol-
lowed by an equally marked lethargy. The specimen took up a posi-
tion in the darkest corner of the tank, where it lay with the body
bent upon itself at-a sharp angle and the tail supported against the
sloping part of the tank. Not until July 22 was the specimen seen, in
repose, away from the wall of the tank when it was found resting with
the end of the tail slightly lifted: the flexure of the body was nearly —
straightened out. It was killed on July 23 the experiment having
lasted 11 days.

In the sections the fiber (in front of the lesion) is found to extend
backwards nearly to the place where the filum terminale was severed.
It is probable, therefore, that the fiber had nearly recovered from the
effect of the operation but the experiment was ruined by an accidental
cut made, far forward in the trunk region, when exposing the spinal
cord. The fiber in the piece examined is much swollen and continu-
ously twisted (text-fig. 2), undoubtedly due to a (backward) re-
traction from this distant cut.

20. The incision was made at 11.15 a.m. on August 3, 1911, and, by
noon, the tail was lifted slightly so that the lower border of the caudal
fin no longer rested upon the tank floor. When disturbed, the fish
swam with a quick wriggling action (cf. 2) and came to rest in a curi-
ous attitude in a corner of the tank, the anterior part of the trunk
being poised vertically, supported by the adjacent walls of the tank,
while the posterior part lay out horizontally upon the tank floor (cf. the
ray, fig. 11). After being again disturbed, it once more came to rest
in this peculiar attitude and so remained until 3 p.m. at which time
it was again compelled to move. It was observed to swim in quick
rushes, even leaping partly out of the water, and the wriggling move-
ment was very noticeable. Ten minutes later it had settled down
with the end of the tail slightly lifted but resting lightly against the
tank wall. It was disturbed yet again and was subsequently induced
to settle well away from the walls of the tank and the tail was then
seen to be held at least an inch and a half from the floor, and it con-
tinued in this attitude until 4.15 p.m. when it was accidentally dis-
turbed. During the next half hour it was repeatedly set in motion
by the movements of another dogfish which shared the tank. It
settled down six several times in the same attitude (with head and tail
lifted) once or twice essaying the half vertical position which it had

THE FUNCTION OF REISSNER’S FIBER 151

assuméd earlier. By 4.45 p.m. the tail was lifted more than two inches
from the floor. The specimen was then driven about the tankand
compelled to swim actively for several minutes and then removed and
killed. There was in this case a well marked reaction which endured
for the entire period of the experiment—53 hours.

In front of the lesion the fiber has completely withdrawn from the
piece of terminal filament and spinal cord examined.

21. The incision was made at 11.40 a.m. August 3. Upon recovery
from the anaesthetic the specimen adopted the normal attitude. It
was killed at 10.30 a.m. August 6, having given no apparent reaction
during the three days of the experiment.

The sections are poor but show that the incision missed the terminal
filament and thus failed to break the continuity of the fiber which is
seen to be of normal diameter and to lie tautly stretched.

22. Within 10 minutes of the operation (performed at 10 a.m.,
August 4) a marked reaction appeared, the lower border of the caudal
fin being lifted a clear two inches from the tank floor. The animal
was sluggish and, after being disturbed, reverted always to this atti-
tude. It was twice photographed later in the afternoon but the reac-
tion had then become less marked (figs. 2, 3) but continued as shown
until the specimen was killed at 5 p.m. Duration of the experiment
7 hours.

The fiber has apparently been withdrawn forward from the lesion
completely beyond the anterior limit of the piece of spinal cord
sectioned.

23. The incision was made at 10.10 a.m. August 4, but was followed
by no apparent reaction and the specimen continued normal until it
was killed on August 22. It was photographed on August 7 (fig. 6).
Duration of the experiment 18 days 8 hours.

The sections show that the cut failed to penetrate the neural canal
and the normal Reissner’s fiber may be seen lying tautly stretched in
the central canal of the undamaged terminal filament.

24. The incision was made at 4 p.m., August 4, and the usual reac-
tion was noticed within half an hour of the operation, the caudal fin
being lifted two inches or more. It was, however, less sluggish than
the subject of the preceding experiment and frustrated all attempts
to obtain a photograph during the early days of the experiment. The
reaction continued uninterruptedly until the evening of August 8,
the photograph (fig. 5) being obtained about midday on August 6.
From the 9th onwards the reaction appeared intermittently and during
the whole of the 10th the caudal fin was observed to be resting lightly
upon the floor of the tank though the head was still somewhat raised.
Late in the evening of the 15th and again at noon on the 18th the tail
appeared slightly lifted for a while, but for the most part the reaction
rarely appeared for any length of time after the morning of the 14th
August (the eleventh day of the experiment). The specimen was
notably sluggish during the later stage of the experiment and passed
most of the time in a corner of the tank, with the tail supported upon

152 GEORGE E. NICHOLLS

the sloping surface there. It was killed at 6.30 p.m., August 22.
Duration of experiment 18 days 23 hours.

The severed (hinder) portion of the terminal filament had not
markedly disintegrated but the short length of Reissner’s fiber sepa-
rated by the incision has altogether disappeared. There is visible some
disorganization of the terminal filament in front of the lesion, but a
little in front of the point where the cut was made the lumen of the
central canal seems to have been widened somewhat, perhaps, to form
a secondary sinus terminalis. Stretching backwards to this point,
there is seen a flimsy wrinkled and fibrillate structure which is, I be-
lieve, the expanded hinder end of Reissner’s fiber.

34. The incision, which was made at noon, August 14, was followed
within a quarter of an hour, by a distinct reaction (fig. 4). This con-
tinued and was well marked at 3 p.m. when the specimen was killed.
Duration of experiment 3 hours.

In front of the region where the experimental incision was made,
the fiber is found retracted and swollen with some spiral twisting.
Behind the point of injury the fiber is markedly swollen and appears
fibrillar.

33. The incision was made at 12.05 p.m., August 14, and by 1 p.m. a
slight reaction had appeared, but for the greater part of the day, the
animal rested with the tail turned over upon its side. The whole
body was strongly curved. During the following day this same atti-
tude was largely maintained but at times the tail was seen to be lifted
considerably. The specimen was killed at 8.45 p.m., August 15, the
duration of the experiment thus being 1 day 82 hours.

Reissner’s fiber is found swollen, retracted forward from the region
of the experimental incision and, at the free end, is slightly spirally
wound. ‘Traced forwardly this spiral becomes a very open one and the
fiber passes into a comparatively straight course. It probably repre-
sents a stage in unwinding.

43. The incision was made at 11.20 a.m., August 18, and was quickly
followed by the usual reaction, the head being well raised. By noon
the tail, also, was well lifted and the head still further raised. At
7.15 p.m. when the specimen was killed, the reaction appeared less
pronounced. Duration of experiment 8 hours.

In front of the lesion, Reissner’s fiber had disappeared entirely from
the length of tail examined. Behind the incision also, the fiber has
evidently contracted, the canal being devoid of fiber nor can the con-
tracted piece be certainly recognized.

44, The incision was made at 11.45 a.m., August 18, but no reaction
appeared either upon this or the following day. The specimen was
killed at 8 p.m., August 19. Duration of experiment 1 day 81 hours.

The fiber though severed has apparently been gripped by the ad-
pression of the walls of the terminal filament and there has been no
retraction of the fiber in either direction (fig. 30).

THE FUNCTION OF REISSNER’S FIBER 153

B. Raia blanda

3. This specimen was one which failed to recover from the anaes-
thetic. Some 2 to 3 hours after the operation it appeared to be dead,
the central nervous system, therefore, was partially exposed and
preserved.

The sections show that the incision severed the fiber but at the
same time apparently pinched together the walls of the terminal fila-
ment sufficiently to hold the cut ends. Behind the incision, therefore,
the fiber is found, stretching backward from the region of the lesion
to the sinus terminalis. It is somewhat swollen, the swelling becom-
ing more marked as the terminal sinus is neared and, actually within
the terminal chamber, it becomes greatly swollen and coiled. The
terminal plug is obscured by this retraction and cannot be certainly
identified (text-fig. 4).

In front of the lesion Reissner’s fiber is found, everywhere in the
length of the terminal filament examined, much swollen and most
remarkably coiled, all the later stages of spiral winding (short of the
production of actual tangles) being found in this short extent of cen-
tral canal (text-fig. 2). Regions in which the fiber is simply twisted
alternate with others in which the coiling is quite complicated and it
is probable that the original (uncontracted) length of the fiber included
in the piece examined was many times that of the length (about #
inch) of the containing central canal. The evidence suggests, there-
fore, that there must have been in progress, at the time the incision
was made, a very definite retraction of the fiber in a backward direc-
tion from a point well in advance of the experimental cut. This cut
clearly checked further retraction behind the lesion, but in front the
retraction probably continued until it was stopped by the hardening
action of the fixing fluid several hours subsequent to the operation.

4. The incision was made at 10 a.m., July 8, and was followed by a
quick recovery. Thereafter, the fish swam about with its tail turned
dorsally. Six hours later, when seen at rest, it was noted that its tail
was turned sharply to one side and that the extremity was raised at
least an inch. The tail was still lifted at 9.30 a.m. next day but later
this peculiarity was less pronounced. The specimen was killed at 3
p.m. Duration of experiment 1 day 5 hours.

The sections are poor and very obliquely cut. A considerable clot
occupies the central canal for some distance in either direction from
the experimental lesion. Behind the region of the incision I have
failed to recognize Reissner’s fiber but in front it can be made out
vaguely, apparently lying somewhat slackly against the epithelial
lining of the central canal and with some trace of irregular swelling
and coiling (fig. 16).

7. The incision was made at 10.30 a.m., July 9, and was quickly
followed by an elevation of the end of the tail, the whole tail being
turned slightly to one side (the left). By July 11 the fish appeared to
have become normal, excepting that it continued to exhibit a prefer-

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THE FUNCTION OF REISSNER’S FIBER ans

ence for the dark corner of its tank and adopted the somewhat un-
usual action in swimming noted in no. 5 (Raia clavata). During the

11 succeeding days, it was observed at frequent intervals but was
apparently normal throughout this period. On July 22 it was killed
Duration of experiment 13 days +.

This tail was examined by means of sections cut transversely which
proved to be quite unsuitable for the purpose of this investigation.
The fiber is found in the more anterior sections, where it appears not
markedly swollen and has apparently nearly made good any retraction
which may have taken place after the operation.

8. The incision was made at 10 a.m., July 13. By 10.30 a.m. the
specimen had completely recovered from the anaesthetic and had at-
tached itself to the (vertical) glass plate forming the front of the
tank, the tail being lifted slightly and turned to the left. This con-
dition persisted for an hour or so but by midday the ray appeared nor-
mal. It was killed at 11 a.m., July 14. Duration of the experiment
25 hours.

There was no considerable retraction of the severed ends of the
fiber, in either direction from the lesion, these being entangled appar-:
ently, in the clot which occupies the central canal for some distance.
From this clot the fiber may be traced tautly stretched and:of normal
diameter.

10. The incision was made at 11.30 a.m., July 13, and was followed
very quickly by a marked uplifting of the tail. An hour after the
operation the ray was found adhering to the wall of the tank with the
tail swung out dorsally and to the left. The reaction continued to be
marked during the three following days. By the morning of July 17,
however, the ray was seen with the tail carried normally and, there-
after, the specimen appeared normal until July 23 when it was killed.
Duration of experiment 10 days.

' For some distance in front of the experimental lesion, the central
canal is found empty of fiber. The free end of the fiber is found,
about half an inch in front of the lesion, swollen and thrown into a
loose tangle (fig. 26), from the anterior end of which the fiber emerges
much less swollen and fairly straight. No part of the fiber shows any
trace of spiral twisting.

11. The incision was made at 10a.m., July 20, with a fine knife from
the right side, care being taken not to penetrate completely through
the tail. The usual reaction did not appear, but there was some dis-
placement of the right pectoral fin which was brought up sharply
dorsally. Next morning the ray appeared entirely normal. Itwas
killed at 4 p.m., July 22. Duration of experiment 2 days 6 hours.

The sections show that the incision missed the filum terminale and
the fiber, therefore, remained unbroken.

19. The incision was made at 4.10 p.m., August 2. The specimen
was kept under observation until it was killed on August 9 but during
the whole of this time nothing unusual in its behavior was noted.
Duration of experiment 6 days 20 hours.

156 GEORGE E. NICHOLLS

The filum terminale behind the lesion appears empty of fiber but
an indistinct mass, which is apparently a tangled heap of fiber is seen
in the sinus terminalis. In front of the lesion the fiber is slightly with-
drawn, the end being swollen and somewhat spirally coiled.

29. The incision was made at 4.20 p.m., August 7, and the ray was
killed at 7.15 p.m. on August 10, no reaction having appeared in the
meanwhile. Duration of experiment 3 days 3 hours.

The sections establish that the incision failed to sever the filum
terminale and the fiber which is unbroken maintains its normal diameter
and is seen tautly stretched.

41. The incision, made at 5.55 p.m., August 17, was followed, very
quickly, by a reaction. The snout was lifted markedly and the whole
body was arched up. The ray was disturbed several times but in-
variably returned to rest in the same attitude. By 8.30 p.m. the
reaction had become less pronounced and by noon next day, when
the specimen was killed, it was much less marked. Duration of ex-
periment 18 hours.

In the terminal piece of the tail, Reissner’s fiber is found slack,
swollen and retracted for some distance from the region of the experi-
mental lesion. Another piece of the spinal cord, taken some consider-
able distance in advance, showed the fiber very slightly slack and
little swollen.

49. The incision was made at 11.15 a.m., August 21. A marked
reaction very quickly appeared, affecting the pose, both in swimming
and at rest. At noon, the snout and tail were down but the body
remained curiously humped up. The specimen maintained this atti-
rude until it was killed at 1245 pm. Duration of experiment 14
houcs.

A conspicuous clot has formed in the region of the lesion and extends
into the central canal both before and behind this point. Reissner’s
fiber is seen extending backwards from this spot as a swollen, loose
and slightly knotted thread. In front of the incision, the fiber emerges
from the clot (fig. 22) markedly swollen and coiled interruptedly, in
which condition it continues throughout the entire length of the piece
of spinal cord examined. The penultimate piece reveals the fiber still
more swollen and more markedly twisted. It is clear, therefore, that
although there has been no withdrawal, in either direction from the
region of the experimental incision, the fiber was, nevertheless, under-
going a marked contraction. The only possible explanation was that
the fiber had been broken farther forward and that a backward recoil
had been set up, that having begun probably at or about the time of
the operation. To test this point, another piece of spinal cord was
taken from a place well forward in the trunk. The sections showed
that, here, the central canal was perfectly devoid of fiber, a flimsy
hollow cylinder of coagulum (?) occupying the center of the canal
(fig. 18).

51. The incision which was made at 11.30 a.m., August 21, was not
apparently productive of any reaction. The specimen was killed at
5 p.m. on the same day. Duration of experiment 53 hours.

THE FUNCTION OF REISSNER’S FIBER Laz

The tail had clearly been truncated earlier in life but had com-
pletely healed and a secondary sinus terminalis had been formed.
The experimental cut failed to break the fiber which is seen of normal
size and tautly stretched.

52.°The incision was made at noon and was followed by a scarcely
perceptible reaction. The ray was killed at 3.30 p.m. Duration of
experiment 33 hours.

The fiber was severed by the incision but the free ends, which are
slightly knobbed and swollen are entangled in a clot and thus, presum-
ably, retraction has been prevented.

53. The incision was made at 12.05 p.m. and was quickly followed by
a fairly definite reaction which, however, was not evident at 2.30 p.m.
when the specimen was killed. Duration of experiment 25 hours.

The fiber is seen cut and slightly slackened but the free ends have
been withdrawn only for a short distance from the lesion.

62. The incision was made at 8.30 p.m., August 21, and was seen
to be followed by a marked swimming reaction but the specimen was
not seen in repose, after the operation. It was killed at 10.30 p.m.
Duration of experiment 2 hours.

The sections are poor but serve to show that the filum terminale
was cut. No fiber can be made out in such parts of the central canal
as I have been able to examine. It is probable that the fiber has re-
tracted forward, beyond the anterior limit of the piece of tissue
sectioned.

63. The incision was made at 11.10 a.m., August 22, and was fol-
lowed by a marked swimming reaction. At 11.40 a.m. it settled down
but the tail was not displaced. It was killed immediately. Duration
of experiment 30 minutes.

The fiber had been cut and had, apparently, retracted forwardly,
completely from the filum terminale in the piece of tail examined.

66. The incision was made at 11.35 a.m. and was followed by a
marked reaction. The ray was killed at 12.20 pm. Duration of
experiment 45 minutes.

The fiber was cut and had retracted some distance forward. In
the sections it may be seen lying slackly in an undulating course but
is not appreciably swollen.

C. Raia clavata

5. The incision was made at 10 a.m., July 8. By 5 p.m. the hinder
part of the tail was seen to be lifted and this reaction was manifested
throughout the evening and became still more marked next day. On
July 11, the specimen appeared very lethargic and, on every occasion
after being disturbed, returned to rest in the darkest part of its tank.
In swimming, the specimen would remain poised nearly vertically,
with a curious hovering movement, for 10 minutes or more at a time,
its tail being turned sharply dorsally. At rest, so far as could be seen,
the tail was disposed normally, but next day it was held uplifted for

158 GEORGE E. NICHOLLS

several hours. On July 13, the tail was seen turned to one side and
supported upon the side of the tank, the animal being very sluggish.
Next morning, appearing normal, it was killed. In this case, then,
the reaction appeared about 6 hours after the operation, was exhibited
continuously for 24 hours and intermittently during the three following
days. Duration of experiment 6 days.

In this case the tail piece was cut transversely, which sections it
was found are quite unsuitable. Behind the incision, the fiber seems
to have largely withdrawn into the sinus terminalis in which a coiled
mass can be recognized. In front of the lesion, the fiber, somewhat
swollen and lying unevenly (fig. 24), appears to extend backwards
almost to the point where it had been broken.

6. The incision made at 9 a.m., July 9, was followed very quickly
by an uplifting of the tail which amounted to as much as 23 inches or
even 3 inches from the tank floor (the specimen being only 9 inches
in length). The head was also raised, the tip of the snout being lifted
at least 1 inch, so that the long axis was very markedly curved. In
addition, there was a transverse flexure of the body, the lateral borders
of both pectoral fins being sharply upturned, also. By 2.30 p.m., the
trunk had become flattened and the ray had settled down normally
excepting that the tail was still raised and at a sharp angle to one
side. Next day the tail had resumed its normal (horizontal) position.
In its marked lethargy and in adopting an unusual attitude in swim-
ming (when disturbed) it resembled the preceding specimen (5). On
July 12, at 10 a.m., the tail was again seen to be well raised: disturbed,
the specimen would rise to the surface and float in a nearly vertical
position for-as many as 20 minutes at a time. On each occasion, it
returned to a vertical position of rest. On July 13, it was observed
at rest upon the floor of the tank with its tail displaced to one side
(the right) but horizontal. Next day it appeared normal and, dur-
ing the morning, was killed. In this instance, there was a well marked
reaction manifested almost immediately after the operation and con-
tinuing for 24 hours or so but thereafter only appearing intermit-
tently. Duration of experiment 5 days +.

The sections are quite unsatisfactory. Behind the lesion, the fiber
appears to have been caught by the pinching together of the walls of
the filum terminale and has not retracted. In front of the region of
the incision, however, the fiber has been withdrawn forward out of the
terminal piece sectioned. Sections were prepared of the penultimate
piece and these revealed a very delicate filament lying slackly near the
anterior end of this piece.

15. The incision was made on August 2, at noon, the needle being
thrust into the very extremity of the tail. There was no reaction and.
the ray was killed at 7.30 p.m. Duration of experiment 75 hours.

In this specimen the sinus terminalis does not extend downward
behind the notochord but lies wholly dorsal to that structure. The
cut, therefore, failed to penetrate the sinus terminalis and the fiber
was undamaged.

THE FUNCTION OF REISSNER’S FIBER 159

16. The incision was made at 12.15 p.m. When the ray was re-
turned to its tank, the tail was seen to float slightly off the floor but
with the visible return to consciousness, the ray took up the normal
position. The specimen appeared very inert and, upon examination
made next day, it was found that the vertebral column had been com-
pletely broken at a point some distance from the end of the tail. The
specimen was killed at 10.30 a.m., August 23, some 22 hours after the
operation.

Sections through the end of the tail showed that the hinder end of
the spinal cord had already largely degenerated, obviously as the re-
sult of the accident which had broken the tail.

17. The incision which was made at 2.30 p.m., August 2, was fol-
lowed very quickly by a curving up, lengthwise, of the body and
snout. This reaction persisted throughout the remainder of the day.
Next morning the specimen was found in the normal attitude. It was
killed at noon. Duration of experiment 215 hours.

The sections show that the fiber had withdrawn wholly from the
terminal piece. The penultimate piece of the tail was subsequently
sectioned but the fiber was absent from the length of spinal cord in-
cluded in these sections, also.

18. The incision was made at 2.40 p.m., August 2, but no reaction
was apparent. The specimen was kept under observation until noon,
August 9, when it was killed. Duration of experiment nearly 7 days.

The sections showed that the fiber was broken by the operation
and has, in the small severed portion of the terminal filament, entirely
disappeared, while this piece of the terminal filament itself appears
to have largely degenerated. In front of the lesion, the fiber stretches
backwards practically to the point where it had been cut. Near its
free end it is, however, slightly swollen and a little slack and its actual
extremity is distinctly fibrillated (fig. 28), the flaring of the extremity
suggesting that a terminal plug was in process of formation. There
was, when the specimen was killed, no new terminal sinus formed. A
little in front of the actual end the fiber is little swollen and runs
nearly truly in the center of the canal, surrounded by an extensive
blood clot which doubtless prevented the retraction of the fiber.

26. The incision was made at 4 p.m., August 7, but no reaction ap-
peared during this or the three following days. The specimen was
killed on August 10, at 6.30 p.m. Duration of experiment 3 days 25
hours.

Reissner’s fiber is seen in the sections as an extremely fine thread
stretching forward tautly from the point of experimental incision and
there has, apparently, been no retraction.

28. The incision was made at 4.20 p.m., August 7,and produced no
apparent reaction. The ray was killed on August 10, at 6.15 p.m.
Duration of the experiment 3 days 2 hours.

The fiber has evidently not retracted, in either direction from the
point of incision being held, apparently, by the adpressed walls of the
terminal filament.

160 GEORGE E. NICHOLLS

32. The incision was made at 7 p.m. on August 10. Upon recovery,
the specimen showed an unusual swimming reaction, then settled down
with the tail lifted dorsally. It was killed at 7.40 p.m. Duration of
experiment 40 minutes.

The sections are practically worthless, merely establishing the fact
that the incision had severed the terminal filament.

36. The incision was made at 6.15 p.m., August 15, and was followed
by a very slight uplifting of the snout. The left pectoral fin was
also raised. Presently the fish settled normally but later the right
pectoral was lifted. The specimen attempted to settle upon the tank
walls but failed to maintain this position. At 7 p.m. the ray appeared
in no way abnormal and at 7.45 p.m., it was killed. Duration of ex-
periment 23 hours.

The fiber was cut, but both free ends seem to be entangled in a
large clot and there is no evidence that any retraction took place.

37. The incision was made at 7 p.m. and was followed, almost at
once, by an elevation of the snout. Like the ray just described, it
appeared to prefer a vertical position but was unable to maintain
itself upon the tank walls for any length of time, invariably sliding
downwards until it was supported by the outwardly (dorsally) bent
tail (fig. 11). Any reaction affecting the tail, therefore, was masked,
if it occurred. The ray was killed at 6 p.m. on August 16. Duration
of experiment 1 day 23 hours.

In front of the place of the lesion a somewhat limited retraction
occurred, but the fiber appears to have become caught in an elongated
clot which extends for some distance*forward along the lumen of the
central canal. From the anterior end of this clot the fiber emerges as
a swollen and indistinctly spirally wound thread (fig. 23).

38. The incision was made at 11 a.m., August 16, but was not fol-
lowed by any visible reaction. The specimen was killed at 11.45
a.m. Duration of experiment 45 minutes.

The tail of this specimen had, at some time, suffered mutilation but
the wound had completely healed and a secondary sinus terminalis
had been produced. The experimental incision had severed the fiber
but the cut ends had not retracted, being held, apparently, by the
pinching together of the walls of the filum terminale.

39. The incision was made at 11.15 a.m., August 16 but produced
no evident reaction. The ray was killed at 5.30 p.m., next day.
Duration of experiment 1 day 6} hours.

There was no forward recoil of the fiber from the point where it
was cut experimentally. During the dissection made to expose the
spinal cord, however, an accidental cut was made far forward in the
spinal cord which evidently broke the fiber in that region and it appears
slack and swollen even as far back as this terminal piece.

40. The cut, made at 12.15 p.m., August 16, accidentally removed
the end of the tail (a piece about one-sixteenth of an inch in length).
The fish took up a vertical position, with the body supported by the
out-turned tail (cf. no. 30) which, as already pointed out, masks the

THE FUNCTION OF REISSNER’S FIBER 161

tail reaction, if that were produced. The ray was killed at 1 p.m.
next day. Duration of experiment 1 day ? hours.

The fiber had apparently retracted forward, completely beyond the
anterior limit of the terminal piece sectioned.

42. In this experiment, also, the cut (made at 6.05 p.m., August 17)
accidentally severed the tail, a piece scarcely one-sixteenth of an inch
in length being removed. The specimen assumed the vertical posi-
tion, both in swimming and at rest. Induced to settle upon the floor
of the tank, it remained in a nearly normal attitude, the snout only
being somewhat raised. It was killed at 4.30 p.m. on August 19.
Duration of experiment 1 day 225 hours.

The sections are poor and very oblique near the hinder end. Reiss-
ner’s fiber cannot certainly be made out near the point where it was
cut. Further forward it is seen here and there and then appears of
normal diameter, lies centrally and is apparently tautly stretched.
Probably no retraction took place, but the cut end was gripped by the
walls of the filum terminale.

45. The incision was made at noon, August 18. The ray rested in a
vertical position but the tail was not deflected from the line of the long
axis of the body. It maintained this attitude and was killed next day,
no reaction being noted. Duration of experiment 1 day 35 hours.

In this specimen, the fiber seems to have been prevented from re-
tracting forward by the grip of the adpressed walls of the filum ter-
minale, while behind the point of injury a clot has formed in the cen-
tral canal and apparently the free end of the severed portion was held
by this clot.

46. The incision was made at 12.10 p.m., August 18, and at 2.30
p.m. the end of the tail was seen to be slightly raised. This reaction
wore off during the afternoon and the ray seemed perfectly normal
next day. It was killed at 3 p.m. Duration of the experiment 1 day
2? hours.

In this ray, Reissner’s fiber is remarkably delicate. It les a little
slackly, apparently, but otherwise shows no sign of retraction. For
some distance it lies embedded in an elongated blood clot.

47. The incision was made at 3.30 p.m., August 19. On recovery
from the anaesthetic, the fish settled in a corner of the tank with its
tail raised several inches and resting against the wall of the tank.
Later it was gently moved away from the vicinity of the wall and it
then brought down the tail to the tank floor. It was killed at 4.45
p.m. Duration of experiment 1} hours.

The short, severed portion of Reissner’s fiber has not retracted
backwardly. In front of the injury, however, the fiber has with-
drawn forward completely out of the length of filum terminale exam-
ined. Sections of a piece of spinal cord, taken from a region well
forward in the trunk, reveal the fiber practically normal in size but
lying slightly slackly and undulating.

48. The incision was made at 4.45 p.m., August 19, and was fol-
lowed by a very slight uplifting of the end of the tail, the lateral bor-

162 GEORGE E. NICHOLLS

der of the pectoral fins being also slightly upturned. By 7 p.m., the
reaction had apparently passed; at 7.30 p.m. the ray was killed. Dura-
tion of the experiment 2? hours.

The sections are very thick and, excepting that they show that the
filum terminale (and, therefore, Reissner’s fiber) was severed, are
practically useless, affording no information as to the effect of the cut
upon the fiber.

54. The incision was made at 12.10 p.m., August 21. There fol-
lowed a marked reaction which was still pronounced at 12.55 when the
ray was killed. Duration of experiment 45 minutes.

The fiber had retracted wholly beyond the anterior limit of that
piece of the tail which was sectioned.

55. The incision was made at 2.40 p.m., August 21. The whole
body of the specimen became slightly lifted, being supported upon the
bases of the pectoral fins. The tail was held out stiffly, unsupported,
in a nearly normal position,its end, however, drooping slightly. This
attitude was maintained until 4.30 p.m. when the specimen was killed.
Duration of experiment 1 hour 50 minutes.

The fiber, in this example, is extremely slight in the tail region. It
was evidently broken by the experimental incision but there seems to
have resulted very slight displacement of the free end; traced forward
from the region of the incision the fiber is seen to lie somewhat slackly
against the wall of the central canal.

56. The incision, made at 2.45 p.m., August 21, was quickly followed
by a very marked reaction. The ray lifted itself well up from the floor
until it was supported only by the lateral border of the pectoral fins
(fig. 14). The tail was turned up sharply dorsally. The specimen was
photographed in this attitude at 3.15 p.m. and was killed at 3.30 p.m.
Duration of experiment 45 minutes. ;

Behind the injury the fiber is seen swollen and, in the terminal
sinus, it is spirally wound but complete retraction was apparently
prevented by the formation of a clot which has entangled the severed
end. In front of the lesion four pieces of vertebral column (including
more than 3 inches of the spinal cord, or half of its entire length) were
sectionized but the fiber had withdrawn forward beyond the most
anterior point examined.

57. The incision was made at 4 p.m. but the reaction was not no-
ticed until 7.15 p.m. when both snout and tail were well lifted. The
ray was killed at 7.25 p.m. Duration of experiment 35 hours.

In front of the point of injury, the fiber was absent in the length
of spinal cord sectioned.

58. The incision, which was also made at 4 p.m., was quickly followed
by a well marked reaction, the whole body being lifted upon the pec-
toral as well as both snout and tail upturned. The ray was killed at
4.50 p.m. Duration of experiment 50 minutes.

The fiber had retracted completely beyond the forward limit of the
piece of the filum terminale examined.

THE FUNCTION OF. REISSNER’S FIBER 163

59. The incision, made at 5.20 p.m., was followed by a reaction as
pronounced as that seen in the subject of experiment 56 (fig. 14).
The ray was still in this attitude at 7.15, when it was killed. Duration
of experiment, a little over 2 hours.

In this example, also, there had been considerable retraction, the
fiber not being found in the stretch of spinal cord examined.

60. The incision was made at 5.15 p.m. Within a quarter of an
hour there appeared a marked reaction which continued until 10.30
p.m. when the specimen was killed. Duration of experiment 3% hours.

Here again, the sections showed the central canal, in the length of
spinal cord sectioned, completely empty of Reissner’s fiber which must,
therefore, have retracted forward out of this region.

64. The incision was made at 11.05 a.m., August 22. There followed
a marked swimming reaction and, when the specimen settled down, the
tail was well raised. The ray was killed at 11.15 a.m. and the partly
dissected specimen was in fixing fluid within 14 minutes of the begin-
ning of the experiment. Duration of experiment 10 minutes.

Behind the region of the incision, Reissner’s fiber is found lying
slackly in the central canal. In front of the injury, there has been
some retraction, the fiber lying in loose wavy curves (fig. 27).

67. An incision was made at 10.45 a.m., August 24, and was very
quickly followed by a marked reaction which persisted until the speci-
men was killed at 12.20 p.m. Duration of experiment 1 hour 35
minutes.

The fiber had withdrawn completely beyond the anterior limit of
the piece of filum terminale examined.

68. The incision was made at 10.45 a.m., August 24. A marked
reaction quickly appeared, the whole body being arched up and sup-
ported only upon the lateral borders of the pectoral fins while the tail
was sharply uplifted. Two photographs (figs. 13, 9) were taken at
ape 11.30 a.m. and noon, respectively. Duration of experiment 13

ours.

In this specimen, also, the fiber has been withdrawn completely
beyond the anterior end of the piece of spinal cord sectioned.

70. The incision was made at 12.30 p.m. A distinct elevation of the
snout appeared when the specimen had recovered from the anaesthetic
but the tail was unaffected. Photographed at 12.45 p.m. (fig. 12),
the specimen was killed at 1.10 p.m. Duration of experiment 40
minutes.

The sections are poor, being thick and oblique. They establish
however, that the incision just failed to break the filum terminale
and, in the one or two sections, in which Reissner’s fiber can be made
out, near the place of the incision, it appears to lie centrally in a fairly
even course. But in a moderately thin section which shows the sinus
terminalis quite clearly there are signs of recent disorganization and
Reissner’s fiber is not present. Much of the lumen of this terminal
chamber is occupied by a clot which is certainly not due to an effusion
of blood resulting from the experimental cut.

164 GEORGE E. NICHOLLS

D. Raia microcellata

61. The incision was made at 7.15 p.m., August 21. At 10.30 p.m
the tail was distinctly raised and remained so until the specimen was
killed at 11 p.m. Duration of experiment 3% hours.

Sections prepared through the tail were useless. The brain was
sectioned, sagittally, and showed the fiber lying in normal position, of
usual size and apparently tautly stretched, so that if retraction of the
fiber took place in the tail region, it had not extended forward to the
head.

In all, serial sections were prepared of sixty-two specimens.’
Of these, the microscopical examinacion showed that in one case
(16) the hinder part of the spinal cord was in an advanced stage
of degeneration due to an accident which must have occurred
at some time prior to the experiment. The sections through
the region including the point of injury were, in five cases (30,
50, 61, 63, 69), absolutely worthless and two others (82, 48)
were somewhat fragmentary and of value only in establishing
that the experimental incision had severed the filum terminale
(and therefore Reissner’s fiber), while in another instance (70)
the sections are, for the most part, very thick and Reissner’s
fiber can be but doubtfully distinguished. In this case the
experimental incision did not penetrate the filum terminale.

Sufficiently satisfactory sections were obtained, therefore, in
fifty-three examples. Of these Reissner’s fiber shows a most
remarkable coiling in two cases (3, 49) which must be attributed
to the breaking of the fiber very shortly before the experiment.
In the former of these, moreover, the specimen never recovered
from the anaesthetic and afforded, therefore, no reaction. Two
experiments (9, 39) were vitiated by an accidental cutting of the
spinal cord very far forward, while the fixation was incomplete
and in both of these cases, also, an interesting spirally wound
condition of the fiber was produced. Apart from these four
experiments, in which there was definite evidence of an interfer-
ence with the condition of the fiber before or after the experi-

2 Four specimens (1, 25, 27, 31) which died during the progress of the experi-
ment had been so long dead, apparently, as to be worthless for the purpose of
this investigation. A fifth specimen (33) was unaccountably mislaid.

THE FUNCTION OF REISSNER’S FIBER 165

ment, there are three cases (19, 46, 55) concerning which I am
in some doubt as to the correct interpretation of the sections.

Excluding for the present these seven experiments which, for
one reason or another, are inconclusive, I have, I believe, very
definite evidence concerning the condition of Reissner’s fiber in
no fewer than forty-six specimens. The conclusions at which I
have arrived are based solely upon the reactions in these speci-
mens, about which there appears to be no question.

The subjects of these forty-six experiments may be classified,
according to the effect of the experiment upon Reissner’s fiber,
in four groups. *

1. Six specimens (nos. 11, 15, 21, 23, 29, 51) in which it was
found that the experimental incision missed the filum terminale
and thus failed to break the fiber.

2. Nine specimens (nos. 8, 18, 26, 28, 36, 38, 42, 44, 45) in
which the fiber, although broken by the incision, failed to re-
tract forward, or in either direction. The severed end (or ends)
were held, apparently, by the adpressed walls of the filum ter-
minale or, in some cases, secured from subsequent slipping by
the clotting of blood which had escaped into the central canal
from the cut meningeal vessels.

3. This, the largest group, includes thirty specimens in which
a more or less extensive retraction of the fiber had followed upon
the experimental incision. While in some individuals (37, 52,
53, 64, 66) this retraction was not very great, in others (10, 17.
20, 22, 34, 35, 40, 41, 48, 54, 56, 57, 58, 59, 60, 62, 65, 67, 68) it
was very considerable. In at least five (4, 5, 6, 7, 24) it may
have been very extensive, also; but, if so, it had been largely
repaired before the termination of the experiment.

4. A single specimen (2) in which the process of regeneration
was apparently almost completed.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

166 GEORGE E. NICHOLLS

VI. THE RELATION BETWEEN THE CONDITION OF REISSNER’S
FIBER AND THE REACTION OBSERVED

1. In the subjects of the experiments

1. Of the six specimens included in the first group two were
dogfish. The duration of the experiment varied from a little
less than 6 hours (51) to nearly 19 days (23). In not one of
these specimens, in which the experimental incision failed to
break the fiber was there any reaction.

2. In the second class come nine specimens in which, although
the experimental incision was successful in breaking the fiber,
this did not undergo retraction forward from the lesion. All
but one of these specimens were rays and the duration of the
experiment varied from three-quarters of an hour (38) to nearly
7 days (18). For the most part, however, the specimens were
killed in the first or second day.

Six specimens were not visibly affected by the operation,
while the remaining three exhibited a scarcely perceptible re-
action. This took the form either of a very slight uplifting of
the tail for a quite brief period (8) or of a trifling elevation of the
snout (36, 42).

It would appear, therefore, that a mere breaking of the fiber
which is, for any reason, not followed by retraction is unlikely
to evoke a reaction and, presumably, does not disorganize the
mechanism of which Reissner’s fiber forms part.

3. A comparison, however, of the records of the experiments
with the evidence afforded by the microscopical examination of
the preserved material in the case of the thirty individuals
composing the third group, suggests that there exists a distinct
connection between the reaction manifested and the retraction
of the fiber.

Thus in certain cases (e.g., 37, 52, 53, 64, 66) in which the
reaction had not been particularly pronounced or prolonged,
there was found to have occurred a comparatively slight retrac-
tion. On the other hand, in a number of experiments (10, 20,
22, 24, 35, 438, 54, 56, 57, 58, 59, 60, 67, 68) in which the reac-
tion had been particularly marked there was found to have

THE FUNCTION OF REISSNER’S FIBER Ta

occurred an extensive withdrawal of the fiber, forward, from the
region of the experimental lesion.

In a couple of instances (47, 64) this relation is less evident,
there having been a somewhat pronounced reaction although the
fiber had not been very greatly retracted. Both of these speci-
mens were the subjects of experiments of quite short duration
(11 hours and 10 minutes respectively) and it is probable that
the fiber would have continued to retract had the experiments
been allowed to proceed for a longer period.

Reissner’s fiber, in three specimens (5, 7, 24), all of which were
the subjects of experiments of prolonged duration, is found to
extend backwards nearly or quite to the region of the experi-
mental incision.

A secondary sinus terminalis is seen in the process of forma-
tion in the last of these and in this case Reissner’s fiber has
become of almost normal diameter but lies freely with a some-
what fibrillated ending in this new terminal enlargement of the
central canal.

The sections through the tails of two other specimens were cut
transversely and the condition of the end of the fiber can not be
certainly determined. In one (7) the fiber has a diameter but
slightly greater than the normal, while in the second (5) it is
quite distinctly swollen.

Another specimen (6) is apparently a normal case in which
there has been a considerable retraction. ‘The terminal piece
sectioned shows that the fiber had withdrawn wholly from that
region. The penultimate piece, however, contains the free end
of the fiber lying slackly and of quite notable slenderness. I
suspect that this may be an early phase of regeneration in which
a delicate new growth of fiber is stretching backward, the usual
simple straightening out of the original thread having, for some
reason, been prevented.

4. Regeneration is seen ina well advanced condition in but
a single specimen (2), a dogfish, of which the condition of the
end of Reissner’s fiber is seen in figure 31. In front of the in-
cision, a secondary sinus terminalis has arisen, the pia mater
having grown around the end of the filum terminale where it

168 GEORGE E. NICHOLLS

was severed to form the delicate hinder wall to this new terminal
chamber. The fiber seems to have flared out into a terminal plug
in which several strands, one somewhat thicker than the normal
fiber, can be distinguished. This les in contact with the menin-
geal wall of this secondary sinus terminalis and was either just
about to become attached to the meninges when the specimen
was killed or, more probably, had actually made its new terminal
attachment.

5. It will now be convenient to consider more fully the con-
dition of Reissher’s fiber in the subjects of eight experiments
(3, 9, 19, 39, 46, 49, 55 and 70) which I have refrained from in-
cluding in either of the four groups, although concerning most
of them I have but little doubt as to which category they really
belong.

Thus in the case of no. 39 which was an experiment of. quite
short duration, the sections show that, although broken by the
experimental incision, the fiber has not retracted forward from
the incision. Since the ray exhibited no reaction after the opera-
tion, it is clear that we have a specimen which should be placed
in the second of my four groups. During the dissection, however,
a slip of the knife inflicted a cut far forward in the spinal cord.
As the result of this post-mortem injury, a retraction of the fiber
took place from before backwards and a simple spiral twisting
has been produced which has affected the fiber back to the region
of the incision.

A similar accident occurred to no. 9 with a similar effect upon
the fiber. In this case, however, the experiment had been one
of considerable duration (11 days) and there had been manifested
a well marked reaction. It is extremely probable, therefore,
that in this specimen there had resulted the usual considerable
retraction of the fiber which had, however, become straightened
out before the specimen was killed. Reissner’s fiber is found in
the sections extending fully to the point where the filum ter-
minale had been severed and, in this resembling the condition
of the fiber in no. 39, it is found twisted into a nearly continu-
ous simple spiral (text-fig. 2). There can be little doubt that,
but for the accidental breaking of the fiber after the death of the

THE FUNCTION OF REISSNER’S FIBER 169

animal, the experiment would have been found to belong to the
third of my four classes.

The case of no. 19 is of a different kind. In this specimen
no reaction appeared as the result of the operation yet, in the
sections, the severed end of the fiber in front of the lesion was
found to be retracted for a short distance, swollen and, near its
free end, spirally coiled. The latter detail probably affords the
clue to what might, in view of the absence of any reaction, ap-
pear as a distinct anomaly. The experiment had continued for
6 days and, therefore, if there had taken place a retraction of the
fiber so extensive that the fiber had not straightened out in that
time, a well marked reaction should have been evident. Spiral
coiling, however, in every other instance known to me, is asso-
ciated, as I shall show, with recent retraction. In this in-
stance, then, there can be little doubt, I think, that the specimen
was one which would in the ordinary way have been included in
the second group—i.e., among those in which the fiber was sev-
ered but failed to retract—the severed end being gripped, prob-
ably, by the compression of the walls of the filum terminale.
During the handling which is unavoidable where a rapid dis-
section is desired, the fiber may have been released and then have
commenced to withdraw. <A disturbance which freed the fiber
from the grip of the walls of the filum terminale doubtless af-
forded, at the same time, ready ingress to the fixing fluid and
thus quickly checked the incipient retraction.

In the last experiment performed (70) there was again an ap-
parent discrepancy. This specimen on recovering from the
anaesthetic, assumed a quite unusual position (fig. 10) which
appeared to be an obvious reaction to the experiment. Subse-
quent examination of the material under the microscope re-
vealed, however, that the experimental incision had just failed
to cut the filum terminale. As it happens, the greater part of
the filum terminale in the piece of tissue sectioned is contained
in one thick section in which, although the presence of Reiss-
ner’s fiber can be ascertained, it is not possible to make out its
condition. Owing to a slight distortion of the material, however,
the sinus terminalis lies in an adjacent section which is moder-

170 GEORGE E. NICHOLLS

ately thin. From this terminal region the fiber is certainly
absent; the sinus terminalis itself shows signs of disturbance
and contains the remains of a clot which is certainly not the
result of the experimental incision, which did not destroy the
pla mater.

The specimen must have been one which had been brought in
recently, probably the previous day, for during my stay at Ply-
mouth I made use of all the moderately small specimens which
were available within a very short time of their capture. Since
in this specimen, the fiber is absent from the region of the sinus
terminalis and the latter chamber itself is somewhat disrupted
(for which injury my experiment was not responsible) it is
almost certain that the ray had been damaged in the trawl,
probably on the previous day.

That there was no reaction in evidence when the specimen was
selected for the experiment is doubtless to be explained by the
fact that the reaction frequently appears intermittently. More-
over, the experiment was the last undertaken and was performed
in some haste so that the usual precaution of keeping the speci-
men under observation for some hours prior to the operation
was not taken in this instance.

The reaction seen in this experiment, therefore, is almost
certainly to be attributed to an accidental breaking of the fiber
at some time prior to the experiment and is in no way due to the
experimental incision which did not disturb the central nervous
system.

Two other specimens (3, 49) reveal the fiber broken as the
result of some accident, but in these cases the snapping of the
fiber must have taken place in the trunk (or head) region and
must have occurred only a very short time indeed before the
operation.

In the case of the former (no. 3, the subject of the first ex-
periment performed upon a ray) an overdose of chloroform was
administered and for some 3 hours following the operation there
was no sign of returning animation. At the end of that time it
was decided to discontinue the experiment and the central ner-
vous system was exposed and hardened. As it happened, this
experiment, while affording no information upon the function

THE FUNCTION OF REISSNER’S FIBER iy gl

of the fiber, provided material which throws considerable light
upon the recoil of the fiber.

A general account has been given above of the condition of
Reissner’s fiber in the hinder part of the spinal cord of this speci-
men. From that description it will be apparent that, since both
before and behind the incision the fiber extends actually to the
severed ends of the filum terminale (text-fig. 4), there could
have been no retraction of the fiber as a sequel to the opera-
tion. Nevertheless, in the condition of the fiber, both in front
and behind the point where it was broken by the operation,
there is very distinct evidence of a recent retraction.

In the terminal (severed) portion of the terminal filament
(text-fig. 4a) the fiber is seen to be considerably swollen, the
swelling becoming more pronounced in the terminal sinus where
the fiber passes into a loose spiral; the terminal plug is not recog-
nizable, having collapsed, presumably, when the recoil began.

Immediately in front of the incision (text-fig. 4b) the fiber is
found in a wonderfully twisted state and continues markedly
coiled to the forward end of the piece of tissue examined. The
torsion is not uniform, short simply-twisted stretches interven-
ing between greatly convoluted lengths of fiber.. As already
pointed out, this short length of terminal filament contains very
many times its length of Reissner’s fiber. It is obvious, then,
that not only must this retraction have been due to a withdrawal
of the fiber from before backwards but, also, that it must have
been in progress prior to the operation, since otherwise it could
not have affected the severed piece of fiber in the region behind
the experimental lesion.

The incipient coiling of the fiber behind the incision is evidence
that retraction had been in progress but for a very short time
when the experimental incision separated this terminal portion
of the fiber and, as it happened, checked further recoil behind
this point. In front of the lesion, however, a gradual retrac-
tion continued during the 3 hours while the specimen lay mo-
tionless,? until a great length of Reissner’s fiber had accumu-
lated in the hinder part of the spinal cord.

3 Doubtless the retraction actually continued until finally BueBE Ce by. the
hardening action of the fixing fluid.

LG GEORGE E. NICHOLLS

That the fiber was broken just prior to the experiment, in
no. 49, also, appears extremely probable. In the trunk region
the central canal of the spinal cord is found empty of fiber,
while in the hinder part of the spinal cord and the filum terminale
the fiber is seen much coiled and swollen throughout the entire
length of two pieces of the tail region which were sectioned.
Here, too, as in no. 3, there was practically no retraction for-
ward from the lesion (fig. 22) so that the whole of the retraction
observed must have resulted from the backward withdrawal of
the fiber towards the tail. Behind the incision the fiber extends
to the severed end of the terminal filament but is loose and un-
dulating, shows some spiral winding and, near the terminal
sinus, is distinctly swollen. As in the previous case, therefore,
we have the evidence of a retraction which has started prior to
the operation and was the result of an accidental breakage of the
fiber far forward in the trunk region. In this case the speci-
men recovered from the anaesthetic and manifested a marked
reaction, not to be attributed to the experimental incision. The
experiment, however, was of much shorter duration than was the
case in no. 3 and the less intricately coiled condition of the fiber
in this specimen is clearly related to the shorter period during
which the fiber was free to withdraw. In nos. 9 and 39, in
which the accidental cutting of the fiber took place after death,
the fiber was free to retract only for the much shorter period
which was required for the penetration of the fixing fluids. In
both of these specimens the fiber has simply undergone a fairly
regular twisting but has not produced the more complicated
secondary spirals seen in no. 49 and, still better developed, in
no. 3.

In both of the two experiments which remain to be considered
(nos. 46 and 55) the fiber appears as an extraordinary delicate
filament lying somewhat slackly but not apparently withdrawn
from the injured place. In neither case was the experiment of
long duration and in both the reaction observed took on a some-
what unusual character, there being manifested a distinct de-
parture from the normal pose but no appreciable deviation of the
long axis from the regular straight line. While this reaction

THE FUNCTION OF REISSNER’S FIBER 16}

was not one which I should be inclined to describe as ‘marked’
it was, nevertheless, too considerable to be attributed to the
scarcely appreciable retraction which has occurred at the severed
end of the fiber.

If, then, I am correct in regarding the occurrence of this ex-
ceptionally delicate fiber as indicative of an early stage in a new
backward growth of the fiber after some unusually extensive re-
traction, the reaction noticed in these two experiments may
perhaps have been the consequence of a renewed disturbance of
the Reissner’s fiber mechanism in specimens in which the repair
of a previous disturbance had scarcely been completed.

The condition of Reissner’s fiber in the subjects of these eight
experiments may therefore be summed up as follows.

One (19) is to be regarded as exhibiting a slight retraction
of the fiber started at the moment of fixation of the material and
quickly checked; two others (9, 39) showed a considerable re-
traction resulting from an accidental cutting of the fiber during
the dissection made to expose the central nervous system. ‘The
remainder are regarded as showing stages in the retraction (or
repair) of the fiber consequent upon a breaking of the fiber prior
to the experiment. This snapping of the fiber may have oc-
curred immediately (8, 49) or some little time (70) or some con-
siderable time (46, 55) before the incision was made. ‘That such
a breakage of the fiber does occur not infrequently in life and
that it may produce a reaction comparable to that induced by
artificial section of the fiber will be seen from the account given
in the following section.

2. Non-experimental material

An attitude similar to that induced in many specimens by the
experimental incision, was occasionally noticed in specimens (not
the subjects of the experiments) confined in the aquarium of the
Plymouth Biological Station.

Of these, one—a dogfish (F)—was obtained during the sum-
mer of 1910. It had been seen in the aquarium at intervals
extending over several days with both head and tail well up.

174 GEORGE E. NICHOLLS

Finally, it was captured and a close scrutiny revealed a slight
external injury to the hinder margin of the caudal fin, which
had a frayed appearance and from which a narrow strip of
tissue (including the extremity of the filum terminale) had been
scraped away. In sections subsequently prepared, it was seen
that the sinus terminalis and the hinder end of the terminal
filament were wanting. Reissner’s fiber had evidently been
broken and had, doubtless, undergone a very considerable re-
traction, but at the time the material was preserved the fiber
had returned almost to normal size and stretched backwards to
the damaged end of the terminal filament. For the most part,
it lay in a fairly even course but near the actual end it was
slack and lay in gentle undulations. The central canal appar-
ently opened freely to the exterior and there were no signs of the
formation of a new secondary sinus terminalis.

During the following summer, several specimens (both rays
and dogfish) exhibiting the reaction (of an abnormal attitude in
repose) which is associated with the broken and retracted con-
dition of the fiber were taken from the tanks of the aquarium.

A piece of the tail (including the terminal portion of the
central nervous system) of several of these specimens and, in
some cases, a piece also of the spinal cord, or the brain, were
sectioned.

The first of these (Raia XIX) was brought in on August 2,
the tail showing numerous abrasions obviously received in the
trawl. The specimen was isolated and next morning was found
showing a well marked reaction. Later that same day (about
24 hours after its capture) it was killed and the nervous system
exposed and preserved in the usual manner.

In the sections, the terminal filament and sinus terminalis
are found apparently undamaged but Reissner’s fiber had cer-
tainly been broken, at or near the sinus terminalis. The free
(broken) hinder end of the fiber was found near the sinus ter-
minalis—almost brush-like (fig. 19). Traced forward from this
point, the fiber is seen slack, swollen and snarled (fig. 25).
It emerges from the anterior end of the tangle in a loosely
coiled spiral, the twisted portion passing into a straight stretch

THE FUNCTION OF REISSNER’S FIBER 175

which continues much swollen to the forward end of the piece
of tissue sectioned. ;

In a second specimen of the same species showing the charac-
teristic reaction (Raia clavata, XX XIX), the free end of the
broken fiber was found near the sinus terminalis swollen and
loosely spirally coiled (fig. 20). In this case I have no note as to
the date of the capture. Almost certainly, however, the specimen
was one of a batch of small rays (of which others were 38, 39,
40) which had been brought in on the previous day. As in
the previous specimen, the end of the central nervous system
appeared quite uninjured (apart from Reissner’s fiber) and in
this case there were no signs of external injury.

In two other rays showing the reaction (XXXV and LX XIII)
there was evidence that the fiber had been broken for, in both,
the fiber was found lying slackly in the region examined (the
filum terminale) and in the case of the former it was distinctly
swollen, but the free end was not found in my sections.

Another ray (X XXIII) and a dogfish (P), although mani-
festing the usual reaction, retained a normal condition of the
fiber in the tail region, of which alone sections were examined.
It is seen stretching apparently tensely and of normal size and
it is probable that the fiber must have been broken in a region
too remote to cause a disturbance of the fiber in the terminal
filament.

VII. DISCUSSION
1. The function and mode of action of the Reissner’s fiber apparatus

In an earlier paper (712, p. 429) I showed that the breaking of
the fiber in life generally resulted in the recoil of the severed
ends, and I concluded that the effect of such a breakage was to
bring about a temporary loss of control over the pose of the body
when at rest (and probably also whilst in motion).

Where the fiber is broken from natural or accidental causes it
appears that retraction of the broken end almost invariably
ensues, but in the subjects of the experiments this retraction
may be, for a while, delayed or even prevented altogether. It

176 GEORGE E. NICHOLLS

was pointed out that in the single example (8) in which the fiber,
although broken, had failed to retract there had been no
marked reaction.

These conclusions, based upon the examination of material
from a comparatively small number of experiments, are strongly
supported by the results of the much more numerous experiments
which were subsequently performed. The examination of the
condition of Reissner’s fiber in several specimens which were
not the subjects of experiment has provided further evidence in
corroboration of the correctness of those conclusions.

The results of this investigation may be said, therefore, to
afford very definite confirmation of Dendy’s suggestion (put
forward in 1909) that the fiber forms part of a mechanism
which is concerned in the automatic regulation of the flexure
of the body. ‘This hypothesis is quite in harmony, moreover,
with certain observations recorded by Sargent (’04), although
that author interpreted the facts in an altogether different sense
(vide infra).

I have been unable, however, to determine whether the re-
action (the assumption of an unnatural attitude at rest and an
abnormal action whilst in motion) is to be regarded as the con-
sequence of the diminution of tension at the sub-commissural
organ due to the slackening of the fiber or whether it is to be
attributed to the putting out of action of a larger or smaller
number of the scattered sensory cells EE in the epithelium
of the central canal.

It has been pointed out that, although there has been found,
in some cases, a quite considerable retraction of the fiber in the
hinder part of the spinal cord, accompanied by much swelling
and spiral winding, yet in the anterior region of the spinal cord
and in the brain itself the fiber may appear to be practically
normal. In such a case it seems improbable that any appreci-
able diminution in the tension of the fiber could have been felt
in the region of the sub-commissural organ. Further, in those
examples in which the slackness of the fiber has extended far
forward, even though it be not accompanied by swelling, it is
inconceivable that it could have taken place without rupturing a

THE FUNCTION OF REISSNER’S FIBER rw!

great number of those delicate component fibrillae (as I believe
them to be) which serve to support and stay the fiber along the
length of the spinal cord.

In the subject of two of the experiments (3, 49) the fiber had
broken very far forward; of these, one failed to recover conscious-
ness and gave no reaction, but it is extremely significant that in
the other (49) the reaction took on a somewhat peculiar form.
It is suggested, therefore, that in this case (where the breaking
of the fiber must certainly have reacted upon the subcommis-
sural organ), the more pronounced reaction was the sequel of
an unusually extensive disorganization of the apparatus.

In this connection, it is interesting to recall what has been
recorded by Sargent concerning his experiments. That author
laid much stress upon the fact that the subjects of his experi-
ments would blunder, headlong, into obstacles (stationary or
other). This behavior, as I have already pointed out (712, p.
420), is to be noted in the lesser dogfish both in normal (con-
trol) specimens as well as in the subjects of the experiments
when removed from the comparatively spacious tanks of the
aquarium to the smaller tanks in which, alone, one can be cer-
tain of keeping them under close observation. Moreover, this
blundering gait disappeared, after a few days confinement in the
more limited space, in the subjects of the experiments as well
as in the control specimens.

_ In the larger sharks of which Sargent made use, and which
were apparently freshly caught specimens, one cannot wonder
at such a result. Moreover, Sargent had no opportunity to ob-
serve the passing of this phase, for his specimens after a day or so
became quite lethargic and died upon the fourth or fifth day of
the experiment. Nor does the failure of the fish to avoid col-
lision with the walls of its cage bear out Sargent’s contention
that there was in these specimens, a delay in the transmission
of the optical stimulus, for such an object, always present,
would be visible for a sufficiently long period to allow any opti-
‘eal stimulus to pass by the ordinary conduction paths. Even
where an obstacle might be interposed with extreme sudden-
ness it would have been scarcely possible to observe any delay

178 GEORGE E. NICHOLLS

in the ‘avoidance reaction,’ for Sargent’s calculations apparently
suggest that, in an animal a metre in length, the passage of a
stimulus along the conduction path alleged to be provided by
Reissner’s fiber might effect a saving of one-fiftieth of a second,
at most.

I believe myself that there is nothing in this behavior but
what may reasonably be attributed to excitement due to the
handling inevitable to the change of accomodation, the strange-
ness of the new environment and the frantic attempt to escape
from the more cramped enclosure. It is interesting, therefore,
to find that in one specimen, described by Sargent as probably
abnormal, because it had been in confinement for a considerable
period (and was therefore accustomed to its enclosure) this
blundering into stationary and movable obstacles (the ‘slow
optical response’) was not observed.

As already remarked, this heedlessness in movement disap-
peared in my specimens (control and experimental) within a
few days when the fish had become accustomed, presumably,
to its new surroundings. In the case of the subjects of Sar-
gent’s experiments (in which the septic condition set up in the
brain, by the operation, brought about a marked lethargy and
speedy death) there was insufficient time for the specimens to
become habituated to an alteration in their environment.

While, therefore, certain of the phenomena observed by Sar-
gent are, in my opinion, to be attributed merely to a change in
the external condition of his specimens or to the ill-effects of the
operation upon the entire organism, others of the reactions, upon
which Sargent has laid little stress, may very well, I think, have
been a consequence of the breaking of the fiber. Sargent noted
that the fish adopted most abnormal attitudes in swimming,
actually turning even and swimming (not floating) ventral sur-
face uppermost. One specimen is described as swimming ‘with
its head curved dorsally’ a reaction clearly suggesting a loss of
control of the posture. In none of my experiments was there
manifested so marked a reaction, but in none of my experiments |
was the fiber cut so near to the subcommissural organ (in the
fourth ventricle).

THE FUNCTION OF REISSNER’S FIBER 179

The less noticeable reaction which I have recorded of so many
of my specimens (viz.: the departure of the long axis of the body
from the normal position in repose) was very probably exhibited
by Sargent’s specimens, also, when at rest. This, however,
would be little likely to attract the attention of an observer who
was viewing the specimens (whether confined in the cage or
free in the pool) from above as they must of necessity have
been viewed.

Such evidence, then, as is available suggests that the nearer
the break in the fiber is to the sub-commissural organ, the more
pronounced is the reaction. On the other hand, there is a
marked reaction in many cases in which there has been no appar-
ent interference with the taut condition of the fiber in the ante-
rior part of the spinal cord and brain and in which, therefore, it
might be supposed that the sub-commissural organ is little if at
all affected.

While, therefore, I am inclined to agree with Tretjakoff (13)
in assigning a considerable importance to the detached sensory
cells distributed along the entire length of the central canal (and
possibly also in the isthmic canal), I think that there can be
little doubt concerning the supreme importance of the sub-
commissural organ as the center of this sensory apparatus.

That Dendy’s suggestion of the manner in which the stimulus
may be supposed to be brought to bear upon its related sensory
cells by alterations in the tension of the fiber is much more prob-
able than Tretjakoff’s view that the stimulus is a result of pres-
sure of the fiber upon sensory cells admits in my mind, of little
doubt. As already pointed out, I believe that Tretjakoff’s
statements upon this point are based upon a study of material
in which a retraction of the fiber had taken place and in which,
therefore, the normal anatomical relations of the fiber are not
seen. I interpret the ‘knobbed ends’ of the sensory processes
as the remains of the broken fibrillae (which had connected
Reissner’s fiber with the sensory cells in the ependymal epi-
thelium of the canalis centralis) retracted to the parent cells.

180 GEORGE E. NICHOLLS

2. The spiral winding of the fiber and the occurrence of ‘snarls’

Examples of the peculiar spiral contraction of the fiber have
been seen in a number of the experiments. Thus numbers 3, 4,
9, 19, 34, 35, 37, 41, 49 and 56 all show the fiber twisted to a
greater or less extent. That the list is not more lengthy is to
be explained by the fact that in a number of cases I have not
cut sections of the spinal cord sufficiently far forward to find the
retracted end.

There is distinct evidence that, in the case of no. 3, this re-
traction (though not the result of the experimental incision)
must have taken place, for the most part, during the three hours
or so which elapsed between the operation and the fixation of the
material. The similar but less extensive coiling which occurred
in no. 49 (again not the result of the experimental incision)
must, likewise, have been produced almost wholly after the fiber
was severed by the operation, for the severed portion of the fiber
behind the incision is but little affected. This specimen was
allowed to live but an hour and a half after the beginning of the
experiment and there is doubtless a connection between the less
intricately coiled condition of the fiber in this specimen and the
shorter period which elapsed between the breaking of the fiber
and fixation.

The case of no. 9 differs from that of the two preceding speci-
mens in that the cut which started the backward recoil was
made after the death of the specimen just as the material was
about to be plunged into the fixing fluid. The simple and con-
tinuous spiral winding which is found in this specimen can have
been produced, therefore, only during the time which was neces-
sary for the fixing fluid to thoroughly penetrate and harden the
material.

In the case of experiments 34 and 56 (both of which were of
short duration) the fiber has retracted (spirally) away from the
lesion and the recoil, therefore, was definitely a consequence of
the experimental incision. The character of the spiral winding
in the fiber in the case of four other experiments (4, 35, 37, 41)
is somewhat different. It is found at the free end but does not

THE FUNCTION OF REISSNER’S FIBER 181

extend for a great distance along the fiber and has a much looser
twist which suggests the uncoiling of a spirally twisted thread.
All four of these experiments had a relatively considerable dura-
tion, the subject being killed towards the end of the first day or
during the second.

Apart from experiment 9 which was vitiated subsequently (by
an accidental cut during dissection), the only case in which even
a slight spiral twisting was observed in an experiment of long
duration is no. 19. The subject was a ray, which, during the
whole time (nearly 7 days) which elapsed between the breaking
of the fiber and the killing of the specimen, gave absolutely no
reaction. I have already suggested that it is probable we are
dealing, in this case, with a slipping and coiling of the fiber which
was started during the dissection by a reopening of the wound
made by the operation but was quickly checked by the rapid
penetration of the fixing fluid.

With but this single possible exception, therefore, every case
of spiral winding of the fiber has been found in the early stages
of the experiment or immediately following an accidental cut;
by the second day this torsion has usually disappeared and,
where it is found at so late a period, is almost certainly in
process of uncoiling.

In confirmation of the view that the spiral coiling is found as
the result of a comparatively recent snapping of the fiber, it
may be noted that, while it has been found frequently in material
in which the spinal cord has been severed during or immediately
prior to fixation, it is more rarely found in specimens of which
the nervous system had been preserved entire.

In many larval lampreys and in adult myxinoids, all of which
_ were preserved entire, I found, it is true, an intricately coiled
mass of fiber which, in some cases, almost fills the sinus termi-
nalis? (12a, figs. 15, 17, 18). In none of these specimens had
there been any attempt to expose the central nervous system

‘That this terminal coiled mass, though so frequently found, is not, as Stud-
ni¢ka (’99) supposed, the normal condition is proved by the fact that Sanders
has seen a taut condition of the fiber in the sinus terminalis (’94, p. 11) com-

parable to that which I have described in the lamprey and other forms (12,
a Draye

5

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

182 GEORGE E. NICHOLLS

and fixation, therefore, had necessarily been slow. I have sug-
gested (12 a, p. 27) that unusually strenuous exertions made by
the animals in their unavailing efforts to avoid capture may be
the cause of the somewhat frequent instances of broken fiber
noted in these cyclostomes.®> Careless handling of the specimens
might be equally responsible for the snapping of the fiber. It
is possible, therefore, that the apparent absence of the fiber
from the spinal cord of specimens which have been some time
dead before fixation may not be due (as I have supposed) simply
to the degeneration of the fiber having already occurred, but
may be owing rather to its having been broken and retracted
entirely beyond the limits of the piece or pieces of tissue
examined.

In two rays which, although not the subjects of experiments,
manifested a well marked reaction a spirally coiled condition was
discovered in the broken fiber. One (Raia XIX) is known cer-
tainly to have been taken some 24 hours before the material was
preserved and it is probable that a similar interval had elapsed
between the capture and preservation of the material in the
case of the second (XXXIX) also. The former bore signs of
recent damage in the tail region evidently caused by the trawl
but the other appeared externally to be undamaged. If, there-
fore, the fiber had been broken at the time of capture, in conse-
quence of the violence of the animal’s struggles to escape, the
fiber might be expected to have become straightened out or to
be in the process of unwinding. In one (XX XIX) the free end
of the fiber is loosely curled (fig. 20) and in the other (XIX) a
tangle remains as evidence of a sharp recoil, but the spiral wind-
ing is found only in the vicinity of the tangle (fig. 25) and has,
elsewhere, disappeared.

That the fiber was liable, in its recoil, to form intricately
tangled knots or ‘snarls’ was first noticed by Sargent (’04, fig.
8). His figure does not suggest the spiral winding which I be-
lieve to be associated with the recent contraction of the fiber
and which is seen in the photomicrograph which I published
in 1912 (’12, fig. 3).

5 The breaking of the fiber, prior to the experiment, in nos. 3 and 49 is prob-
ably to be attributed to this cause.

THE FUNCTION OF REISSNER’S FIBER 183

At the commencement of this investigation I inclined to the
idea that such a knot would be invariably produced when the
broken fiber retracted, and supposed that the spiral winding,
extending more or Jess uniformly along a great length, would be
found only in those cases where a gradual process of fixation
prevented the more sudden recoil. The results obtained from
a large number of experiments indicate, however, that this sud-
den contraction may be of much less general occurrence than was
supposed and that the withdrawal of the fiber is brought about
usually, by the simple spiral twisting of the fiber.

The tightly knotted tangle of fiber present in Raia XIX, just
above mentioned, is the only example of this condition which I
have encountered in the course of this investigation. There is
reason to believe that in this case the fiber may have broken
some 24 hours before the material was preserved. In another
specimen (10) in which the fiber was broken by the experi-
mental incision made some 10 days before the fixation of the
material, there is found a loosely twisted skein of fiber, a small
part of which is represented in figure 26. That this condition
had been preceded by the tightly knotted condition is very prob-
able, this knot having doubtless served to hold the broken end
and thus prevent more extensive retraction, for the tangle is
found at no great distance (about half an inch) from the point
of injury. It must be supposed, therefore, that during the 10
days of the experiment the spiral torsion had disappeared and
the process of disentangling the snarl had been proceeding.
The fiber, except at its immediate hinder end, had become
nearly normal in size, but whether the tangle would have been
smoothed out eventually, if the experiment had been prolonged,
or whether a new delicate growth from the hinder free end would
~ have followed, I have no evidence to decide.

8. The duration of the reaction and the problem of regeneration

It is not quite obvious why the reaction appears to be so
variable in its duration. If the assumption of an abnormal atti-
tude in repose is, as I believe, a consequence of the disorganiza-
tion of the mechanism of which Reissner’s fiber forms part, we

184 GEORGE E. NICHOLLS

should expect that the reaction would continue until the fiber had
reéstablished its attachment to the walls of the sinus terminalis
and had once more attained to its normal tension.

The reaction did persist, indeed, in some specimens until the
attachment was practically made good (2) or until the termi-
nation of the experiment (9). Occasionally the reaction was
manifested intermittently for several days (5, 6) while in one
case (24) it reappeared after several days of apparent normality.
On the other hand, a reaction was sometimes marked during the
early hours of an experiment but was not noticed subsequently
(7) although the sections showed that the fiber had been broken
but gave no indication that the new terminal attachment was
completed.

While, then, it is quite possible that in some specimens (in
which the reaction had seemingly disappeared and which were
killed very soon after the operation) the reaction might have
reappeared at intervals had the experiment been prolonged, it
is probable that in many the reaction would have apparently
completely vanished (as in 7).

Nevertheless, it seems unlikely that the effect produced by
the breaking and retraction of the fiber can really altogether
disappear until the tension of the fiber has been restored.

The more obvious irregularities of the pose are possibly soon
corrected, to a large extent, by the aid of the other senses, no-
tably that of touch, and these corrections would be likely to
become more exact as time passed, thus accounting, In some
measure, for the gradual diminution in the magnitude of the
reaction. It is, however, extremely probable that there may
have been other reactions which persisted long after the speci-
mens seemed to me to be normal. Especially may this have
been the case with minute irregularities in action, in swimming,
for whilst in motion the correcting influence of the sense of
touch, at least, would almost certainly be eliminated. The
motion of the animal is particularly difficult to observe closely
and defied my attempts at analysis so that, although at times
I felt convinced that the action was not exactly that which is

THE FUNCTION OF REISSNER’S FIBER 185

usual, yet it was extremely difficult to decide wherein the
difference lay.

There is yet another possible explanation. It has been seen
that, where retraction did not follow the breakage of the fiber,
there was no obvious reaction. It has been assumed that this
was due to the maintenance of the tension of the fiber by the
firm grip of the adpressed walls of the filum terminale upon the
severed end of the fiber. Not only, however, might the tension
be maintained but the connections with the numerous sensory
cells in the central canal, whose filamentous processes con-
tribute to the substance of the fiber, are preserved intact. The
absence of the reaction may be partly or wholly attributable
to this latter fact for, although the terminal plug at the hinder
end of the fiber is clearly to be regarded as the principal insertion
of the fiber, yet there can be little doubt that the attachmenis
of the fiber by the component fibrillae throughout the length
of the spinal cord must afford a very considerable support.
Such evidence as these experiments have afforded suggests
that the greater the extent of the retraction forward the
greater is the degree of the reaction. It may well be, then, that
as soon as the forward retraction is checked and the repairing
process has brought about the unwinding and siraightening
out of the fiber, the component fibrillae may forthwith begin
to renew their attachment to the fiber. In this way while
they may assist in restoring the tense condition of the whole
fiber a constantly increasing number of sensory cells may be
coming into action again, the diminution of the visible reaction
being attributable to the restoration of these connections.

Sargent has stated (04, p. 230) that “sharks have shown
almost no capacity to heal wounds or regenerate skin.”” During
the progress of this investigation several rays were taken in
which there was evidence of the loss of part. of the tail but the
stump had healed perfectly. While it may be that, as regards
this power of regeneration, rays differ from sharks and dogfish,
it must be remembered that my rays were, in general, quite
small specimens and it is exceedingly likely that the injury had
been inflicted when the specimens were very small, indeed.

186 GEORGE E. NICHOLLS

In young animals the recuperative powers are frequen ‘ly much
greater than in aged specimens and it may prove that the
restoration of the normal (functional) condition of Reissner’s
fiber after injury may be effected much more quickly in some
specimens than in others.

In this connection the condition of Reissner’s fiber in the
dogfish (F) is of interest. This specimen, it will be remembered,
was one which was seen in the large aquarium tank exhibiting,
very markedly, the reaction which is associated with the broken
and retracted fiber. The fish had certainly been in confine-
ment for some time and there was reason for connecting the
injury to the hinder border of the caudal fin with damage in-
flicted by the trawl. The injury was, therefore, probably of
long standing and the reaction had almost certainly persisted
for a considerable time. In the sections the fiber was found to
be somewhat slack, lying near its free end in loose undulations
and there were no indications that regenerative processes were
at work. The specimen was unusually large and presumably
an old individual. There seems, therefore, to be in this case
a connection between the size (age) of the specimen, the lack of
regenerative powers and the continuance of the reaction.

As already noted, I have but one example of undoubted re-
pair of the mechanism (fig. 31) after the experiment. This is
seen in a dogfish (2) which was killed sixteen days after the
operation.

In several cases, however, the fiber had clearly undergone a
considerable retraction but had straightened out again before
the specimens were killed. In the sections, therefore, it is seen
to extend almost or quite to the spot where it had been broken
by the experimental incision. Examples of this phase of repair
are to be seen in nos. 5, 7, 8 and 24. The disentanglement
of the snarl (10) and the unwinding of the spirally twisted
fiber (37, 41) must be regarded as preliminary stages in the proc-
ess of repair. In no. 18, where there had been no retraction,
it would seem as though the end of the fiber was flaring out in
preparation for a new terminal plug (fig. 28).

THE FUNCTION OF. REISSNER’S FIBER 187

Whether the delicate fiber found extending backward slackly
in some specimens (46, 55) almost to the region of the incision,
is to be regarded as yet another early stage in regeneration is less
certain. Possibly it is a backward growth from the free end of
the fiber in a snarl which has altogether failed to become dis-
entangled. On the other hand the fineness of the fiber may be
nothing but an individual peculiarity.

It is noteworthy that, where this abnormally fine fiber was
found, it had retracted but little when cut and the oe
reaction had proved to be but slight.

In view of the fact that a case of complete repair had been
obtained in but a single experiment (2), it has not been possible
to determine the period within which regeneration might nor-
mally be expected to take place. If the incision has completely
divided the filum terminale (in practice an inevitable conse-
quence of any attempt to cut the fiber) even though this be very
far posteriorly, it is almost certain that regeneration cannot be
effected until a new (secondary) sinus terminalis has been formed.
It is unlikely that this comes into existence earlier than the end
of the second week, although the exact time would depend upon
the regenerative powers of the tissue of the individual and would
be likely to take place more quickly in young and rapidly grow-
ing specimens. The new attachment of the fiber may, however,
be even then delayed if the retraction has been very consider-
able, has resulted in a tangled knot, or if the fiber has broken
very far forward.

In several of my experiments the fiber had returned nearly
to its normal diameter and had pushed backwards to the region
of the incision within a week of the operation, while the spiral
twist appears to be straightened out during the second day under
ordinary circumstances. A complicated tangle evidently re-
quires a considerable period in which to become resolved and
in such a knot it is probable that the spiral twisting may per-
sist rather longer. In my experiments, however, only a short
length of the fiber was actually separated and consequently no
great length of new growth, if any, was required to enable the
fiber to extend to the newly formed sinus terminalis. Where,

188 GEORGE E. NICHOLLS

however, the fiber may have been broken by an accident at a
point very far forward it may be supposed that a relatively
‘long period may elapse before the new growth has pushed back
to the terminal sinus.

But regeneration might, under such circumstances, take place
much more quickly when (unlike the condition after the experi-
ment) the walls of the central nervous system had remained
intact and there was no need for the production of a new terminal
sinus.

Specimens 38 and 41 are instances in which regeneration must
certainly have occurred, both of these rays having suffered the
loss of the hinder part of the tail (and therefore of the sinus
terminalis) earlier in life. |

VIII. SUMMARY

1. Reissner’s fiber, if severed, will generally be withdrawn
in both directions from the lesion, the retraction being apparently
effected by a spiral winding of the fiber which attains a greatly
increased thickness as the withdrawal proceeds.

2. In dead or dying material, this retraction may continue,
if not checked by prompt fixation, until the whole of the fiber
has withdrawn to its points of attachments; in living specimens
there may be produced at the broken ends a tangle or snarl
which doubtless serves to prevent such extensive retraction.

3. In individual rays or dogfish in which such retraction of
the fiber has taken place there is manifested a distinctive re-
action; the specimen assumes an abnormal posture while at rest
and probably, also, exhibits an unusual action while in motion.

4. This reaction becomes apparent very shortly after the
return to consciousness (of the animal anaesthetized for the
operation), may be intermittent, and is manifested by different
specimens for widely different periods. Probably there is a
connection between the degree of the reaction and the extent
of the retraction of Reissner’s fiber.

5. This reaction is not observed in those individuals in which
the fiber has been broken but has, for any reason, failed to
retract.

THE FUNCTION OF REISSNER’S FIBER 189

6. The time required for regeneration is probably not less
than a week, even when the retraction has not been extensive
and the filum terminale and sinus terminalis are undamaged;
in the case of the experimental material probably several weeks
are required. Regeneration commences with the uncoiling
of the fiber, which may be complete in a couple of days. The
fiber extends backwards more or less slackly, becomes less swollen
and probably by further growth, comes once more into contact
with the hinder wall of the sinus terminalis (original or second-
ary) into which 1t becomes inserted.

7. It would appear, therefore, that, as suggested by Dendy,
the fiber serves to control automatically the flexure and pose of
the body. While it is probable that the related sensory cells
are largely concentrated in the sub-commissural organ, it is
equally probable that many other such sensory cells are scattered
in the ependymal epithelium of the canalis centralis throughout
the length of the spinal cord.

190 GEORGE E. NICHOLLS

IX. LITERATURE CITED

ALLEN, W. F. 1916 Studies on the spinal cord and medulla of cyclostomes
with special reference to the formation and expansion of the roof
plate and the flattening of the spinal cord. Jour. Comp. Neur.,

vol. 26.
Ayers, H. 1908 The ventricular fibers in the brain of myxinoids. Anat.
Anz., Bd. 32.

Bearp, J. 1896 The history of a transient nervous apparatus in certain Ich-
thyopsida. Zool. Jahrb., Bd. 9.

Denpy, A. 1902 On a pair of ciliated grooves in the brain of the ammocoete,
apparently serving to promote the circulation of the fluid in the brain
cavity. Proc. Roy. Soc., vol. 69. ‘

1909 The function of Reissner’s fiber and the ependymal groove.
Nature, vol. 82.

1912 Reissner’s fiber and the sub-commissural organ in the verte-
brate brain. Brit. Assoc. Adv. Sce., 1912.

Denby AND Nicuouis 1910 On the occurrence of a mesocoelic recess in the
human brain and its relation to the sub-commissural organ of lower
vertebrates, with special reference to the distribution of Reissner’s
fiber in the vertebrate series and its possible function. Proc. Roy.
Soe., Lond., vol. 82.

Epincer, L. 1892 Untersuchungen iiber die vergleichende Anatomie des
Gehirnes. Abhandl. Senck. Naturf. Gesells. Frankt., Bd. 18.

1908 Vorlesungen tiber den Bau der nervésen Centralorgane des
Menschen und der Tiere. Vergl. Anat. des. Gehirnes, Bd. 2, 7 Aufl.,
Leipzig, 1908.

Gapvow, H. 1891 Végel., Bronn’s Klassen und Ordnungen des Thierreichs, Bd.
6, Abth. 4.

Horstey, V. 1908 Note on the existence of Reissner’s fiber in higher verte-
brates. Brain, vol. 31. :

Houser, G. L. 1901 The neurons and supporting elements of the brain of a
selachian. Jour. Comp. Neur., vol. 11.

Karperuan, F. 1900 Uber das Riickenmark der Plagiostomen. Ein Beitrag
zur vergleichenden Anatomie des Centralnervensystems. Zeitschr.
f. ges. Naturwiss., Bd. 73.

Kouurker, A. 1902 Uber die oberfliichlichen Nervenkerne im Marke der
Vogel und Reptilien. Zeitschr. f. wiss. Zool., Bd. 72.

Koutmer, W. 1905 Zur Kenntniss des Riickenmarkes von Ammocoetes. Anat.
Hit;, ba..29) Abth. 1.

Kurtscain, O. 1863 Uber den Bau des Riickenmarks von Neunauges. Kasan,
1863. Abstract by Stieda, Arch. mikr. Anat., Bd. 2, 1866.

Nicuouts, G. E. 1909 The function of Reissner’s fiber and the ependymal
groove. Nature, vol. 82.

1910 See Dmenpy AND NICHOLLS.

1912 An experimental investigation on the function of Reissner’s
fiber. Anat. Anz., Bd. 40.

1912 a The structure and development of Reissner’s fiber and the
sub-commissural organ. Quart. Journ. Micr. Sc., vol. 58.

THE FUNCTION OF REISSNER’S FIBER 191

Nicuoutts, G. E. 1913 An experimental investigation on the function of
Reissner’s fiber. Jour. Marine Biol. Assoc., vol. 9.

Retssner, E. 1860 Beitrage zur Kenntniss vom Bau des Riickenmarks von
Petromyzon fluviatilis. Arch. f. Anat. u. Physiol., Jahrg. 1860.

Rowon, J. V. 1877 Das Centralorgan des Nervensystems der Selachier.
Denks. Akad. Wiss. Wien. Math. naturw. K1., Bd. 38.

SanpeErs, A. 1878 Contributions to the anatomy of the central nervous sys-
tem in vertebrate animals. Ichthyopsida. Pisces. Teleostei. Phil.
Trans. Roy. Soc., Lond., vol. 169.

1894. Researches in the nervous system of Myxine glutinosa.
London.

Sarcent, P. E. 1900 Reissner’s fiber in the canalis centralis of vertebrates.
Anat. Anz., Bd. 17.

1901 The development and function of Reissner’s fiber and its cellu-
lar connections: a preliminary paper. Proc. Amer. Acad. Artsand Sc.,
vol. 36.

1903 The ependymal grooves in the roof of the diencephalon of verte-
brates. Science, n.s., vol. 17.

1904 The optic reflex apparatus for short-circuit transmission of
motor reflexes through Reissner’s fiber; its morphology, ontogeny,
phylogeny and function. Part I, The fish-like vertebrates. Bull.
Mus. Comp. Zool. Harvard.

Srizpa, L. 1868 Studien tiber das centrale Nervensystem der Wirbelthiere.
Zeits. f. wiss. Zool., Bd. 19.

1873 Uber den Bau des Riickenmarkes der Rochen und der Haie.
Zeits. f. wiss. Zool., Bd. 23.

Streeter, G. L. 1902 The structure of the spinal cord of the ostrich. Am.
Jour. Anat., vol. 3.

SrupniéKa, F. K. 1899 Der Reissner’schen Faden aus dem Centralkanal des
Rickenmarks und sein Verhalten in dem Ventriculus (Sinus) ter-
minalis. Sitzb. k. bohm. Ges. Wiss. math. nat. Kl., Prag.

1900 Untersuchungen itiber den Bau des Dadi der nervosen
Centralorgane. Anat. Hft., Abth. I, Bd. 15.
1913 Das Extracellulare Protoplasma. Anat. Anz., Bd. 44.

Tretsaxorr, D. 1909 Das Nervensystem von Ammocoetes. I. Das Riicken-

' mark. Arch. f. mikr. Anat., Bd. 73. ;
1913 Die zentralen Sinnesorgane bei Pétromyzon. Arch. f. mikr.
Anat., Bd. 83.

ViauLT, F. 1876 Recherches histologiques sur la Structure des Centres ner-

veux des Plagiostomes. Arch. Zool. expér. gen., Tom. 5.

PLATE 1

EXPLANATION OF FIGURES

1 Seyllium ecanicula. A photograph of a normal specimen upon which no
experiment had been performed. The lower border of the caudal fin is seen
lightly resting upon the floor of the tank.

2 <A photograph of the subject of experiment 22, taken nearly five hours after
the operation, showing a moderate reaction. *, indicates the region of the
incision.

3 A photograph of the same dogfish, taken half an hour later, the tail being
rather more lifted.

4 A photograph of the subject of experiment 34, taken about two hours
after the operation, showing a well marked reaction.

5 <A photograph of the subject of experiment 24, taken upon the third day
of the experiment, and showing a well marked reaction of the tail.

6 A photograph of the subject of experiment 23, the specimen being seen re-
posing in the attitude adopted by normal dogfish. The photograph was taken
upon the fourth day of the experiment, no reaction having appeared.

192

THE FUNCTION OF REISSNER’S FIBER PLATE 1
GEORGE E. NICHOLLS

PLATE 2

EXPLANATION OF FIGURES

7 Photograph of a normal ray (not the subject of an experiment), showing
in side view, the attitude of repose which is normal in these animals.

8 A photograph of the subject of experiment 31, showing a reaction affect-
ing the tail only. *, indicates the region of the incision.

9 A photograph of the subject of experiment 68, taken about two hours after
the operation. (Cf. figure 13, a photograph of the same specimen taken half
an hour earlier. )

10 A photograph of a normal ray (not the subject of an experiment), show-
ing the natural position of the snout in the ray when at rest.

11 A photograph, taken several hours after the operation, of the subject of
experiment 30. This ray was one which, while it occasionally showed the typical
reaction, rested for the most part in the attitude shown.

12 A photograph, taken a quarter of an hour after the operation, of the sub-
ject of experiment 70. The head is seen well raised but the tail appears unaf-
fected.

13 A photograph of the subject of experiment 68, taken an hour and a half
after the operation. The tail is seen, somewhat indistinctly, well raised and
turned to the left.

14. A photograph of the subject of experiment 56, taken half an hour after
the operation, showing an extreme reaction, the body being raised completely
from the floor and supported only upon the lateral border of the pectoral fins.

15 A photograph of a ray (XXXIII), not the subject of an experiment, but
in the attitude which results from the breaking and retraction of Reissner’s
fiber. This photograph was taken after the ray had been kept for four days
under observation.

194

THE FUNCTION OF REISSNER’S FIBER PLATE 2
GEORGE E. NICHOLLS

PLATE 3

EXPLANATION OF FIGURES

16 Raia blanda (4). Part of a sagittal section through the filum terminale
showing the free end of Reissner’s fiber in front of the lesion (the posterior end
to the left, in the figure). > 240.

17 Part of a transverse section through the spinal cord of the cat, showing
a much swollen Reissner’s fiber, nearly 10 in diameter, embedded in a mass
of coagulum which almost blocks the central canal. In the center of the fiber
ean be seen the cut end of what appears as an axial thread or core. X 240.

18 Raia blanda (49). Part of a sagittal section through a piece of the spinal
eord from the anterior part of the trunk, from which region Reissner’s fiber had
wholly retracted (caudally). The middle of the canal is occupied by a filmy
strueture (x) which is probably coagulum. XX 240.

19 Raia clavata (XIX, not experimental). Part of a sagittal section through
the end of the tail showing the broken end of Reissner’s fiber in the (secondary)
sinus terminalis. The terminal neural pore is almost choked by a mass of débris
and coagulum. X 240.

20 Raia clavata (XXXIX, not experimental). The broken end of Reiss-
ner’s fiber is seen loosely coiled near the end of the central canal, the anterior
piece of fiber depicted having been added from an adjacent section. The (pri-
mary) sinus terminalis is fairly typical and extends downwards, in normal
fashion, behind the end of the notochord. 240.

21 Raia blanda (11). A length of Reissner’s fiber from the fourth ven-
tricle, to show the brittle condition of the preserved fiber, which has splintered
upon the microtome knife. X 240.

22 Raia blanda (49). Part of a sagittal section through the filum terminale,
immediately in front of the lesion. Reissner’s fiber is seen entangled in a clot
from whick there has been no apparent retraction forward. Nevertheless, the
whole length of fiber in the piece examined is spirally coiled, this being the re-
sult of a backward recoil from some point in the spinal cord. X 45.

23 Raia clavata (37). Part of a sagittal section through the filum ter-
minale, in front of the lesion. MReissner’s fiber is seen swollen and irregularly
coiled. 240.

ABBREVIATIONS

c.c., central canal of the spinal cord nch., notochord
(and terminal filament) R.f., Reissner’s fiber
cg., coagulum, practically filling the s.t., sinus terminalis
central canal (fig. 17) i.n.p., terminal neural pore
cl., blood clot, in the central canal v.c., vertebral column
e., epithelium lining the central canal ** indicate the region of the incision

f.t., filum terminale

196

THE FUNCTION OF REISSNER’S FIBER PLATE 3
GEORGE E. NICHOLLS

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pine.

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THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

PLATE 4
EXPLANATION OF FIGURES

24 Raia clavata (5). A transverse section (shghtly obliquely cut) through
the filum terminale, at a point a little in front of the sinus terminalis. Several
fibrillae are seen which have apparently broken free from the displaced and
slack Reissner’s fiber. X 320.

25 Raia clavata (XIX, not experimental). Part of a sagittal section through
the filum terminale. Posteriorly the fiber is seen swollen but fairly regular.
It passes into a tightly tangled knot, from the anterior end of which it emerges,
loosely coiled. (Posterior end to the left, in the figure.) > 240.

26 Raia blanda (10). Part of a sagittal section of the hinder end of the
spinal cord, some half inch in front of the lesion. The posterior end of an ex-
tensive but loosely tangled skein of Reissner’s fiber (of nearly normal diameter)
is seen in the central canal which is cut obliquely. (Posterior end to the left
in the figure.) X 320.

27 Raia clavata (64). Part of a sagittal Section through the filum ter-
minale in front of the lesion. Reissner’s fiber is very fine and, to the left (ante-
rior) of the figure is seen in apparent contact with the wall of the central canal.
Behind this point it lies slackly, well away from the wall of the canal. X 320.

28 Raia clavata (18). Part of a sagittal section through the filum ter-
minale, immediately in front of the lesion. Retraction of the fiber was pre-
vented by the formation of an extensive clot (lying more anteriorly and not
shown in the-figure). The severed end of the fiber is seen fibrillated and flaring
as though to produce a new terminal plug. X 240.

29. Salamandra maculosa. Part of a transverse section through the spinal
cord. Reissner’s fiber apparently receiving three or four constituent fibrillae.
Near the dorsal line there projects a conical process which is probably the apex
of a sensory cell. X 340.

30 Seyllium canicula (44). Part of a sagittal section through the filum
terminale showing the unretracted fiber, with what are apparently constituent
fibrillae in situ. > 340.

31 Seyllium ecanicula (2). Part of a sagittal section through the filum ter-
minale at the point where this was severed by the experimental incision. The
cut end has become rounded off and the meninges have grown around it to com-
pletely enclose the secondary sinus terminalis. The end of Reissner’s fiber is
seen flaring somewhat and has, apparently, made good its new attachment.
x 340.

ABBREVIATIONS

c.c., central canal of the spinal cord mn., meninges, forming the hinder
(and terminal filament) wall of the sinus terminalis’

d.b.v., dorsal blood vessel R.f., Reissner’s fiber

e., epithelium lining the central canal s.p., sensory process (?)

fb., fibrillae of Reissner’s fiber in the s s.l., secondary sinus terminalis
central canal ** indicate the region of the incision

{.t., filam terminale

LYS

THE FUNCTION OF REISSNER’S FIBER PLATE 4
* GEORGE E. NICHOLLS

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STUDIES ON THE OLFACTORY BULBS OF THE ALBINO
RAT—IN TWO PARTS
I. EFFECT OF A DEFECTIVE DIET AND OF EXERCISE
II. NUMBER OF CELLS IN BULB
CAROLINE M. HOLT
From The Wistar Institute of Anatomy and Biology!

FOUR PLATES

PARTI. EXPERIMENTS TO DETERMINE THE EFFECT
OF A DEFECTIVE DIET AND OF EXERCISE UPON
THE WEIGHT OF THE OLFACTORY BULBS

CONTENTS
Pp nO GUC lO Mess cet ae Clk alee Aussi S's 2, ws GROMER AM GAIA eg oc dolteh rene ees 202
PE Wetectuve diet experimenter ti. 4.2.2... eames sae ee AO. os 204
1. Previous experiments on the effect of starvation upon the central
NELV OUR SV Scena OF “Ghe Tats es. cen. Ae oe tate eee 204
2. Series A: Aj, rats on defective diet from time of weaning at eighteen
to twenty days, and Az, at thirty to thirty-two days........ 205
Bite WECTHOGNG sien Sood tte ehh: CS TRG ERS See SRR AEE A tM te, 205
Dae sUled sate ays the Ai eeace ee. cts sk dt ee ee eee ean en ee 207
General morphological, and physiological modifications. ... . 207
Effect on brain and olfactory bulbs......................-. 209
3. Series B. Rats on defective diet from birth...................... 210
ce UC t NO Gieie cnn RO Say dh pe eo An, Rebel wera eave Mn hers 210
peeve Slts Oitn GF teesd | aan Wiese See emert tsb. se beksenmen. ay t,t edie: 214
ih PSETRISS ( CISUIS TIC. Uh 7: SR ae eR de me eR ge, E(t ae 215
SUMINE SUGGES Sere ei gaw a cae AL ae ne ee occ er ey cate ereiels eee. t, 215
5. Summary and conclusions: Defective diet experiments........... 217
Ty xe reise ex peninentses td smi ee BoM ON eae Ly 219
1. Previous experiments on the effect of exercise upon the albino rat.. 219
2. Description of experiments: Series D and E...................... 220
3. Series D: Rats in revolving cages for thirty days................ 221
Be RESIS PINS soo, 2 ine sch Awe ARM Se Ln SO SS 221

‘Thesis presented to the Faculty of the Graduate School of University of
Pennsylvania in partial fulfillment of the requirements for the degree of Doctor
of Philosophy.

201

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

202 CAROLINE M. HOLT

4. Series E: Rats in revolving cages for ninety-eight to one hundred

and three vdavs hss ts2G doa ee eee ie tenet ie eee 222

De SRESIILGS ey. 8. ais ee Serkan Ae ask the Le AER etn 212 Se ORO rea 223
Generalsbody growth: ...c-m-ceack. cckeaeeee (soe tee ere 223

Activaty ofexercised animals::.:5..cdassee ee ie aoe 224

Possible effect: oni fertility. 222: .ehGk sete ee one eee 225

Effect on brain and olfactory bulbs........................ 226

Eph SUUDAAIND TM pee eee oc ss. 3) co nay eM cee Re EI REE ene i es ae 233

IV. (‘Conclusiongtateers.|.. : os .. ee ee Gee er eh ee ee oe 233
Vi. Mbiterature wetted .). !. 62.08 oh eee Oe CE eer Cee oe eee 234

I. INTRODUCTION

The various members of the mammalian series show con-
siderable variation in the relative development of all parts of
the central nervous system, but probably no part of the encepha-
lon shows so great a degree of variability as does the rhinencepha-
lon. Of this portion of the brain, the olfactory bulbs are, with-
out doubt, the most variable in size. Thus we have the very
large bulbs of the opossum and the ant-eater; the almost rudi-
mentary bulbs of the ape and of man; extreme reduction of
these organs in the Cetacea, with their complete disappearance
in the dolphin. Not only do we find variation in size of the
olfactory bulbs among the different orders of mammals, but
we find that there is a considerable degree of variability within
each order and even among the members of the same species.

This variation in size and weight of the olfactory bulbs within
a species is well illustrated by observations upon the rats in the
colony of The Wistar Institute. The domesticated albino rats
exhibit a considerable range in the development of this part
of the brain. But while we find an appreciable difference in
the bulb size of rats of different litters even under like environ-
mental conditions, the individuals of a given litter usually show
a more uniform development of the olfactory system. Some
wild Norway rats examined at The Wistar Institute a few years
ago had olfactory bulbs heavier in proportion to total brain
weight than the bulbs of the albino. In the course of the present
study, observations made upon some thirty wild Norway rats
caught at different places in Philadelphia suggested that this
difference between the two strains is not a constant one, for,

OLFACTORY BULBS OF THE ALBINO RAT 203

while the olfactory bulbs of these animals were much heavier
than those of the albinos, the ratio between bulb weight and
brain weight in this series was about the same in the two forms.

Inequality in the size of the two bulbs in the same individual
appears not infrequently in the albino and when it occurs it
will often be found in several, and occasionally in all, members
of the same litter. For this reason, in selecting material for
these experiments, we discarded all litters in which cases of
asymmetry were observed among the initial controls.

Observations made from time to time by Dr. Donaldson,
indicated that rats born in the early summer differ from winter-
born rats in the relative size of the olfactory bulbs; also that
there might be a difference between rats reared on a restricted
diet, such as is frequently used in colonies, and those fed on the
table-scrap diet adopted for the Wistar colony. Moreover,
cases had appeared in which the bulbs of sick rats were appar-
ently smaller than those of healthy individuals.

All these facts suggested that there might be factors in, the
living conditions of the rats which would account for the varia-
bility of this portion of the nervous system, the growth of the
bulbs being retarded or arrested in rats reared under unfavor-
able conditions, such as the intense heat of the summer, or a
monotonous diet, or in those suffering from the various infections
which may attack the rats from time to time.

It was, therefore, with the hope of throwing some light upon
the question of the effect of environmental conditions upon the
olfactory bulb of the growing albino rat, that, at the suggestion
of Dr. Donaldson, the present experiments were undertaken.
The problem resolved itself into two questions—Can the growth
of the olfactory bulbs of the stock albino be modified (1) by
underfeeding or (2) by exercise?

The writer wishes here to express her deep gratitude to Dr.
Donaldson for his unfailing helpfulness and encouragement,
and her appreciation to Dr. Stotsenburg and Dr. Heuser, and
to the other members of The Wistar Institute who did much to
aid in the course of the experiments which have extended over
the past two years.

204. CAROLINE M. HOLT

Il. DEFECTIVE DIET EXPERIMENTS

1. Previous experiments on the effect of starvation upon the central
nervous system of the rat

There have been several previous studies upon the effect of
underfeeding and of starvation upon the central nervous system
of the albino rat. In 1904, Hatai reported experiments on
‘partial starvation’ for twenty-one days. He fed large quantities
of starch with some fat but no proteids of any kind. He obtained
a deficiency in body weight of 27 per cent in females and 32
per cent in males. Taking the values from the initial controls
for a standard, the brains of the test rats showed, at the end of
twenty-one days, a deficiency of 2.8 per cent for the females
and 5.8 per cent for the males. Thus the treatment produced
not only an arrest of brain growth but a loss in absolute brain
weight. This experiment was followed by a series in which the
animals, after a defective diet (Oswego starch), were returned
to a normal diet. Here Hatai (07) found that the effect of
twenty-one days of partial starvation was eventually compen-
sated for, so far as brain weight was concerned, but the central
nervous system had suffered some change in its chemical com-
position. The following year (08), Hatai published the results
of further experiments, this time in quantitative underfeeding
with an adequate ration, in which he concludes that growth
in the stunted rats is just as normal as in the controls; 1.e., all
parts are proportionately stunted.

In 1911, Donaldson published an account of the effect of
underfeeding, with a quantitatively deficient, but adequate
ration, on the percentage of water, on the ether-alcohol extrac-
tives and on medullation in the central nervous system of albino
rats, showing a slight diminution of percentage of water, slight
increase in percentage of ether-alcohol extractives, and no not-
able difference in medullation.

Jackson (715) found, in young rats maintained at a constant
weight on a diet of bread and milk, that the relation between
body weight and brain weight remained unchanged. The brain

OLFACTORY BULBS OF THE ALBINO RAT 205

ceased to grow simultaneously with the body, while the cord
increased somewhat in weight during underfeeding.

Although there are, at present, no data by which it is possible
to make a definite comparison between the effect of a defective
diet and that of an adequate, but quantitatively insufficient
diet, it is important to bear in mind in each case the method by
which growth has been retarded or arrested.

Two series of experiments upon the effect of underfeeding
were undertaken—one upon rats which had been reared to the
time of weaning (at three or at four weeks of age) by well-fed
mothers, Series A; the other, upon rats reared to the time of
weaning by underfed mothers, which meant rats underfed
practically from birth, Series B.

Observations were also made upon a few sick animals, Series C.

2. Series A. Ax, rats underfed from time of weaning at eighteen
to twenty days, and A», at thirty to thirty-two days

a. Method. As has been previously stated, while there is a
considerable range of variation between litters in the matter of
the relative size of the olfactory bulbs, yet within a given litter
the size is fairly uniform. For this reason, so far as possible,
control and test animals were taken from the same litter. This,
of course, made it necessary to select fairly large litters in order
to have several animals for initial and final controls, and also
for experiment. The litters were always taken from healthy
stock animals.

For the first few individuals experimented upon, no initial
controls were examined, but the results of these experiments
made the advisability of such controls apparent and subsequently

each litter was weighed and divided into three groups; so far as

possible, equivalent in sex, weight, and bodily condition. All
these rats were ear marked and a card filed for the data upon
each animal.

The first or initial control animals were at once etherized,
weighed, measured, eviscerated, and the brains removed. One
olfactory bulb was cut off from each brain, in the following man-

206 CAROLINE M. HOLT

ner. The brain was placed, ventral side down, on the dissect-
ing board. Then with a thin, sharp scalpel held in a position
perpendicular to the plane of the board and at right angles to
the plane of the median longitudinal fissure, the bulb wassevered
just below the anterior limit of the cerebrum.? The bulb, with
the remainder of the brain was then placed in a covered weighing
bottle and the weight of both the entire brain and of the severed
bulb ascertained.

The final controls were weighed and placed under the normal
living conditions of the colony: 1.e., housed, in long wooden
cages with wire fronts, thick shaving-covered floors, and paper
nests, and given plenty of fresh water with a carefully super-
vised scrap diet. The test rats were weighed and placed in
adjoining cages under exactly the same conditions as the final
controls, save for the diet. The food given the test rats con-
sisted of an unlimited amount of whole corn, usually fed on the
cob, save in case of very young animals, or those weak from a
long period of underfeeding. In such cases, the corn was shelled
as the animals were not able to remove a sufficient amount for
themselves.

Both control and test animals were weighed from time to
time and the weights recorded. Note was also made of any
irregularities, such as a temporary change in diet, etc.

At maturity, a certain number of test and of control animals
were mated in order to find out whether underfeeding affected
the fertility of albino rats.

In the case of relatively small litters in which the members
were usually well grown and in good physical condition when
weaned—and especially if weaning was delayed until the rats
were four weeks old—it was possible to keep the test animal on
a corn diet for a month or more with practically no difficulty.

2 Small bulbs tend to differ characteristically in shape from large ones. On
section it is seen that the cap of gray substance extends somewhat further
caudad on the ventral surface of the small bulb than it does in the case of the
large bulb. The weight of gray substance thus lost in the case of the small bulb
is a very small fraction of the total weight of the bulb but a much larger fraction
of the gray cap. Care must, therefore, be taken to include this portion when
the number of cells of the gray substance is to be detérmined.

OLFACTORY BULBS OF THE ALBINO RAT 207

With the rats weaned at three weeks or in case of small rats
from very large litters, there was a good deal of trouble in keep-
ing the animals on the corn diet for so long a time, and of course
the difficulty increased as the period of underfeeding was
prolonged. )

At first an attempt was made to keep animals from several
litters in one cage with the result that after a short time, the
less well grown rats were killed and eaten by the stronger indi-
viduals. Then the plan was adopted of having members of
only one litter in a cage. This worked successfully up to the
time when the animals began to weaken. Then the males
frequently killed and ate the females. So finally, for prolonged
experiments, it was found safer to place only animals of the same
sex, approximate weight and physical condition together, but
even this precaution was not always sufficient.

In most cases, for the first few weeks, there was a very slow
gain in weight or the weight was just maintained. But in every
case when an animal began to lose or became very feeble, a
dose of condensed milk was fed. One or two doses were usually
sufficient to restore the animal to equilibrium and there was not
infrequently a sudden temporary gain in weight, doubtless due
to increased appetite and the consequent gorging of the ali-
mentary tract with corn.

In a few cases where the underfeeding had gone on for several
months, it became necessary to administer small doses of con-
densed milk more frequently—in two cases, practically every
day—in order to keep the animals from losing weight.

At the end of the experiment, both test and final control ani-
mals were killed, weighed, measured, eviscerated, and brains
and bulbs weighed as in the case of the initial controls. One
bulb with a part of the cerebrum was preserved for histological
study. A record was kept of any signs of disease or other
abnormality. The weighing was done in closed bottles and all
weights of brain and of olfactory bulbs were made to 0.1 mgm.,
but recorded here in milligrams only.

b. Results. General morphological and physiological modi-
fications. A summary of the data from observations upon 108

208 CAROLINE M. HOLT

individuals of Series A is given in tables 1 to 8. The complete
tables with the records for each individual rat of this, as well as
of the other series, are deposited at The Wistar Institute. Of the
two litters weaned at eighteen and twenty days, only three
individuals survived to be killed; the others died in the cages
and the brains were not weighed. The records for the three
rats Just named have been included in tables 3 and 7, and their
controls, with the corresponding controls. The size and body
weight of rats weaned at the end of the third week and placed
on a corn diet indicated clearly that under like conditions, rats
weaned at three weeks are considerably more sensitive to adverse
conditions than are those weaned at four weeks.

For every individual of Series A; and A, (tables 1 to 8), the
stunting effect of the corn diet was apparent almost from the
first. During the early weeks of underfeeding the test rats
appeared rather more lively than the controls. Later this
activity decreased, the gait became unsteady, and the animals
appeared stupid. They were often unable to find the dish of
condensed milk by themselves, whereas control rats would
go to it immediately. This suggests that the underfed animals
lacked an acute sense of smell and perhaps did not see clearly.

In every one of the test animals of which there are complete
records, the general bodily growth was arrested by a diet of
corn. This agrees with the observations of Osborne and Men-
del (13). These rats remained like young animals in appearance
as well as in size. The earlier weaning took place and the corn
diet was begun, the more complete the stunting.

The skeleton became modified and somewhat distorted owing
to imperfect calcification. The growth of the long bones was
not quite so completely arrested’as that of the rest of the skele-
ton. The skull, sternum, and sometimes the ribs, became like
parchment. In two cases the pressure of the heart upon the
sternum had formed a sort of pocket out of that structure, which
appeared like a tumor on the ventral side of the rat. The
vertebral column became somewhat bowed, giving to the rat a
‘humped’ appearance and making it necessary to stretch the
animals when measuring body length. One to four months of

OLFACTORY BULBS OF THE ALBINO RAT 209

underfeeding, following the first month under normal con-
ditions, left the rats but slightly longer (4 to 10 mm.) than the
initial controls measured at thirty days. The average increase
in weight was in about the same proportion. Compare tables
2 to 8, for body weight and body length.

All the rats showed extreme emaciation but this condition was
largely masked by the condition of the coats. The hair re-
mained short and soft, with a fluffiness which gave even to mature
rats the appearance of plump young animals. Such emaciation
was, of course, accompanied by great muscular weakness.
Rats kept for long periods on the defective diet became unable
to remove corn from the cobs. They walked with a tottering
gait and moved about but little.

The cyanosed condition of these animals was clearly indicated
by the blue color of all exposed parts of the body—nose, ears,
feet and tail. In protracted cases of underfeeding, a chronic
palpitation of the heart developed which increased in violence
as time went on. As a result of this, the whole body shook
constantly. :

All animals kept on corn up to maturity failed to breed or to
show any sexual instinct whatever.

Effect on brain and olfactory bulbs (compare tables 1 to 8).
In Series A, both A; and A, show a slight increase in brain weight
during the period of underfeeding. Under normal conditions,
as the rat grows, the brain becomes relatively lighter in pro-
portion to body weight. In the underfed rats the brain forms
practically the same proportion of the total body weight as in
the initial control rats (agreeing with Jackson’s results (’15)),
which of course indicates in the cases where growth has taken
place that the brain has not been as much arrested in its develop-
ment as has the rest of the body.

After four to eight weeks of underfeeding, the rats of Series
A, and A; had olfactory bulbs which, taken together, formed
about the same proportion of the total brain weights as did the
bulbs of the initial controls of the same series, showing that the
relation of these parts of the brains had not been changed during
the experiment. But normally the olfactory bulbs grow faster

210 CAROLINE M. HOLT

during this period than the rest of the brain so that at eight
weeks, for example, the bulbs should form a considerably greater
percentage of the total brain weight than at thirty days. The
average absolute weight of the bulbs of the test animals was
equal to but 70 to 81 per cent of the average weight of the bulbs
in the control animals of the same series. It is therefore evi-
dent that the retarding effect of underfeeding has been greater
upon the olfactory bulbs than upon the other parts of the brain,
which had 85 to 90 per cent of the weight of the brains in the
control series.

If the relative weight of the bulbs in Series A; and A, is de-
termined for the test group as contrasted with the final control
group, we obtain the following relations:

TABLE 1
PERCENTAGE
GROUP AGE WEIGHT OF
OLFACTORY BULBS
days

Table 3..........] Test rats, defective diet 60 Beg
Mails raver cis Final controls 60 3.99
ab lesa sere i Test rats, defective diet 79 3.39
MOEN yaeocweee Final controls 79 4.16
Rablewereter cs... Test rats, defective diet 118 3.83
Mahler Sw wees Final controls 128 4.30

This arrangement of the results shows clearly that in each of
the three sets, grouped according to age, the olfactory bulbs of
the underfed rats are significantly lighter in proportional weight
than those of the controls. We may, therefore, conclude that
the relative weight of the olfactory bulbs is reduced by the
form of defective feeding employed in this experiment.

The details are given in tables 2 to 8, which follow.

8. Series B. Rats on deficient diet from birth

a. Method. Since it was evident that the earlier the animals
were weaned, the greater the stunting effect of a qualitatively
inadequate diet, it occurred to the writer that it would be inter-
esting to try underfeeding from birth, by underfeeding the

TABLE 2. SERIES A
Initial control animals
In all of the tables the averages are weighted for the number of animals in each

entry
OLFAC- pet
PALS aan een CaNere SanreHe Gee anne se eae
WEIGHT WEIGHT
days gm. mm. gm. gm.
Asmmale sae cere ae oak 30 44.1 118 | 1.4389} 0.051) 3.52 3.01-3 .97
iiefemales sta eats cae oe 30 | 43.5 | 116 | 1.409} 0.050) 3.53 | 2.41-4.19
Averages for males and
females'..0 eee eee: 43.8 | 117 | 1.426] 0.050) 3.53 | 2.41-4.19

TABLE 3. SERIES A
Test animals

Stock albinos kept on corn diet for twenty-nine to forty-two days after weaning
at three to four weeks

OLFAC- peer
bars naz |,20P%,|, BOD | BRAIN, | TORE loon or] ANGE
DOYS GEELE | eee
days gm. mm. gm. gm.
SPIN ALES eerie taseeedoe ee vie 60 99).9 126 | 1.505) 0.052) 3.45 2.38-4.53
i Pferiall Osirapcpdecs: Pe sets es 60 53.9 126 | 1.502) 0.054) 3.62 2.68-4.21
Averages for males and
femalesec qacoct ey see vor 54.8 | “126 | 1.504] 0.053] 3.52 2.38-4.53

TABLE 4. SERIES A
Final control animals

Stock albinos kept on normal diet for twenty-nine to forty-two days after weaning
at four weeks

OLFAC- res
EONS sNGHS emus ‘aie mreHt eens aac ER
WEIGHT WEIGHT
days gm. mm. gm. gm.
LOimalestee ee aan Ol 127.4| 167 | 1.668) 0.066] 3.95 2.76-4.62
females) eee eee 60 95.1} 154 | 1.606} 0.065) 4.04 3.66—-4.53
Averages for males and
females: =. ass eee ee 118.8} 164 | 1.651] 0.066) 3.99 2.76-4.62
Summary sae a, Vee a wh) 91 81
Control

AP CAROLINE M. HOLT

TABLE 5. SERIES A
Test animals -
Stock albinos kept on corn diet for forty-nine days after weaning at four weeks

OLFAC- se
RATS Aas) | crea |eunere Warare| unas |CoNT OF] | aaNGal
WEIGHT Babee ci
days gm mm gm gm
PRIN ALES sa) eed |e 47.8 126 | 1.502) 0.055} 3.63 3.31-3.93
I sfemale sree regen setae oi. chav ons 78 37.8 116 | 1.458} 0.042} 2.89
Averages for males and
FEMALES SE EEE cis t:- sick 124 | 1.487) 0.050} 3.39 2.89-3 .63

TABLE 6. SERIES A
Final control animals
Stock albinos kept on normal diet for forty-nine days after weaning at four weeks

OLFAC- gd es as
nas no | Ropy,| pope | PRawn| TORY loewroe| RANE
WEIGHT WEIGHT
days gm. mm. gm. gm.
Dimnalesaeee eo. nic haere ee ell oO 155.0} 178 | 1.727) 0.068) 3.93 3.72-4.14
iGremalettees a) hee ies 78 151.2} 183 | 1.703] 0.079) 4.63
Averages for males and
FeIMalesiate swie ss cae eae ae 153.8} 180 | 1.719) 0.072) 4.16 3.72-4.63
Test 7
Summary ae es ee nooo oes Ie 69% 78% 70%

Control

TABLE 7. SERIES A
Test animals

Stock albinos kept on corn diet for fifty-nine days or more, after weaning at
four weeks. (One rat weaned at eighteen days)

OLFAC- giclee
BopY | Bopy | BRAIN| TORY
EBT AGE |wricHt|LENGTH|WEIGHT| BULBS |CENT OF TLS Ket
weiqaut| BRAIN
WEIGHT
days gm. mm, gm. gm.
A TOALES: na. oa ss Ce 120 | 47.1 118 | 1.463} 0.057} 3.86 3.47-4.19

6 females.>) reys*.. eee 115 | 54.3 | 126 | 1.594) 0.060) 3.79 | 3.55-4.29

Averages for males and
females:;: : acc... Ree 50.5 | 122 | 1.524) 0.058) 3.88 | 3.47-4.29

OLFACTORY BULBS OF THE ALBINO RAT Zils

TABLE 8. SERIES A

Final control animals

Stock albinos kept on normal diet for fifty-nine days or more, after weaning at
four weeks

OLFAC- eee
nas non | BOBE,| BORE, | Beare | TORE, loss or] RANGE
Vrnikenste WEIGHT
days gm. mm. gm. gm.
Grmiales.-... cease. cae. | 2 200 4 199) FA s80602078| 4:29. (381-4773
AREMB eB... Bete see 1361| 157.0] 181 | 1.706) 0.074) 4.32 | 4.03-4.62
Averages for males and
Git Las AMER by BHC oe 186.7| 193 | 1.772} 0.076) 4.30 | 3.81-4.73
Test
Summary = es oss de. 63% | 85% | 77%
Control

1 This higher average age for the female controls is due to the fact that one
female was kept for breeding purposes until two hundred and thirteen days old.
As all her measurements were practically identical with those of another female
of same litter, one hundred and fifteen days old, the record was included in the
table.

mothers which were bearing or nursing the young to be tested.
Consequently nine pregnant females were selected. A few
were put on a corn diet several days before the birth of the young,
but most of them began the corn feeding on the day of the birth
of the litter. The young rats were weaned at three weeks and
fed exclusively on corn.

It is intended to carry out this experiment more extensively
at some future time but enough amimals were tested to give
significant results. It was found very difficult to raise such
litters, for two reasons. In the first place, after the young reached
an age to leave the nest, the mother was very apt to kill the
entire litter. This, apparently, was not because of hunger, for
in all but two cases in which the young rats were partially eaten,
the animals were mutilated only to the extent of a bite through
the cerebellum, and sometimes through the front of the throat.
It has been suggested that the increasing demands of the young,
coupled with an inadequate milk supply, may have been the
cause of this unnatural behavior of the mothers.

214 CAROLINE M. HOLT

But the chief reason for the difficulty in raising these rats was
their lack of vitality. Although very active and playful, these
animals were extremely frail little creatures, so weak that the
slightest disturbance was likely to prove fatal. For example,
an unusually active and promising test rat of fifty-three days, was
carried from the colony to the laboratory for examination. As he
appeared much excited, the carrier cage was set aside for an hour.
The rat was heard running about for a time but at the end of the
hour was found dead. The body weight of this rat was that
of an animal fifteen days old and the brain weight was scarcely
more. Young rats might appear lively and in every way normal
in a late afternoon and be found dead in the cage next morning,
for no reason to be discovered even after careful examination.
Of nine such litters only two survived to the time of weaning,
and these were kept with much difficulty.

A litter of ‘runts’ was also included in this series. This was a
litter of rats, all of which failed to grow normally, presumably
because the mother had an insufficient supply of milk. They
appeared in every way like the rats which had been stunted by
underfeeding the mothers.

b. Results. The general results of underfeeding in Series B
were essentially the same in character as in Series A but they
were considerably more marked (tables 9,10 and 11). The body
length and general appearance of seventy-seven day rats, under-
fed from birth, were practically the same as in normal three-
weeks-old rats, save for the extreme cyanosed condition.

From a comparison of Series Ai, As, and B, it becomes evident
that it is easier to retard the growth of an eighteen day rat than
of a rat thirty days old, and still easier to stop the growth of a
rat at about the size of an eighteen day individual if the under-
feeding is begun at birth. Moreover, it is obviously far more
difficult to rear these animals underfed from birth than rats
which have been allowed to get a good start of thirty days under
favorable conditions and are therefore much more resistant to
the deleterious effects of partial starvation.

Effect on brain and olfactory bulbs. Series B shows brains
actually lighter in weight at twenty-four to fifty-three days of

OLFACTORY BULBS OF THE ALBINO RAT 215

age than normal brains of seventeen days (The Rat, table 74).
Rats seventy-seven days old had brains weighing practically
the same as those of normal female rats of forty-two days.

Bulbs of rats twenty-four to fifty-three days old, actually
weighed only 70 per cent as much as those of normal rats of
thirty days (table 2) and only 52 per cent as much as bulbs of
normal rats eight weeks old (table 4).

Rats eleven weeks old (table 11) gave bulbs of the same abso-
lute weight as those of control rats of thirty days (table 2).
In both cases the olfactory bulbs formed a smaller per cent of
the total brain weight than appeared among the controls of
like age in Series A, as the following arrangement of the data
shows:

TABLE 9
PERCENTAGE
GROUP AGE WEIGHT OF
OLFACTORY BULBS
days
Maile Ose. sana. Test rats, defective diet-
Series B. 24-53 33, 11a
Mable as4. 028s: Control. 30 3) 4
Ho) Kee [Ree Test rats, defective diet-
Series B 77 3.48
May lewa ne Fo. - Control 60 3.99
Meatblen Gienasecie.. Control 79 4.16

The details for these series are given in tables 10 and 11 which follow.

4. Series C. Sick rats

In the course of the experiments a number of sick rats came
under observation. Eleven of these were examined to determine
whether the brain, and especially the olfactory bulbs, showed any
effects of the diseased condition. Three of these rats were the
sole survivors from a group of twelve attacked by a serious
bowel trouble which killed the other nine occupants of the cages.
At the time of the onset of the illness, the rats were about eighty
days old. After about ten days, these three seemed to recover
and were kept until they were about a hundred and thirty-five

216 CAROLINE M. HOLT

TABLE 10. SERIES B
Test animals

Stock albinos underfed from birth. Under two months old

RATS AGE CENT OF RANGE

WEIGHT WEIGHT

days gm. mm. gm. gm.
Grmaless es eee | cael eee 86 1.114} 0.036} 3.19 | 3.06-3.38
ASTemaAles: toe eee ae 24-53] 15.6 79 1.062} 0.033} 3.08 2.11-3.82
Averages for males and

fomiBlosaserss cet ese fake 18.1 |; 83 | 1.091) 0.034) 3.14

TABLE 11. SERIES B 1.
Test animals

Stock albinos underfed from birth. Over two months old
BODY BODY BRAIN TORY

tas NER WEIGHT|LENGTH| WEIGHT! BULBS pe ei BENGE

WEIGHT WEIGHT

days gm. mm, gm. gm.

SMOMBLER =. 2 tks 6h ohare sizes 77 | 31.2 | 102 | 1.437) 0.050) 3.48 | 3.15-3.93

days old when they were killed and examined. The other
eight sick rats of this Series C were individuals showing a con-
siderable infection of the lungs, and one of these (No. 20) had,
in addition, a large abscess of the liver.

All of these rats were examined in the same way as those of
Series A.

a. Results. In the group of sick animals, those with the
intestinal infection had, at one hundred and thirty-four days,
bulbs which averaged 0.050 gram or 3.02 per cent of the total
brain weight (table 12, group 1) while a set of normal individuals
of practically the same age gave an average of 0.073 gram or
4.32 per cent of the total brain weight (see table 20, group of
females). These results seem especially interesting because
here the adverse conditions appeared only after the rats were
well grown—eighty days old—and lasted only about ten days.

The remaining two groups of sick rats all had infected lungs
and were very old when killed. The two males had bulbs

OLFACTORY BULBS OF THE ALBINO RAT vi LP

averaging 0.037 gram, or 2.08 per cent of the total brain weight
(table 12, group 2); while for the four females, the bulbs averaged
0.033 gram, 1.89 per cent of the total brain weight (table 12, group
3). For these last two groups there are no data of normal
individuals for comparison but the percentage for the bulbs
is strikingly low. Some unpublished data in Dr. Donaldson’s
hands show, however, that while the relative weight of the
olfactory bulbs tends to increase up to about one hundred and
fifty days of age, in older rats there is a tendency to decrease so
that some of this decrease observed in the old sick rats (groups
2 and 3) may be due to normal age changes. But the remark-
ably small proportional weight of the bulbs here examined is
probably due chiefly to the effect of disease.

In this connection may be mentioned two young rats of litter
PR (group 2), killed at seventy days. Each had infected lungs.
These rats came from parents with infected lungs and had lived
since birth in a dark damp cage. One had very small unequal
bulbs which were not weighed. The other had bulbs weighing
only 0.019 gram or 1.30 per cent of the entire brain weight.
This pair of bulbs were the smallest observed in the whole series
of experiments. It seems quite evident that the bulbs are
abnormal and quite probable that this abnormality is due to
disease.

5. Summary and conclusions. Defective diet experiments

1. General bodily growth in the albino rat is arrested by an
exclusive ration of corn which constitutes a defective diet (Os-
borne and Mendel).

a. The skeleton is poorly calcified and somewhat distorted.

b. The muscular system is greatly reduced.

The coat has the appearance of that of a young animal.

. Functional disturbances follow the arrested development.
. There is increasing muscular weakness.

. An increasing palpitation of the heart.

The animals appear cyanosed.

Sos wa

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

218 CAROLINE M. HOLT

TABLE 12. SERIES C
Sick animals
Females

Group 1. Three albino rats from a lot of twelve controls for revolving cage
experiment. At about eighty days all contracted a severe bowel trouble from
which these three recovered.

BULBS
ANIMALS AGE BODY BODY BRAIN pagent 2 PER CENT
WEIGHT LENGTH WEIGHT WEIGHT packer)
days gm mm gm gm
Digs NAS EOC 135 128.5 165 1.607 0.033 2.03
Vise Ome cea: 135 150.5 180 1.534 0.034 2.24
bets ss ae em ee 134 157.9 182 1.803 0.082 4.55
Average females....|.........| 145.6 176 1.648 0.050 3.02
Males
Group 2. Two, old, with infected lungs.
PR eee er teins, os get 70 88 148 1.489 0.019 1.30
Dal Sota ose A a Ba 70 90 155 1.538 | unequal
NGS ARIE. es. h tele old 213 202 1.759 | 0.050 2.81
INGO OS 2 is: Seed es 365 214 195 1.791 0.024 1.35
Average Numbers
DE G9) eer! Flin D0 SS 21825 198 1.775 0.037 2.08

Females
Group 3. Infected lungs, old age, and in case of No. 20, bad abscesses on liver.

INO eee ic atch cas ie 340 143.0 173 1.673 0.027 1.63
Fei ke eee 340 146.1 187 Lol 0.032 1.79
DUO. lee tia ee hs ass 370 186.0 186 1.687 0.051 3.01
DO: fees oa ho 240 218.3 ig) 1.794 0.021 1.16

Average females.... 173.4 186 1.726 0.033 1.89

d. The sense organs become dulled after prolonged defective
feeding—the animals respond but slowly to stimulations of
sound, or light or smell.

e. Defectively fed animals fail to breed.

3. The effect of defective feeding on the brain and olfactory
bulbs is less than upon the rest of the body, but is, nevertheless,
very marked. The olfactory bulbs are stunted and to a con-

OLFACTORY BULBS OF THE ALBINO RAT 219

siderably greater degree than is the entire brain. When de-
fective feeding is begun in rats about thirty days of age, the
bulbs of rats thus experimentally stunted form about the same
percentage of the total brain weight as do the bulbs of rats of
the same litters killed at the beginning of the experiment.
Whereas, under normal conditions, the bulbs of older rats (up
to one hundred and fifty days) are considerably heavier in
proportion than those of the young animals. With prolonged
defective feeding the proportional weight of the bulbs tends to.
become slightly greater.

4. Sick animals, especially those with lung infection, show a
marked diminution in the relative weight of the olfactory bulbs,
accompanied by a certain amount of loss in total brain weight.

Ill. EXERCISE EXPERIMENTS
1. Previous experiments on the effect of exercise upon the albino rat

Several investigators have worked upon problems connected
with the changes in the albino rat occasioned by an increased
amount of exercise. J. R. Slonaker in 1907 published observa-
tions upon four rats of different ages kept in revolving cages
for a short period. In 1912, the same author published an ac-
count of further experiments along the same line, and although
this time, also, the work was with a small group of rats, yet the
experiment was continued during the natural life of the animals.
Slonaker was working chiefly upon the problem of normal activity
in its relation to age and sex but, incidentally, he made some few
observations upon the comparative development of ‘exercised’
and normal rats. He found that “exercised rats are more active,
more alert, and brighter in appearance than the control ones,”
but that “the control males reach their maximum weight at
an earlier age than exercised males, and also greatly excel them”’
and that “control rats live longer than exercised rats.” No
observations were made on the effect of exercise upon any of the
internal organs.

Donaldson, in 1911, conducted a series of experiments to
ascertain the effect of exercise upon the central nervous system

220 CAROLINE M. HOLT

of the albino rat, using the same sort of apparatus—a revolving
cage with cyclometer attachment—employed by Slonaker. He
found that there was a slight increase in brain weight (2.4
to 2.7 per cent) to be attributed to the effect of exercise. This
was what was to be expected in view of the heavier brain to be
found in the wild Norway rat. The cord showed no effect.
The olfactory bulbs were not weighed separately.

Hatai (15) published a series of observations based upon his
own experiments and upon those of the present writer, showing
the rather marked effect of the same exercise conditions upon
the weight of the internal organs. In these experiments, the
brains of the test animals showed an excess of 4 per cent over
the controls with no effect upon the cord.

2. Description of Experiments. Series D and E

As it had thus been demonstrated that the brain of the albino
rat could be modified by exercise in the revolving cage, it re-
mained to determine whether, under such conditions, the ol-
factory bulbs would show a more marked variation than the
brain as a whole.

For this work, also, large litters of stock albinos, were chosen.
Each litter was weaned and divided into three groups when
about thirty-five days old. One group constituted the ‘Initial
Controls,’ and these were killed and examined as in the previous
experiments. The second lot, the ‘Final Controls,’ was set aside
in cages under the normal living conditions of the colony. The
third group was used for the experiment. Each of these test
animals was placed by itself in a wire revolving cage such as
had been used by Slonaker, and later by Donaldson and Hatai.
Each cage was 5 feet in circumference with an open nest box
fastened to the central fixed axis. From this axis the food was
suspended so that, theoretically, the rat must descend to the
floor of the cage to eat. Practically, some rats soon learned
to avoid this and so escaped a considerable amount of enforced
exercise.

Each cage was provided with a cyclometer. Readings were
made and recorded six times a week. These cyclometer read-

OLFACTORY BULBS OF THE ALBINO RAT 221

ings showed only the activity of the rats when the cage revolved
and were therefore incomplete, since some rats learned to play
from side to side of the cage and keep it from revolving, while
others learned to run up the middle of the sides in such a way
as to hold the cage at rest. But most of the rats soon learned
to run the cages and appeared to enjoy it.

The rats were fed on the same diet as the controls and all the
animals were weighed at intervals of about two weeks.

3. Series D. Rats in revolving cages for thirty days

There were but two litters in this series. One litter was
weaned and set aside at thirty-five days of age and the other
at forty days. Both litters were subjected to exercise in the
revolving cages for a period of only thirty days. All were
killed at the end of the thirty days of exercise.

a. Results. The exercised males of these two litters gained
more rapidly in both weight and body length than did the
controls, while the females fell behind. The superior growth
of the test males was sufficient to bring the averages for both
males and females up to 113 per cent of the weight of the con-
trols and to 104 per cent of the length (tables 13 and 14).

The records of the activity of Series D were accidentally
destroyed, but as these were for a period of but thirty days,
they would be of little value save in adding further evidence
that the female rat becomes active sooner than the male.

While, on the average, there is no difference in the absolute
brain weight of the test rats in Series D from that of the controls,
when both are compared with the reference table values in The
Rat (Donaldson, 15), according to the method there suggested
(pp. 4 and 5), yet I believe the bulbs do show, even after this
short period, some effect of the unusal activity (tables 13 and 14).
In the females, the bulbs make up 4.46 per cent of the brain
weight in test rats as compared with 4.36 per cent in the controls.
With the males, the difference was more marked—4.55 per cent
in tests to 4.20 per cent in controls, making a joint average for
males and females of 4.51 per cent in tests against 4.32 per cent

222, CAROLINE M. HOLT

TABLE 13. SERIES D

Test animals
Albino rats kept in revolving cages for thirty-three days after weaning

Males

OLFACTORY

OLFACTORY BULBS:

ANIMALS sce | werent | canara | werner | BULBS | PER CENT

WEIGHT

days grams mm. grams grams
PRG tee ee ee 70 88 148 1.489 0.0191 1.30
We Pam eh ce, St 68 eH 7 170 1.871 0.079 4.21
Teak os 1 Sat iteah oO ey 68 140.9 176 1.831 0.085 4.63
PR; 70 alee 185 1.784 0.085 4.79
Average males....... 148.6 LTE 1.829 0.083 4.55
Females
hey EE ere 68 102.9 158 1.744 0.079 4.53
Le ade ne) ean Sick 70 116.0 162 1.653 0.073 4.39
Average females...... 109.5 160 1.698 0.076 4.46
Average males and

femaleseys [nen yee 132.9 170 1.776 0.080 4.51

1Tungs infected. Rat undersized in every way, therefore not included in
averages (Series C, Sick rats, p. 218).

in controls, the olfactory bulbs of the former being, therefore,
7 per cent heavier than those of the latter.

4. Series E. Rats in revolving cages for ninety-eight to one hundred
and three days

The test animals of this group were kept in the revolving cages
for fifteen weeks. At the end of that time, three pairs of test
animals and one pair of controls were mated (brother to sister
in each case). Some digestive trouble appeared in the cages of
control rats rather early in the experiment and most of the rats
died, while the remaining animals failed to attain a normal
growth, so that satisfactory final controls were lacking for this
group. But the rest of the test animals and the surviving con-
trols were killed at the end of the fifteen weeks, measured,

OLFACTORY BULBS OF THE ALBINO RAT pepe

TABLE 14.. SERIES D

Final controls

Males
OLFACTORY
OLFACTORY BULBS:
soars non | mor, | zope, | pean | Sous | eae con
WEIGHT
SP i taage Dl) gram) | me lprams.” | grame
ARG eens Gis Rear cessed 70 100.7 154 1.636 0.061 3.72
eerie ete ti ckvotane cis 70 90.0 155 1.5388 Bulbs
unequal!
MAM ore od cc Sareayaktate 68 132.4 170 1.802 0.084 4.64
Average males....... 116.6 162 1.719 0.072 4.20
Females
aU 3 A Rls A NR ee ae 68 110.8 159 1.780 0.081 4.52
Meena Se use. ac gece cs Shs 68 115.6 165 1.774 0.077 4.32
ARR hee hs ve ae iy soll) oy 70 130.5 170 1.704 0.074 4.32
Average females...... 119.0 165 1.753 0.077 4.39
Average males and
femalesn. 945 nyt 118.0 164 1.739 0.075 4.32
Test Bs
Cet ea ee 103.7%} 102.1%| 106.8%

1 Lungs slightly infected. Not included in average.

weighed, examined, and bulbs preserved exactly as in the under-
feeding experiments.

The mated animals were kept about one hundred days longer
to see whether the exercise of the previous weeks would show
any effect upon fertility.

a. Results. General body growth. We find by examination
of the records of body weight taken at two-week intervals during
the experiment, that up to the time the larger set of control
rats fell sick, the exercised animals were gaining less rapidly
in weight than were the controls. From the time of the illness,
some five weeks after the beginning of the experiment, the control
rats fell off in weight, and with a single exception, they never
recovered. Litter W escaped the infection and the weight
records for the six rats composing it are as follows:

224 CAROLINE M. HOLT

TABLE 15

Record for Litter W, Series E, showing gain in weight for individuals in revolving
cages and for centrols

TEST RATS CONTROL RATS
We (m) Ws (f) Wi (f) We (m) Wz (m) Ws (f)
Initial weight........| 44.0g.} 40.5¢.|] 50.5¢.| 438.5¢.| 50.0¢g.| 40.5¢.
2 weeks weight...... 60.4 62.0 65.6 63.2 68.7 69.2
4 weeks weight...... 97.0 82.0 103.0 107.0 120.0 89.0
6 weeks weight....... 139.0 124.0 142.2 139.0 148.8 119.0
9 weeks weight...... 186.0 142.0 170.5 194.0 212.0 148.0
30 weeks weight...... 210.0 148.0 187.0 205.0 224.0 150.0
inal genet neers et: 209 mm.| 182 mm.} 198 mm.} 207 mm.| 200 mm.} 190 mm.

The test rats from Litter W were, on the whole, slightly longer
and lighter in weight than the control animals. The majority
of individuals in Litter W proved to have abnormal brains—
one or both olfactory bulbs being very much undersized. The
brains, therefore, could not be used for comparison and the
litter was excluded from the tables. For comparison with the
rest of the litters of Series E, it was necessary to use other stock
litters, as will be described later (tables 18 to 25). The compari-
sons are not, therefore, of as much value as they would be were
the controls from the same litter. On the average we find body
length slightly more, and body weight slightly less, in test ani-
mals (table 25). I think we may conclude that these results
agree in general with those of previous investigators indicating
that exercise has but a slight effect, if any, upon either body
weight or body length.

The size of the viscera was considerably modified. These
results have been incorporated in the report by Hatai (15).

Activity of exercised animals. These rats showed great
individual difference in the amount of activity and in the age
at which they became most active (tables 16, 19, 21, 25). In
these respects, there was also a considerable difference in litters
as shown by the following record.

If we take the record of these same rats for ninety-three days
we get an average of 5.76 miles per day for males, and 5.96 miles

OLFACTORY BULBS OF THE ALBINO RAT 225

TABLE 16

Activity record of rats in revolving cages. for one hundred and three days. Series E

A Ces Lata ints sine canes 914.5 8.9 aI is. a ees os. 559.7 5.4
Vise eee ee i ei GD Z3M... 476.3 4.6
OLY et eres Oe ee es Pa" oS 0 XeM.. 470.8 4.6
BY Gear care nats ofl ODES 6.8 XoF... 458.7 4.5
A ana oe: 2 a ee 689.0 Gerd Lig Rab Need Oe 457.1 4.4
1, | ROC, «SOS 577.8 5.6 Tighe. # Mic ede ke 446.6 4.3
Average for males...| 614.7 5.96

Average for females} 593.7 5.76

for females, and if we go back still further we get a still higher
average for the females and lower for the males. The males
were slow to begin to run the cages. An extreme example, Y,
of the present series, ran less than 2 miles during the first five
weeks in the cage, but became extremely active during the last
four or five weeks making a final average of 5.4 miles per day,
a record almost equal to the average for the entire lot of males.
The females soon learned to run the cages and became very
active at an early age. During the last weeks of the experiment,
the activity of practically every female in the series was on the
decline. I think from a study of all the records it may be con-
cluded that while, in the revolving-cages, the females reach the
period of greatest activity earlier than do the males, yet in the
long run, the records of a large number of males and females
would average about the same.

Possible effect on fertility. There is some indication that the
fertility of the albino rat is increased by exercise. In the cases
of the three pairs of exercised rats which were mated, the fol-
lowing record of offspring was obtained, together with the record
of one control pair.

The average size of litter for normal stock albinos has been
found to be between 6 and 7 individuals (Donaldson 715). This
is about the average for the control pair, while the averages for
the three test pairs is considerably higher—13, 10.5, and 9.

226 CAROLINE M. HOLT

TABLE 17

TEST PAIRS CONTROL PAIR

W, 21st day after mating, 12 young Ws 24 days after mating, 3 young

and 61st day after mating, 11 young |and 50 days after mating, 12 young

W. 102d day after mating, 16 young W. 102 days after mating, 0 young
pregnant not pregnant

Y, 22d day after mating, 9 young

and 67th day after mating, 9 young

Y; 102d day after mating, 0 young
not pregnant

Zs 22d day after mating, 12 young

and 88th day after mating, 9 young

Z; 102d day after mating, 0 young
not pregnant

It is significant also that the pair making the record of an average
of 13 per litter for three successive litters, and the control pair
are from the same original litter. Of course the numbers here
are too few to enable one to draw conclusions but it would not
be surprising to find some correlation between the greater weight
of the sex organs in the exercised rats (Hatai 715) and the fertil-
ity of these animals.

Effect on brain and olfactory bulbs. It has already been
noted that most of the control rats of this series were lost through
disease. For comparison with the exercised rats, a set of con-
trols used in Series A of the defective feeding experiment was
chosen (table 20). These rats seemed better suited for the
purpose than any others because they had been born at the
same season as the test animals and reared in the same laboratory,
so the food from day to day was the same for the two sets of
rats. Among these, it was possible to find records of eight
rats of almost the same body length and weight and of approxi-
mately the same age as the exercised rats killed at the end of the
experiment. For the four which were mated and not killed
until they were two hundred and thirty-eight days old, it was
not possible to get controls of the same age, the four oldest of
the controls (table 22) averaging only one hundred and sixty-
nine days and the body length being 6 per cent less than that
of the test animals. But as these are beyond the one hundred
and fifty day limit, up to which time the bulbs increase in rel-

OLFACTORY BULBS OF THE ALBINO RAT pipet

ative weight, the difference is not so serious a matter as it would
be were the rats younger.

The set of test animals killed at the end of one hundred and
three days of exercise, gave bulbs averaging for the males 4.28
per cent of the entire brain weight, and 4.60 per cent for the
females—an average of 4.41 per cent for the entire set (table 19).
When these results are compared with the controls (table 20)
we find that while the test animals were 1 per cent shorter than
the controls and had brains 2 per cent lighter in weight, the
olfactory bulbs were 3 per cent heavier. These results seem to
indicate that the olfactory bulbs of the test animals have been
affected by exercise.

An examination of the records for the initial controls of the
litters concerned seems to give additional weight to this supposi-
tion. See table 23 below.

‘TABLE 18. SERIES E

Initial control animals

Males
OLFACTORY
OLFACTORY BULBS:
ANIMALS sce | werent | umnata | weronr | BOERS | PER cENT
WEIGHT
days grams mm. grams grams
Wi 30 26 99 1.338 0.052 3.89
Yenils Sees ae teeta 30 36 104 1.254 0.052 4.18
Wire teet ce cuatiey csthowsnte chs 30 Al 109 1.472 0.056 3.83
ate ies: St es ops ty | bt 30 41 112 1.444 0.050 3.46
Tpit os Ste IO A a A 30 46 12 1.452 0.056 3.87
Average males....... 38 109 1.392 0.053 3.84
Females
Vie ee eee a inate ae ah Ree 30 29 96 al 0.043 3.57
BCs OA ee 30 24 96 1.280 0.045 3.52
RP occs aiclosnhans 30 28 101 1.338 0.034 2.51
Wiser oro eka ees 30 34 107 12335 0.043 See
Wish ae Fe one eels 2 30 43 114 1.432 0.048 3.34
Average females...... 31 103 1.319 0.043 3.27)

Average males and
femalesteeeenes ...- 35 106 1.356 0.048 3.54

228 CAROLINE M. HOLT

TABLE 19. SERIES E
Test animals
Albino rats kept in revolving cages for one hundred and three days after weaning

Males :
OLFAC-

OLFAC- TORY AVERAGE

ANIMALS acu | weionr | canara | waren | uum |rmsomne| amas

WEIGHT BRAIN PER DAY

WEIGHT
days ‘ grams mm. grams grams
Xe 134 233 193 1.927 | 0.079 | 4.12 4.6
Vit asec cn aR eo 135 188 195 1.693 | 0.070) 4.16 5.4
ET RN penta 135 226 198 1.849 | 0.080) 4.30 5.6
Y7 135 237 205 1.875 | 0.085 | 4.54 8.9
Average males....... 221 198 1.836 | 0.079 | 4.28 6.1
Females
XEN al RPI 10s testo oeeve 134 151 177 1.661 |unequal 4.5
BY cot ene Pee ere is sccke pee 135 157 181 1.655 | 0.081 | 4.80 aD
Lge tee oto ME 135 162 180 1.796 | 0.078 | 4.32 4.4
WG 135 169 191 1.693 | 0.079 | 4.64 6.8
Averages females.... 162 184 1.715 | 0,079) 4-60 5.8
Average males and

females. seas ce- 196 192 1.784 | 0.078 | 4.41 5.95

As we see, the brains of the initial controls for the test animals
(X, Y, Z) averaged but 91 per cent of the weight of the initial
controls for the final controls (L, N, O, T, U, V); the olfactory
bulbs but 89 per cent. Since it has been found that brain and
olfactory bulb weight are pretty uniform for any given litter,
and that when we find light or heavy brains or bulbs in the initial
controls, we are fairly sure of finding the same relative develop-
ment in the adult animals of the same litters, it seems fair to
assume that normal adult individuals of litters X, Y, Z, would
have had relatively lighter brains and bulbs than were found
in adults of litters L, N, O, T, U, and V._ If this assumed relation
were true, then the results given in tables 19 and 20 doubtless
would fall into line with those of previous experiments in which
exercised rats showed an increase in brain weight over the

OLFACTORY BULBS OF THE ALBINO RAT 229

TABLE 20. SERIES E

Final control animals!

Males
OLFACTORY

OLFACTORY BULBS:
ANIMALS sce | werenr | uenorm | wercnr | 2UEBS | PeR cer
WEIGHT

days grams mm. grams grams
Ming eeeeperse le cc eae EN 8 160 223 202 1.890 0.085 4.51
Wie aici ara tise: 146 228 202 1.987 0.081 4.06
1 ee eee ee es 121 203 205 1.825 0.072 3.93
Dia Sache arte Hetceeela 157 274 218 2.021 0.077 3.81
Average males....... 232 206 1.931 0.079 4.08

Females
lige noticed Sa ae 93 1 172 1.559 0.063 4.03
Rirtrrticra conic e ty: 124 144 Wee 1.672 0.067 4.03
Meee cs SOM Saarinen 213 183 187 1.801 0.082 4.56
Oe ae eee 115 175 188 1.792 0.083 4.62
Average females...... 157 181 1.706 |. 0.074 4.32
Average males and
femalesi.ic: 3.) es 195 194 1.818 0.076 4.20
Test
Series A see 99% 98% 103%
Control °

1 Data from stock Albinos used for controls in Defective Feeding Series A
and again used for comparison here, since the original controls died early in the
experiment. ~

controls, and would indicate an even greater gain in bulb weight
for the test animals than is indicated in the tables.

In the same way, we may compare the initial controls for the
mated test animals and those for Series A used for a standard
(tables 21 and 22). We find the initial relations practically
the same as for the group just discusesd.

In the final results (tables 21 and 22) we see that although
the test rats were older, with bodies 6 per cent longer, the brains
were actually 5 per cent lighter in weight. Here again, examina-
tion of the initial controls suggests that in all probability there
was not an actual loss of brain weight in the exercised animals.

230 CAROLINE M. HOLT

TABLE 21. SERIES E
Test animals

Albino rats kept in revolving cage for one hundred and three days after
weaning. At end of that time mated and allowed to rear 2-3 litters. Age, when
killed, about eight months.

Males
OLFAC-
OLFAC- TORY AVERAGE
soos Roe ea, |e Rees eS eee
ys WEIGHT BRAIN PER DAY
WEIGHT
days grams mm. grams grams
Dine scrva anne cto Be 238 299 P| 2.018 | 0.092 | 4.54 4.6
Wats dnc h tere se 238 311 228 1.842 | 0.094) 5.10 6.7
Average males....... 305 PAS) 1.930 | 0.093 | 4.81 5.7
Females
7 cs Oa ences A et 238 216 202 1.777 | 0.080 | 4.58 4.3
Ye 238 156 203 1.654 | 0.080} 4.81 a0
Average females..... 186 203 1.716 | 0.080 | 4.66 Od
Average males and
femaless-eeec ack 246 214 1.823 | 0.086} 4.74 on

TABLE 22. SERIES E
Control animals
Stock albinos used for control in defective feeding experiment, Series A.
The four oldest of this set chosen for present tests since original controls died
early in the experiment.

OLFACTORY
OLFACTORY | BULBS PER
ayant sce | werour | uexora | werenr | BULBS | CENT oF
WEIGHT
| Or tr) eee 146 228 202 1.987 0.081 4.06
| DS 2 db aie 157 274 218 2.021 0.077 3.81
yses 24: oe eee 160 223 202 1.890 0.085 4.51
fb ee Pm Pais, 9 213 183 187 1.801 0.082 4.56
AV CTAZC....0 cae Papal 202 1925 0.081 4.23
ehat Test :
Series A 106% 95% 106%

Control |"

OLFACTORY

BULBS OF

TABLE 23

INITIAL CONTROLS FOR TEST ANIMALS

THE ALBINO RAT

INITIAL CONTROLS FOR CONTROL ANIMALS.
(DEFECTIVE FEEDING EXPERIMENT)

Average

Average
Average Per cent Average Per cent
Litters brain BlRaEry of brain Litters brain pliantory: of brain
weight weight | weight weight weight weight
grams grams grams grams
DN A AC e se 1.3805 | 0.047 3.60) |L,.N; O}) 1.431 0.058 3.69
405 Wh, We
Test
———............ ss 91% 89%
Control 2 g
TABLE 24
INITIAL CONTROLS FOR CONTROL ANIMALS
HSER (CONIA O} ES IAC alsin! Lengel cues, (DEFECTIVE FEEDING EXPERIMENT)
Average ay oto Per cent Average pvereee Per cent
Litters brain cenibe of brain | Litters brain Opelika Y! of brain
weight weight weight weight woicht weight
grams grams grams grams
XGA) Vereen asa eek 1.340 0.051 Bie eb, WU 1.458 0.055 3.76
and T
Test
———_............... 92 92
Control 7 7

But, be this as it may, we find the bulbs of these test animals
actually 6 per cent heavier than those of the controls, the bulbs
making 4.74 per cent of the total brain weight, while those of
Series A controls were only 4.23 per cent of the total weight of the
brain.

Since we have no true control series for comparison, we can
not, of course, draw conclusions as to the absolute gain in brain
weight after exercise. But of the gain in olfactory bulb weight
in exercised animals, there seems to be no doubt.

When we turn to table 25 and note that the average percent-
age weight for the bulbs in case of 29 normal rats (59 to 88 days
old) is 4 per cent, while a study of table 13 shows there was no
rat there recorded (save one sick one) in which the per cent fell
below 4.21 per cent, while the average was 4.51 per cent, we
must be convinced, I believe, of the reality of the effect of exercise.
For the older rats, likewise, when we compare tables 19 and 25,

HOLT

CAROLINE M.

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OLFACTORY BULBS OF THE ALBINO RAT 233

we see that the average for 12 controls (90 to 160 days old) was
4.26 per cent while only two test animals fell as low as this ,
(one of these was of abnormally light body and brain), and the
averages were 4.41 per cent and 4.74 per cent for four and one-
half months and eight months respectively.

5. Summary

1. The results of the present experiments agree with those of
previous investigators in that they show no marked effect of
exercise either upon body length or body weight in the albino rat.

2. The female albino becomes very active earlier than does
the male but the activity of the male later increases to such an
extent that the total activity for the two sexes for long periods
is probably about equal.

3. These experiments suggest that there is an increase in
fertility correlated with increase in the size of the reproductive
organs.

4. The brain weight is slightly increased by exercise.

5. The weight of the olfactory bulbs of albino rats exercised
in revolving-cages for periods of from thirty to one hundred days,
is considerably increased. The bulbs of such rats form from
4.41 to 4.74 per cent of the total brain weight as compared with
4.20 to 4.32 per cent in rats reared under normal colony conditions.
These bulbs show an increase of 5 to 11 per cent over and above
the increase in weight manifested by the entire brain.

1V. CONCLUSIONS

From the preceding observations we may conclude that we are
able to modify the olfactory bulbs of the rat by changing the
conditions under which it lives and to modify them to a con-
siderably greater degree than we can change the rest of the brain.
In eases of stunting, the bulbs tend to overcome the effect, to
a certain extent, as time goes on. With exercise the effect seems
to increase with age. Yet the bulbs respond more markedly
to the stunting effect of defective feeding or sickness than to
the stimulating effect of exercise.

A histological study of these modified bulbs will be presented
in the second part of this paper.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

234 CAROLINE M. HOLT

V. LITERATURE CITED

Donatpson, H. H. 1911 On the influence of exercise on the weight of the
central nervous system of the albino rat. Jour. Comp. Neur., vol.
21, pp 129-137.
1911a The effect of underfeeding on the percentage of water, on
the ether-alcohol extractives, and on medullation in the central nerv-
ous system of the albino rat. Jour. Comp. Neur., vol. 21, pp. 189-145.
1915 The Rat. Memoirs of the Wistar Institute of Anatomy and
Biology, No. 6.

Harat, 8S. 1904 The effect of partial starvation on the brain of the white rat.
Am. Jour. Physiol., vol. 12, pp. 116-127.
1907 Effect of partial starvation followed by a return to normal
diet, on the growth of the body and central nervous system of albino
rats. Am. Jour. Physiol., vol. 18, pp. 309-320.
1908 Preliminary note on the size and condition of the central nerv-
ous system in albino rats experimentally stunted. Jour. Comp. Neur.,
vol. 18, pp. 151-155.
1915 On the influence of exercise on the growth of organs in the
albino rat. Anat. Rec., vol. 9, no. 8, pp. 647-665.

Jackson, C. M. 1915 Changes in young albino rats held at constant body
weight by underfeeding for various periods. Anat. Rec., vol. 9, pp.
91-92.
1915 a Changes in the relative weights of the various parts, systems,
and organs of young albino rats held at constant body weight by
underfeeding for various periods. Jour. Exp. Zool., vol. 19, pp. 99-
1565

Osporne, T. B. anp Menpet, L. B. 1911 Feeding experiments with isolated
food substances, Part 2.

SLonakeER, J. R. 1907 The normal activity of the white rat at different ages.
Jour. Comp. Neur., vol. 17, pp. 342-359.
1912 The normal activity of the albino rat from birth to natural
death, the rate of growth, and the duration of life. Jour. Animal
Behavior, vol. 2, pp. 20-42.

PART II. ON THE NUMBER OF NERVE CELLS IN
LARGE AND SMALL OLFACTORY BULBS

CONTENTS
1. Introduction. Preliminary experiments on the effect of certain fixa-

DIVERSION Ger Tat eOEa Us Sao. kos! soe) eae ee ee Sea ese ee ke 235
lieeihe problem, Of size diterences2y5 2: H..¢4). Lag eet eis th oak 236
iieehechniqte-and methods or study... 2j5..5. bck eter eee ela bate 237
IPE Te pArablONyOlSCCMOMS 2:2... asa ae Te ear es Ate cata 237

Dee Net hodstormscudiynn cue eee he ae ee tees aes cre ee entree 238
iiVerGeneral differences in sizes) ts 7.4. 425.02 ee ih See Ath Ate Ry Sa 238
Vie Comparison: of .cells:of ray layer n...cosee eee ee gees es 240
1. Size and number of small cells in molecular layer................ 240

Je Size and miimper or misral cells. scat te oo se ee ee ce oe t,o 243

3. Study of the small cells in the gray layer............ Pod Cs aE te 248

Nile (Conclusions soe a ed. |. UR Re: WR DR ott Sete dees 250
Welle mlihervuire Cubed ee cat et ee hoe 5 5,3 \< 54 oN a os ae oe oad 251

I. INTRODUCTION

The foregoing studies have shown that it is possible to change
the relative weight of the olfactory bulbs in the albino rat
(Holt, ’17). This relative weight is decreased by a defective diet
and increased by exercise. Such being the case, it seemed very
desirable to make a histological comparison of the bulbs which
had been stunted by a defective diet or enlarged by exercise, with
those of rats reared under normal colony conditions. For this
purpose it was of course essential to find a method of fixation and
treatment which would give uniform results. Fixation in Ohl-
macher’s solution as recommended by King (’10) for the study of
cortex cells, gave very satisfactory results, but her statement that
“various individuals react differently although subjected to the
same course of treatment,” and her tables (loc. cit., p. 231)
showing a variation in shrinkage ranging from 2 to 18 per cent
in brains so fixed, suggested that it would be best to examine
this method a little more in detail. Unless uniform results
could be obtained by it, this method would, of course, be un-

235

236 CAROLINE M. HOLT

suited to comparative study of the size of bulb elements. Ac-
cordingly, the method was further tested, and at the same time
an examination was made of the effect of Miiller’s fluid and of
Orth’s Formol-Miiller solution upon the various parts of the
brain.

A long series of experiments demonstrated quite conclusively
the following points which have an important pean ae upon the
present investigation.

1. Of the three fixing fluids tested, Ohlmacher’s solution causes
the least change in weight in brain tissue.

2. Orth’s solution (cold) causes a slight increase in weight.

3. Miiller’s solution causes a very considerable increase in
the weight of brain tissue as has already been noted (Donaldson,
’94).

4. Olfactory bulbs, fixed in Ohlmacher’s solution, reach a
state of equilibrium at the end of twenty-four hours; fixed in
Formol-Miiller, they reach this state at about the end of one
week; fixed in Miiller’s solution alone, changes continue from
six weeks to two months.

5. There seems to be no appreciable individual variation in
the reactions of albino rat brains of like age to Ohlmacher’s
solution, to Miiller’s fluid, or to the Formol-Miiller solution.
The results obtained by Dr. King are due apparently to the
fact that the brains which were weighed in her experiments had
been fixed for varying short lengths of time and the initial changes
in weight were so rapid that there appeared to be a considerable
difference in the way the various brains reacted to the fixative,
when in reality, had all the brains been fixed for exactly the same
length of time, no such large disagreement would have been
found.

ll. THE PROBLEM OF SIZE DIFFERENCES

Although under normal conditions, there is a good deal of
variation in the size of the olfactory bulb of the albino rat,
we have found that it is possible, experimentally, to increase
this range of variation to a very considerable degree. The
question next arises as to the structural cause of the difference

OLFACTORY BULBS OF THE ALBINO RAT 237

in size. Is it one of size of elements or of their number? Have
we more cells and fibers in the heavier bulb or are the cells and
fibers merely of a larger size? The present paper deals only
with the question of cells. The fibers have yet to be examined.

Ill. TECHNIQUE AND METHODS OF STUDY

Since experiments on the effect upon the rat brain, of Ohl-
macher’s, Miiller’s, and the Formol-Miiller solutions have demon-
strated that for brains of like ages there is a definite, practically
unvaried, swelling or shrinking reaction for any given fluid,
the brains of the rats used in the defective diet and exercise
experiments were fixed in these several solutions for histological
comparison. For the present cell study the method adopted was
that recommended by King (’10) for study of the cortex, namely
fixation in Ohlmacher’s solution for twenty-four hours, followed
by one hour in 85 per cent alcohol, three to four days in iodized
70 per cent alcohol, double embedding in celloidin and paraffin,
and staining in carbol-thionin and eosin. However, after the
first trials, this method was varied in the matter of embedding.
For such small objects as the olfactory bulbs, paraffin proved
more satisfactory when used alone. Sections were cut 8yu
thick and mounted serially. A rather deep thionin stain gave
the best results for cell enumeration.

1. Preparation of sections

At first, some bulbs were cut sagittally and the largest sections
compared. In the study of these sections the number of cells
in the gray layer of the different bulbs was found to be so nearly
identical that it was decided to attempt a thorough study of cell
number.

In dissecting a rat brain into its parts, the bulbs are cut from
the brain in such a way as to leave an appreciable portion of the
bulb attached to the cerebrum. The method followed was to
place the brain, ventral side down, on a flat surface and with a
knife held in a plane perpendicular to the table, to sever the bulb
at the point where it disappears beneath the cerebrum (plate 1).

238 CAROLINE M. HOLT

This is the part of the bulb which is weighed, and since all bulbs
are removed in the same way, it has been assumed that we have
corresponding portions for comparison. Because the recorded
weights represented only this portion of the bulbs, it seemed
advisable at first to compare the cell elements of these parts only.
Accordingly cross sections of the bulbs were made but unfortu-
nately, as appeared later,most of the test series were not complete
for the portion of the gray substance beneath the hemispheres.
The method of meeting this difficulty will be described later.

2. Methods of study

The study of sections was made largely with the aid of the
Edinger projection apparatus. Cell counts were made by pro-
jecting the sections onto white wrapping paper, outlining the
area, and punching the image of each cell nucleus with a tally-
ing register fitted with a sharp prong in place of the usual blunt
register arm (Hardesty ’99). The hole punched by this prong
insured against counting the same cell twice. It also left a
permanent record of any particular region which could be re-
examined later. In some cases the count for each section was.
recorded; in others, the whole number of sections to be counted
were registered consecutively and no record made until the end.
Occasionally a section was recounted—to serve as a check on the
work.

IV. GENERAL DIFFERENCES IN SIZE

Most of the comparisons of size and the determinations of
cell number have been made on bulbs stunted by a defective diet,
and on their respective controls. Only two bulbs of the exercise
series have yet been examined.

The general differences in size between young and mature,
stunted and normal olfactory bulbs are very well illustrated
by the sections shown in Plates 1, 2, and 3. Figure 1 of plate 1
is a camera drawing of a median sagittal section through the
bulb of a rat stunted by feeding for thirty-one days on a corn
diet. The body length was 138 mm.; brain weight, 1.547 grams
and the weight of the corresponding bulb which was removed and

OLFACTORY BULBS OF THE ALBINO RAT 239

weighed, was 0.020 gram—the approximate weight then of the bulb
shown in the drawing. Figure 2, plate 1, is a median sagittal sec-
tion through the control bulb. The rat from which this was taken
was 166 mm. long, with brain weight of 1.698 grams, and the
weight of the corresponding bulb was 0.032 gram. Like the bulbs
of very young rats, the gray layer of the stunted bulbs extends
somewhat further, in proportion, beneath the cerebrum than in
the case of normal older individuals.

When the stunted bulb is compared with its control, there
appears to be a rather uniform size difference involving all parts
of the bulb. It is hard to compare the outer fiber layers owing
to the difficulty in removing the bulbs perfectly from the skull.
The anterior end of the fiber layer is very likely to be entirely
torn away and sometimes the ventral side also suffers. However,
it is plain that the glomeruli of the larger bulb are much larger
and more open; the granular cells are not so closely packed to-
gether; and the gray layer is usually broader in the larger bulb
and the inner granular area considerably more extensive. These
differences between the peripheral portions are illustrated by
the more highly magnified mid-dorsal areas S and S of figures 1
and 2, shown in figures 3 and 4 of plate 2.

Plate 3 shows three cross sections; through Q;, a thirty-day
control bulb (fig. 5); M,, a sixty-two-day stunted bulb (fig. 6);
and M;, the sixty-two-day normal control (fig. 7), for M,. These
sections were made through the region where the bulbs are
usually cut from the brain. The figures illustrate another
typical difference. The normal bulb (figs. 5 and 7), as it grows,
elongates more rapidly than it increases in thickness and it
tends to grow faster dorso-ventrally rather than laterally. In
these figures, the outer fiber layer is probably complete at the
sides. Ventrally it has doubtless been torn away to some extent
in all three bulbs. The difference in size of the glomeruli is
well shown here, but while there is a greater area of gray matter
in figure 7 than in the other two, the gray layer seems narrower
than in M, (fig. 6). The companion bulb of Q,;, (fig. 5) weighed
0.024 gram, that of Mg, (fig. 6), 0.025 gram while M; weighed
0.037 gram (fig. 7). The portion of Q; anterior to the section

240 CAROLINE M. HOLT

illustrated, was about 1350. long, while M, had 1500, anterior
to the section, and M;, 2000u. The differences in size are
confined to no one region but are distributed somewhat pro-
portionally through the different layers.

V. COMPARISON OF CELLS OF GRAY LAYER
1. Size and number of small cells in molecular layer

It has been the general impression that, within certain limits,
the size and weight of the brain are indices to its functional
capacity. In the phylogenetic series, of course, it is, with one
or two exceptions, true that increase in intelligence is accom-
panied by increase in the relative size of the brain. So within
any given species of mammals, it has been assumed that the
more efficient brain is the larger and heavier.

The question as to whether, within such a group, increase
in size of the brain means an increase in the number of nerve
‘elements or in the size of the elements themselves, becomes an
“important one. For an increase in the number of elements
should give increased functional possibilities. ‘So, if we find in
comparing large and small brains or divisions of brains from
closely related animals, that the larger structure contains a
greater number of cells and fibers, then we have reason to expect
from the larger and more complex structure the greater degree
of efficiency.

If, on the other hand, the number of elements is found to be
uniform for the part under consideration, then we must conclude
that the large and the small brains have potentially the same
ability to function, save as their efficiency may be affected by
the size or degree of development of the individual elements.

The small cells of the molecular layer (mo, fig. 2) show more
cytoplasm; or perhaps we may say that it is possible to distin-
guish more cells with cytoplasm in the molecular layer of large
bulbs than of small ones. For example, the section of F; shown
in figure 1 shows 68 cells between mitral layer and glomeruli,
in which cytoplasm may be distinguished, while the control,
F;, shows 158 such cells. Corresponding sections through M,,

OLFACTORY BULBS OF THE ALBINO RAT 241

a thirty-day control, and Cs, a sixty-day underfed bulb, show
108 and 103 cells with cytoplasm.

Although a difference in cell size appeared, there seemed to be
little difference in numbers of cell elements in the gray layer.
Although it is not always possible to distinguish between the
nuclei of very small cells and possible cross sections of fibers
under the conditions used for counting—yet the error due to
this difficulty is probably negligible. A preliminary count was
made of all elements, having the appearance of nuclei in the
largest sections of the bulbs F,, C;, F;, Fs, and M:, with the
following results.

TABLE 1

INITIAL CONTROL TEST FINAL CONTROL

Bulb | Num- Bulb | Num- Bulb | Num-
Bulb Age weight |ber cells} Bulb Age weight |ber cells Bulb Age weight |ber cells

days grams days grams days grams
Mie ees oO) | "0029) 2172)" By 62 | 0.020} 2569] F; 61 0.032) 2569
C; 59 | 0.021} 2604] Fe 61 0.033} 2693

These counts for the test and final control bulbs suggested so
strongly that the number of cells is the same for bulbs of different
sizes that attention was turned entirely to the investigation of
this point. At first longitudinal sections were used, but these
were soon abandoned for two reasons. First, it seemed desirable
to be able to count the cells of just that portion of the bulbs
corresponding to the part weighed; and second, the longitudinal
sections presented so many irregularities that 1t was necessary
to count many more sections to approximate the true average
than in the case of the cross sections. Counts were made of all
elements in the gray layer outside the mitral layer between the
tip of the bulb and the point at the proximal end where the gray
layer is first interrupted on the dorsal aspect of the bulb (see
figs. 5, 6, 7). These counts consumed a vast amount of time
and when completed seemed to disagree with the observations
already made upon the longitudinal sections (table 2).

A first glance at the table would indicate that the small bulb
has fewer cells and would suggest that this difference in cell

242 CAROLINE M. HOLT

TABLE 2
BULB AGE HISTORY BRAIN WEIGHT |WEIGHT 1 BULB Ae eae
days grams grams
Dla Abe 30 | Control 1.338 0.017 636,656
NERS: 62 | Underfed 31 days 1.461 0.025 662,982
Gi... 62 | Underfed 31 days 1.543 0.027 601,982
Gerace 61 | Normal control 1.630 0.031 675,305
Mcgee 62 | Normal control Peale 0.037 716,582

X,5....| 184 | Revolving cage 104 days 1.927 0.040 789,680

number is one of the factors in bulb size. But corresponding
sagittal sections had given fairly close agreement in numbers
and the study of sagittal sections made it more and more evident
that these counts of cross sections could be taken only to compare
the parts commonly considered the bulb and not for an enumera-
tion of the cells in the whole bulb. The difference in shape in
the large and small bulbs made it apparent that a true count
must be made either from sagittal sections or from cross sections
cut through the entire length of the gray matter covering the
bulb. Comparison of such sections as figures 1 and 2 made
it clear that if we had, in reality, a constant number of cells in
the gray layer, the numbers in the regions here designated as
the ‘bulb’ could scarcely be expected to show any closer agreement
than we find in this table, and would probably have the relations
there given. For the larger and better developed the bulb,
the greater the proportion. of it lying anterior to the cerebrum,
while the young or the stunted bulb runs somewhat further back
beneath the hemisphere and so some of the cells escaped enumera-
tion. For example, M;, a bulb of 0.037 gram, has 271 sections
containing mitral cells in the portion of the bulb beneath the
cerebrum. Mg, the test bulb of this litter, which weighed but
0.025 gram had 336 sections in this region. Taking these facts
into consideration, the table in question pointed to a uniformity
rather than variation in numbers corresponding to size. Later
we shall see how, in the light of the study of the mitral layer,
a part of this table can be shown to closely conform to this
supposition that the number of cells in the entire gray layer is
approximately constant for olfactory bulbs of different sizes.

OLFACTORY BULBS OF THE ALBINO RAT 243

2. Size and number of mitral cells

The cells of the mitral layer show a good deal of variation in
size and shape and there is much difference in these respects in
different regions of the same bulb. This makes the comparison
of the size of the mitral cells in large and small bulbs rather
difficult.

But if all the mitral cells of a section from a small bulb are
drawn with a high magnification by means of camera lucida or
projection apparatus and those cells arranged side by side with
a series from a corresponding section of a large bulb, drawn to
the same scale, it is possible to make a general comparison. In
this way the mitral cells have been compared, and there is no
doubt I think that the mitral cells of large bulbs are larger and
better developed than those of small bulbs.

Details of technique and examination. It was sometimes quite
difficult to determine whether a cell should be counted or not.
For instance, when counting mitral cells it was hard to know
at times whether a cell was a mitral cell or a brush cell, as many
cells occur in the mitral layer which are exactly like those large
cells occurring in the molecular layer but which lack the typical
mitral form. On the other hand, typical mitral cells occur not
infrequently out in the molecular layer or even among the
granules on the inner edge of the glomerular layer. For this
reason and in order that there might not be any unconscious
influence in deciding whether cells should be counted, an at-
tempt was made to vary the order of procedure for each successive
count.

A rather complete count was made of the mitral cells of four-
teen bulbs and of the small cells of the gray layer in four bulbs.
Eight of these were cut longitudinally and six cut transversely.

The first series counted were those of X,, Initial control,
Series E and X,, Test, Revolving Cage Series. In both series
every other section was counted for the region anterior to the
cerebrum—corresponding to the portions of these same bulbs
in which the small cells of the gray layer had been counted. The
result was 64,470 cells for X,, a 0.017 gram. thirty-day control

244 CAROLINE M. HOLT

bulb. The number for X.s, whose weight was 0.040 gram,
was 73,950. ‘To see whether there were any virtue in making
so thorough a count of cross sections, the total number was
computed from a recount of every 10th section, excepting at
the most anterior end where every cell was counted in every
section, until the sections showed a single layer of mitral cells.
By this method the number obtained for X, was 64,775 cells,
making a difference of only 0.4 per cent. For X, the count was
73,324, which was 0.8 per cent smaller than the more exact
count obtained by counting half the sections. These differences
were so small as to make the more exhaustive count seem un-
necessary. X, gave an almost complete series through the
entire gray layer so the count was completed, giving for the entire
bulb 80,114 cells. The count of the mitral cells in X; could not
be completed as the series had been cut, unfortunately, with the
idea of comparing only the parts of the bulbs whose weights we
knew, and which, therefore, extended back but a short distance
under the cerebrum. ‘The cells of these few sections were, how-
ever, counted, giving a total of 71,914.

The number of mitral cells in Gs was computed from absolute
counts of anterior and posterior ends of the series and by count-
ing every tenth section through the rest of the series. M,, M;
and @; ran so evenly that here in the middle portion of each
series, only every twentieth section was counted; on either side
of this portion, every tenth section, and all cells of all sections
at either end.

With the sagittal sections, the task was more difficult and the
results, I believe, less reliable for this reason: toward the sides
of the bulbs, especially the median side, the sagittal series may
give tangential sections of the mitral layer so that a single section
may yield a count of 1500 cells whereas a section two or three
removed on either side might have but 300 or so mitral cells.
It can be easily seen that if the section to be counted, happened
to fall in such a region, or entirely skipped such a region, the
count would be considerably modified. Some of the bulbs
gave no trouble of this kind while others were hard to count for
this reason. G,; was an interesting example of the way this

OLFACTORY BULBS OF THE ALBINO RAT 245

may work out. A count was first made of all cells at either end
of the series and those in every tenth section through the middle
portion. The result when computed was 95,993 mitral cells.
Then the middle section of every ten was counted with a total
result of 83,974 cells. Two other series were attempted but
abandoned as the bulbs were so irregular that an accurate
count would have required the enumeration of the cells of at
least every alternate section. The other bulbs, except Cs
for which every fifth section was counted, were fairly regular
so that the mitral layer offered no such complications. For
these, the method of counting all cells at either end of the series
and those of every tenth section through the median portion
was followed. The sequence of counts was varied with each
bulb, and the records kept in various ways and not infrequent
recounts made. The recounts were surprisingly close to the
original, for, as has been stated, it is not always easy to decide
whether or not a cell should be counted, and in focusing as one
counts, a granule lying below or above a portion of a mitral
cell sometimes looks very like a nucleus, but the error due to
this cause is probably too small to be considered.

Details of counts of the different bulbs, arranged in the order fol-
lowed in table 3.

Bulb X,, thirty day, initial control. Cross sections. Mitral cells
counted in every section of anterior end back to the first section in
which the mitral cells appeared in a single layer. From this point,
counts were made for every tenth section back to the cerebrum. By
computation, the total number of mitral cells was 64,775. By a
recount in which the mitral cells of every other section were enumer-
ated the computed number was 64,470 making a difference of only
0.4 per cent in the two counts. The series of sections for the region
beneath the cerebrum was incomplete, the posterior portion not having
been preserved. A count was made, however, of the sections which
were present. This number added to the number already counted
by the second method, brought the total up to 71,914 cells.

Bulb E,, Test, defective diet series. Sixty-two days. Sagittal
sections. Mitral cells counted in all sections at either end of the series
and for every tenth section between.

Bulb C; Test, defective diet series. Fifty-nine days. Sagittal
sections. Counts made as in F;.

Bulb Q;, thirty day, initial control. Defective diet series. Cross
sections. Mitral cells counted in allsections at both ends of the series.

246 CAROLINE M. HOLT

TABLE 3
Giving number of mitral cells*in one olfactory bulb of the albino rat

Arranged according to bulb weight

RAT AGE BODY BRAIN | WEIGHT! NO. MITRAL

LENGTH|WEIGHT| 1 BULB CELLS LEER NESS
days mm. grams | grams

X; Control.....) 30] 101 | 1.338] 0.017} 71,914 | Very incomplete. Up to
point of union with cere-
bellum 64,470 cells, cross
section

By Mest. cee 62 | 138 | 1.547) 0.020) 71,527 | Sagittal section

Gerlest sete 59 | 142 | 1.482) 0.022) 79,165 | Sagittal section

Q; Control 30 97 | 1.316] 0.024) 82,192 | Probably about 300 more
cells. Cross section

C@emlestveca. oe 59 | 150 | 1.578) 0.024) 70,625 | Sagittal section

M, Test... 62 | 109 | 1.461} 0.025) 76,611 | Cross section

Ger Testes). 759 61 | 136 | 1.456) 0.027) 83,974 | Sagittal section

Op Restidn aa: 115 | 121 | 1.556} 0.029} 71,663 | Sagittal section

Gs Control..... 61 | 166 | 1.630) 0.031] 81,638 | Cross section

BeiControle ss. 61 166 | 1.698} 0.032) 71,468 | Sagittal section

C; Control..... 62 | 174 | 1.709) 0.036) 79,839 | Sagittal section

M; Control.... 62 | 169 | 1.711] 0.037] 76,596 | Cross section

X- R. C. Test..! 134] 193 | 1.927] 0.040} 80,114 | Up to point of union with

cerebrum 73,950 cells,
cross section
O; Control.....; 115 | 175 | 1.792] 0.041] 72,333 | Sagittal section(X, omitted

from average)

Average..... 76,749

Standard deviation ¢ = 4564
Probable error of the mean +855

Through the middle region only every twentieth section was counted
as the sections were extremely uniform. Through the two regions
between this middle portion and the ends in which all cells were counted
the mitral cells for every tenth section were counted.

Bulb Cs, test, defective diet series. Fifty-nine days. Sagittal
sections. Mitral cells counted for all sections at both ends of the
series and every fifth section of the rest of the series.

Bulb Mg, test, defective diet series. Sixty-two days. Cross sections.
Mitral cells were counted as in Q;. Count was made also of all cell
elements in every alternate section in the gray layer back to the anterior
end of the cerebrum. Computation was made for entire series.

Bulb G3, test, defective diet series. Sixty-one days. Sagittal
sections. Mitral cells were counted for all sections at ends of series
and for every tenth between. The computed result was 95,993. The
middle sections between every tenth were then counted, giving a

OLFACTORY BULBS OF THE ALBINO RAT 2AT

TABLE 4
Giving number of mitral cells in one olfactory bulb of the albino rat
Arranged by litters

PERCENTAGE

WEIGHT NUMBER
AGE RAT lee | GoRDAD, GEINC DIFFERENCE OF REMARKS
TEST
days grams

62 | Fi Test 0.020) 71,527 (S.) |+ 0.08

62 | F; Control | 0.032] 71,468 (S.)

115 '| O; Test 0.029] 71,663 (S.) |— 0.9

115 | O, Control | 0.041] 72,333 (S.)

62 | M, Test 0.025] 76,611 (C.) |+ 0.2

62 | M; Control] 0.037| 76,596 (C.)

59 | Cs Test 0.022| 79,165 (S.) |— 0.9 ee

59 | C; Test 0.024) 70,625 (S.) |—12.0

62 | C7 Control | 0.036} 79,839 (S.)

30 | X, 30d. T.| 0.017] 71,914 (C.) Very incomplete

134 | X.-R.C.T. | 0.040] 80,114 (C.) Slightly incomplete (not
in average)

61 | G3; Test 0.027| 83,974 (S.) |+ 2.9

61 | Gs Control | 0.031] 81,638 (C.)

30 | Qs 30d. C. 0.024] 82,192 (C.) Probably 300 more cells

Average per cent difference of test....]|— 1.8

(S.) = Sagittal section.
-(C.) = Cross section.

count for every fifth section of this region. The computation then
gave a total of 83,974. Two other bulbs of this litter were also cut
in sagittal sections and an attempt was made to count the mitral cells
but the bulbs were so irregular that it would have been necessary to
count practically every section, so these counts were abandoned.

Bulb O;, Test, defective diet series. One hundred and fifteen days.
Sagittal section. Counts made as in FE).

Bulb Gs, control, defective diet series. Sixty-one days. Cross
section. Counts made as in &}.

Bulb F;, control, defective diet series. Sixty-one days. Cross
section. Counts made as in FE}.

Bulb C;, control, defective diet series. Sixty-two days. Sagittal
sections. Counts as in F}.

Bulb Ms, control, defective diet series. Sixty-two days. Cross
sections. Counts as in M, and computation of all cell elements in
gray layer made for entire series.

Bulb X., test, revolving-cage series. One hundred and thirty-four
days. Cross sections. Mitral cells counted by both methods de-
scribed for X;. Also all cell elements of the gray layer computed for
the entire series as in My, and Ms.

Bulb O,, control, defective diet series. One hundred and fifteen
days. Sagittal section. Mitral cells counted as in Ej.

248 CAROLINE M. HOLT

Table 3 gives the results of the counts of the mitral cells of
fourteen olfactory bulbs, arranged according to bulb weight.
It is obvious that there is no correlation between bulb size and
the number of the mitral cells or between age—within the limits
taken—and number of cells. The numbers range from 70,625
to 83,974 with an average of 76,750 cells for 13 bulbs, X, being
omitted from the average. I am inclined to think 83,974 cells
is too high a count for G; and that still closer enumeration might
yield a lower number. <A recount was made for C; counting
every fifth section, as this was a somewhat irregular bulb and
it was thought that might account for the variation of this bulb
from the rest of the litter. But the recount gave practically
the original number.

Table 4 which is arranged by litters indicates a striking
agreement between the members of the same litter. With the
exception of litter C, in which C; falls 12 per cent below the con-
trol in number of mitral cells, there is extremely little difference
between test and control bulbs of the same litter. So we find
that whether the bulb has been stunted by a defective diet, or
enlarged by exercise, the number of mitral cells is practically
constant for any given litter. The factors which have brought
about a change in size of the olfactory bulbs have failed to affect
the number of mitral cells, at least in the gray layer. The test
and control counts are, with one exception, extremely close.
This is a fresh example of the similarity in structure among
members of the same litter—a relation which is continually
appearing in the study of this animal.

3. Study of the small cells in the gray layer

The sections of bulbs Mu, Ms, Xi, and X, are all series in which
a count was made of the small cells of the gray layer in the ante-
rior portion of the bulb as well as of the mitral cells. Assuming
that the relation between the number of mitral cells in two given
sections of a bulb would be the same as that between the small
cells of the gray layer in these same regions, computation was
made of the total number of small cells in the gray layer of Mag,

OLFACTORY BULBS OF THE ALBINO RAT 249

M;, and Xs. Bulb X, was too incomplete to make such caleula-
tion possible. Let us take M,. The small cells were counted
back to section 4, 1/1. We have the total number of mitral
cells and also the number of mitral cells back to section 4, 1/1.
This gives us the data for computing the total number of small
cells as follows:

MuatralaceliisroteVicntorse ction Axailt/ilmee ce 2). l aeeene ene hens a Ser ue ne 50,729
Mota lemantralecelillistaec sisters Mets Ue tiat ios lake cea eetee PRAY «cure uel do. 76,611
66 per cent of mitral cells in anterior portion.

NumibenusmiealllcellsSitomsectiomed lll se ae ee ee eer 662,982

If this number equals 66 per cent of the total number, then the total number
of small cells in the gray layer would be 1,004,518.
If we treat M; in the same way we have:

IMGnaesll- Cabls tro. SeCunom 3), GPs 8025 ne deee can tossosndencectoaene BURUUO
MO uallenmitraleCellllister teat weet tens sine Misntn on sic Soe SR eNn eon geet oop Sears 76,595
73 per cent of mitral cells in anterior portion.

Niumberssmallizcelliss torsectlomes- 16/12... see see ees ae eee 716,382
Rhenyiotalenumlyerssnialllecelllichesere iss sels ee eee, eee 981,619

According to this computation the test bulb would have two per cent more

cells than the control.
Now if we treat X, in like manner we have the following:

Mitral celiiskionsecinonko sll pane eee ene eee ee 27000)
Mo tallenniGrealecelll Sis settee wae ee kG AS cae a ee Oa Te Hd, 80,114
77 per cent of mitral cells in anterior portion.

Suigllll Calle wo, SECO IY Gee oles cea tle boss he satan seen 789,680
{lever THON aayoramloere our Siooellll Celle occ one eomenebocbumeoncocuce 1,020,258

These total numbers are strikingly close. The number of
bulbs is too small to warrant us in drawing general conclusions
but I think the results certainly point to close agreement in
number even of the small cell elements in the gray layer.

While there is a constant increase in the number of myelinated
fibers correlated with age, as has been demonstrated by Green-
man (713) for the peroneal nerve, Boughton (’06) for the oculo-
motor, Hatai (02) and (’03) for both dorsal and ventral roots
of several spinal nerves, and Dunn (12) for the ventral root of
the second cervical nerve; there is very little evidence of any
true increase in the number of cells in the central nervous system
after the first few days after birth. Allen (’12) found dividing
cells in the cerebellum up to twenty-five days and in the cere-
brum up to twenty days, with a few along the lateral walls of

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 2

250 CAROLINE M. HOLT

the lateral ventricles until the end of the second year. Hatai
observed an increase in number of cells in the spinal ganglia,
corresponding to increase in age but this increase was attributed
in part, at least, to failure to count all the ganglion cells in
very small animals. Ranson (’06) in a study of the second cervi-
eal nerve found no correlation between the number of cells and
the number of myelinated fibers, neither did he find the number
of cells to vary with the age of the rat.

The results of the present investigation of the number of cells
in the olfactory bulb help to confirm the impression that the
number of cells in the central nervous system becomes fixed at
an early age so that after the first three or four weeks at least,
there is no material change in the cell number.

This study also gives us reason to believe that the number
of small and of mitral cells in the gray layer of the olfactory bulb
is very nearly the same for all individuals with especially close
agreement between individuals of the same litter. Itseems fairly
evident that while external conditions may modify to a con-
siderable extent the size of the brain of the albino rat and espe-
cially the size of the olfactory bulbs, the only effect is upon the
relative development of the individual cells. The number of
cells remains the same. The fibers have yet to be examined.

It is important to bear in mind in a determination of this
sort—e.g., the number of mitral cells—that a fixed number,
in the physical sense, is not to be expected, for all organisms are
normally variable in all of their parts, variability being an es-
sential character for living things; so the number which is ob-
tained gives a mean value which we take to be characteristic
for the species under the present conditions, but around which
equally characteristic variations also occur.

V1. CONCLUSIONS

1. For bulbs of different ages and sizes, the regions anterior
to the cerebrum, which are commonly considered the bulbs, are
not strictly homologous, since, in the brains of young or stunted
rats, a larger proportion of the bulb lies beneath the cerebrum
than in the ease of the better developed brains.

OLFACTORY BULBS OF THE ALBINO RAT 251

2. All layers of the olfactory bulb are about equally concerned
in the increase in size or in the arrest of development of the
bulb.

3. The small cells of the molecular layer show a larger amount
of cytoplasm in large bulbs than in small ones.

4. The number of small cells in the molecular layer, appar-
ently, is not correlated either with age of the rat or size of the
bulb. The entire computed number for a small, medium, and
large bulb was found to be approximately 1,000,000 cells + 2
per cent.

5. The mitral cells of small bulbs are smaller, on the average,
than those of large bulbs.

6. Within the limits here taken the number of mitral cells is
not affected by the age or the size of the bulb.

7. There seems to be some variation between litters in the
number of the mitral cells. The average number of mitral
cells for 13 bulbs was 76,750, the lowest number being 70,625,
and the highest 83,974. The standard deviation o is 4564 and
the probable error of the mean + 855.

8. When members of the same litter are compared, bulbs
stunted by a defective diet or enlarged by exercise show practi-
cally the same number of mitral cells as do their controls. The
mean difference is —1.8 per cent for the tests.

9. The olfactory bulb size is correlated with cell size and not
with cell number.

VII. LITERATURE CITED

ALLEN, E. 1912 The cessation of mitosis in the central nervous system of
the albino rat. Jour. Comp. Neur., vol. 22, no. 6, pp. 547-568.

Boucuton, T. H. 1906 The increase in the number and size of the medul-
lated fibers in the oculomotor nerve of the white rat and of the cat
at different ages. Jour. Comp. Neur., vol. 16, pp. 153-165.

Donaupson, H. H. 1894 Preliminary observations on some changes caused
in the nervous tissues by reagents commonly employed to harden
them. Jour. Morph., vol. 9, pp. 123-166.

Dunn, E. H. 1912 The influence of age, sex, weight, and relationship upon the
number of medullated nerve fibers and on the size of the largest fibers
in the ventral root of the second cervical nerve of the albino rat.
Jour. Comp. Neur., vol. 22, pp. 131-157.

252, CAROLINE M. HOLT

GREENMAN, M. J. 1913 Studies on the regeneration of the peroneal nerve of
the albino rat: number and sectional areas of fibers: area relation
of axis to sheath. Jour. Comp. Neur., vol. 23, no. 5, pp. 479-513.

Harpesty, 1. 1899 The number and arrangement of the fibers forming the
spinal nerves of the frog (Rana virescens). Jour. Comp. Neur., vol.
Ono dep: 104.

Harar, 8. 1902 Number and size of the spinal ganglion cells and dorsal root
fibers in the white rat at different ages. Jour. Comp. Neur., vol. 12,
pp. 107-124.
1903. On the increase in the number of medullated nerve fibers in
the ventra’ roots of the spinal nerves of the growing white rat. Jour.
Comp. Neur., vol. 13, pp. 177-183.

Hout, C. M. 1917 Studies on the olfactory bulbs of the albino rat. I. Experi-
ments to determine the effect of a defective diet and of exercise upon
the weight of the olfactory bulbs. Jour. Comp. Neur., vol. 17, pp. 201-
234.

Kine, H. D. 1910 The effects of various fixatives on the brain of the albino
rat, with an account of a method of preparing this material for a
study of the cells in the cortex. Anat. Rec., vol. 4, pp. 214-244.

Ranson, 8S. W. 1906 Retrograde degeneration in the spinal nerves. Jour.
Comp. Neur., vol. 16, pp. 3-31.

ABBREVIATIONS

s, areas in figures 1 and 2 enlarged in mi, mitral layer
figures 3 and 4. g, granular layer
fi, outer fiber layer f, inner fiber layer

gl, glomeruli c, cerebrum

mo, molecular layer
PLATE 1
EXPLANATION OF FIGURES

Median longitudinal section through olfactory bulb of Fy, section 3 1/6.
F;, underfed 31 days. Fina! brain weight, 1.5470 grams, bulb weight, 0.0203 gram.
Defective diet. Magnified 24 diameters.

Median longitudinal seetion through olfactory bulb of F;, section 5 5/4. Fs,
control for Fy. Brain weight, 1.6984 grams, bulb weight, 0.0315 gram. Magni-
fied 24 diameters.

OLFACTORY BULBS OF THE ALBINO RAT PLATE 1
CAROLINE M. HOLT

~ ae

SSises eens
Agee Screen “ene

PLATH 2
EXPLANATION OF FIGURES

Portion of section of F,; area 8, in figure 1. Magnified 172 diameters.
Portion of section of F;; area 8, in figure 2. Magnified 172 diameters.

c

FOR ooo npn gor 09 24%00 9 eee
ly © bag M60 ge? w 06%, oe go Op Upp v0 AP

gone athens, ceca dt ote ta ais
WR CA ae ei ot Oe

90 8 ap FPP PO ny O 0, yOW
BaH20 yg SOW gf FP po cued OME 06 2 apgesifeco “EH o

0

ALBINO RAT

7)
u

OLFACTORY BULBS OF THI

CAROLINE M. HOLT

PLATE 3
EXPLANATION OF FIGURES
Cross section of Q;, section 2 3/3, cut at region where bulb joins cerebrum.
Q;, 30-day control rat, killed when weaned. Brain weight, 1.3164 gram; bulb
weight, 0.0238 gram. Magnified 30 diameters.
Cross section of My, section 4 1/1, cut same region as figure 5 above. Mas,
underfed 30 days. Brain weight. 1.4613 grams; bulb weight, 0.0251 gram. De-
fective diet. Magnified 30 diameters.

256

PLATE 3

OLFACTORY BULBS OF THE ALBINO RAT

CAROLINE M. HOLT

Raee
Sap arabe ce

Pg

oe

PLATE 4
EXPLANATION OF FIGURES

Cross section of M;, section 3 6/12, cut same as figure 5 above. Ms, control
for Ms. Brain weight, 1.7110 grams; bulb weight, 0.0374 gram. Magnified 30
diameters.

ae

al

PLATE 4

OLFACTORY BULBS OF THE ALBINO RAT

CAROLINE M. HOLT

259

[Dedicated to the memory of my friend Mrs. Susanna PHELPS GAGE]

FURTHER CONTRIBUTIONS ON NEUROBIOTAXIS

Ix. AN ATTEMPT TO COMPARE THE PHENOMENA OF NEUROBIOTAXIS
WITH OTHER PHENOMENA OF TAXIS AND TROPISM. THE
DYNAMIC POLARIZATION OF THE NEURONE

C. U. ARIENS KAPPERS

From The Central Institute for Brain Research, Amsterdam

SIX FIGURES

CONTENTS
Neurobiotaxis and) itsyselective) character, ... 5540. siodudseios selon sis Balaets 261
Bok’s researches: the stimulogenous formation of the axon................ 267
Experiments concerning phenomena of tropism and taxis in plants and

animals: , Katgphoretic phenomena.-.. os. ios2 sys sso pee wal alen seins 270
Application ef these experiments to the growth of the neuroblast. The

TORU APLANOL CHELAKOM. Sh ..,. 8 ead cele +. 3 «08S SUA ERE oe eke SE Meee balou ee 25
The formation and contraction of dendrites. The final shifting of -the

perikaryon 5 US SRE IOs COLES C Te EI EERE cis An ih AR ROR NA A 279
Monoaxonismvand poly dendritism sx. sss ir: 4) ats eg neues ae ects toe. sla B's) 3ts 4s 282
The selectivity in the processes of neurobiotaxis in harmony with psychologi-

Gaal a oy Scie Rte cca erela Sem ewe pera, oth 4) ce een A Une ras Marsha) Sine a 284
Fasciculation of axons. Improvement of the nervous path............... 287
aie forma tion-or the medullary sheath «<p. .5)sih. sce cre treble ls shales chet oe 290
EN eSTETHC A Gi COC USL Aes tabs te fate =, Bes ete ced eacicet eT ya edareh | SOURIe ES tee wie 293

NEUROBIOTAXIS AND ITS SELECTIVE CHARACTER

In various articles,! first in 1907, I have published observations
concerning the shifting of nerve cells in the central nervous
system, which could be shown by the different places that

1 The principal points are mentioned in:

Die phylogenetische Verlagerungen der motorischen Oblongata-Kerne, ihre
Ursache und ihre Bedeutung. Neurologisches Centralblatt, 1907, and Rapport
du Congrés international de Psychiatrie et de Neurologie, Amsterdam, 1907.

Weitere Mitteilungen iiber die Verlagerungen der motorischen Oblongata-
Kerne: der Bau des autonomen Systems. Folia Neurobiologica, Bd. 1, 1908.

Specially in: Weitere Mitteilungen tiber Neurobiotaxis. Die Selektivitit der

261

5

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3
APRIL, 1917

262 Cc. U. ARIENS KAPPERS

the motor nuclei of the oblongata exhibit in the series of
vertebrates.

Ontogenetically the same phenomenon could be stated. Since
it was most evident that the shifting of these central groups
took place in the direction of the point whence the majority
of stimuli proceeded to their cell:, we apparently had to do with
a phenomenon of taxis or tropism, which I called Neurobiotaxis,
because it occurs in the nervous system during life (in its phylo-
genetic and ontogenetic development) and I did not know where

Zellen-Wanderung. Die Bedeutung synchronischer Reizverwandtschaft, etc.
Folia Neurobiologica, Bd. 1, 1908.

Uber die Bildung von Faserverbindungen auf Grund von simultanen und
sukzessiven Reizen. Bericht des III. Kongresses fiir experimentelle Psycho-
logie in Frankfort am Main, 1908. ;

Further anatomical details are found in:

Weitere Mitteilungen iiber Neurobiotaxis, Il. Die phylogenetische Entwick-
Jung des horizontalen Schenkels des Facialiswurzelknies. Folia Neurobio-
logica, Bd. 2, 1908.

Weitere Mitteilungen iiber Neurobiotaxis, ITI. Uber den Hinfluss der Neurone
der Geschmackskerne auf den motorischen Facialis und Glossopharyngeuskern
und ihr Verhalten zur Radix descendens Nervi quinti. Folia Neurobiologica,
Bd. 3, 1909. p

Weitere Mitteilungen iiber Neurobiotaxis, IV. The migrations of the abducens
nucleus and the concomitating changes of its root-fibers. Psychiatrische en
Neurologische Bladen, Amsterdam, 1910.

The migrations of the motor cells of the bulbar Trigeminus, Abducens, and
Facialis in the series of vertebrates and the differences in the course of their
root-fibers (counted as Mitteilung V.). Verhandelingen der Kon. Akad. v.
Wetenschappen, Amsterdam. Tweede Sectie, Deel 16, Nr. 4, 1910.

Weitere Mitteilungen tiber Neurobiotaxis, VI. The migrations of the motor
root-cells of the vagus group and the phylogenetic differentiation of the hypo-
glossus nucleus from the spino-occipital system. Psychiatrische en Neurologische
Bladen, Amsterdam, 1911.

Weitere Mitteilungen tiber Neurobiotaxis, VII. Die phylogenetische Ent-
wicklung der motorischen Wurzelkerne in Oblongata und Mittelhirn. Folia
Neurobiologica, Bd. 6, Sommergiingzungsheft, 1912.

Weitere Mitteilungen iiber Neurobiotaxis, VIII. Uber den motorischen
Facialis und Glossopharyngeus Wurzel bei niederen Vertebraten. Folia Neuro-
biologica, Bd. 9, 1912.

The structure of the autonomic nervous system compared with its functional
activity. Journal of Physiology (England), vol. 37, 1908, p. 139. ,

Phenomena of neurobiotaxis in the central nervous system. Section Anatomy
and Embryology, of the XVITth International Congress of Medicine, London, 1913.

NEUROBIOTAXIS 263

to classify it under the phenomena of galvanotaxis, chemo-
taxis or other processes of taxis or tropism known at that time.

This phenomenon of shifting is clearly shown by figures 1 and
2, where the dorsal position of the abducens nucleus in the
shark with its huge fasciculus longitudinalis posterior (f.l.p.,
fig. 1) strongly contrasts with the ventral position of the same
nucleus in a bony fish (fig. 2), where the fasciculus longitudinalis

Fig. 1 Acanthias vulgaris, showing the dorsal position of the abducens
nucleus. /f.l.p., fasciculus longitudinalis posterior; Nuc.VI, abducens nucleus;
N.VI, abducens nerve; 7.V/J,m., motor facialis root. After Van der Horst.

posterior is much smaller, but where the ventral set of central
afferent tracts which influences this cell group is much more
strongly developed (tr. tecto-bulbares ventrales, tr.t.b.v., fig. 2).

The first way in which I formulated this law was thus: When
from different places stimuli proceed to a cell, its chief dendrite
grows out and its cell-body shifts in the direction whence the

264 Cc. U. ARIENS KAPPERS

majority of stimuli proceed.2. The truth of this was soon con-
firmed also in other parts of the cerebrum, by Tretjakoff,*
Herrick,‘ Bartelmez,> Obenchain,é Bok, Van der Horst? and
others. .

I observed, however, on an increase of afferent stimuli in a
given center, that not all the neighboring cells approach this
center, but that only certain cells proceed to that center which
apparently had a certain relation to it, while other cells (even
lying nearer by) did not migrate into the direction of the increased
sensory field, because evidently they had nothing to do with it
and did not stand in relation to it.

Further researches convinced me that the functional relation
which appeared to be the condition for the approach was shown
to be a correlation depending on simultaneity of function—of
stimulation.

So the abducens nucleus shifts from one center of visual co-
ordination fibers (the f.l.p.) to another set of visual co-ordination
fibers (the tr. tecto-bulbaris) if the latter increase; but an increase
of the taste fibers for instance, does not have any effect upon it.

* Later I found that a similar observation had been already made by Strasser
(92) and by Cajal (99). Compare: Strasser, Alte und neue Probleme der Ent-
wicklungsgeschichtlichen Forschung auf dem Gebiete des Nervensystems.
Ergebnisse der Anatomie und Entwicklungsgeschichte, Bd. 1, 1892, p. 721.
Cajal, Textura del sistema nerviosa del hombre y de los vertebrados, vol. I,
1899, p. 560. See also Cajal, Algunas observacionas favorables a la teoria neuro-
tropica. Trabajos, vol. 7, 1908, p. 63. Both, however, failed to see the corre-
lative character in this process, and Cajal ascribes a great influence to the spon-
gioblasts (ependyma and glia) in the secretion of attracting chemicals for the
axones, in which I do not at all agree with him.

> 'Tretjakoff. Das Nervensystem von Ammococtes, I]. Das Gehirn. Archiv
f. mikrosk. Anat., Bd. 75, 1909.

* Herrick. The morphology of the forebrain in Amphibia and Reptilia.
Jour. Comp. Neur., vol. 20, 1910.

° Bartelmez. Mauthner’s cell and the nucleus motorius tegmenti. Jour.
Comp. Neur. vol. 25, 1915.

*Obenchain (with Herrick). Notes on the anatomy of a cyclostome brain,
Ichthyomyzon concolor. Jour. Comp. Neur. vol. 23, 1913.

7 Van der Horst. De motorise¢he kernen en banen in de hersenen der visschen,
hare taxonomische waarde en neurobiotactische beteekenis. See also: Tijd-
schrift der Ned. Dierk. Vereen, 1917.

NEUROBIOTAXIS 265

Then I found—though not starting my work with a psychologi-
cal secope—that the anatomical relations of the dendrites and the
cells in the nervous system were regulated in accordance with the
law which, in psychology, is known as the law of association,
in which law (in all the different forms’ in which it may appear)
the simultaneity of stimulations or residua of stimulations is
the essential part.

Fig. 2. Tetrodon, showing the ventral position of the abducens nucleus.
f.l.p., fasciculus longitudinalis posterior; Nuc.VI, abducens nucleus; N.VJ,
abducens nerve; r.V/7,m., motor facialis root; tr.t.b.v., tractus tecto-bulbaris
ventralis. After Van der Horst.

This anatomical observation, first made on motor cells, led
me to study more carefully the courses of several axon-tracts,
sensory tracts, as well as the so-called ‘‘central motor tracts,”
such as the pyramids, and it soon appeared to me that a criti-

8 Those forms are simultaneity, successivity, similarity and contrast. In the
three first named forms the presence of one stimulus, or remains of a stimulus,
while the other is added, is obvious. The association by contrast is also due in
the first place to simultaneity of impression since the simultaneous or successive
contrast makes us discriminate things: black and white, father and mother, etc.

266 Cc. U. ARIENS KAPPERS

cal study of their relation showed most clearly that the same
law of neurobiotaxis, the simultaneous relationship in their
stimulative function, had been the cause of their final arrange-
ment.® So I was able to formulate the phenomena of neuro-
biotaxis in the following words:

I. If in the nervous system several stimulation-charges occur,
the growth of the chief dendrite, and eventually the displace-

Centre
Whence the sti-
mult
proceed

Axiscylinder

B

Fig. 3 Showing that, while the axis-eylinder runs with the direction of the
nervous current, the dendritic outgrowth and the final shifting of the cell body
occur against the nervous current. A, giant dendrites grown out towards the
center cf stimulation. B, the cell body (perikaryon) has shifted toward the
center of stimulation; the axis-cylinder is consequently elongated.

ment of the cell-body itself, takes place in the direction, whence
the majority of stimuli proceed to the cell.

II. Only between correlated centers does this outgrowth or
shifting take place.

III]. The growth of the axis-cylinder (.e., its final connec-
tion) is not primarily regulated by motor centers,!? but also here
synchronic or successive stimulation (correlation) acts a part."

"Folia Neurobiologica, Bd. 1, 1908.

10 Not by some undefined transcendental willing (teleologically).
11 That is, it is defined by correlation.

NEUROBIOTAXIS 267

While, however, it was evident that the approach of the
dendrites and nerve cells to a territory (fig. 3) took place towards
the center of the stimulation (as a stimulopetal or centripetal
tropism), that is, against the nervous current of stimulation pro-
ceeding from this center, the problem became much more diffi-
cult to explain how the connection between correlated centers
was effected by the axis-cylinder, since it was obvious that the
axis-cylinder does not grow towards the stimulation (stimulo-
petal) to meet it, but moves in the same direction as the stimu-
lus-irradiation (stimulo-fugal or centri-fugal).

BOK’S RESEARCHES: THE STIMULOGENOUS FORMATION OF THE
AXON

That the axis-cylinder. really grows with the current and
that the irradiation of this current plays an important part in
its growth has been proved and very carefuliy examined in this
Institute by S. T. Bok, who got highly important results.

Bok” found that when an axis-cylinder or a bundle of amye-
linated nerve-fibers grows out and passes nerve cells on its
way, these nerve cells can be activated to send out an axis-
cylinder of themselves in a region perpendicular to the activat-
ing axon or bundle (fig. 4).

This fact was found with the fasciculus longitudinalis posterior
in such a form as left no doubt, since it appeared that the motor
nuclei which undergo the influence of this bundle were only
activated according to the degree in which the fasciculus longi-
tudinalis posterior had grown out. So the axons of the tri-
geminal" cells first grow out, then follow the axons of the faci-
alis cells, then those of the glossopharyngeus and vagus.

The same was seen in the activation of the oculomotorius,
abducens and hypoglossus nuclei which are activated by another
influence of the same character. .

2 Bok. Die Entwicklung der Hirnnerven und ihrer Zentralen Bahnen. Dei
Stimulogene Fibrillation. Folia Neurobiologica. Bd. 9, 1915. See also Bok,
Stimulogeneous Fibrillation. The cause of the structure in the nervous system.
Psych. en Neurologische Bladen, Amsterdam, 1915.

13 Concerning the Trochlearis. See the first-named original.

268 Cc. U. ARIENS KAPPERS

Bok, considering the fact that the formation of the axis-
cylinders in those cells took place under the influence of the
current irradiating from the primary activating axis-cylinder,
ealled this stimulogenous fibrillation, following the direction of
that current in contrast to the outgrowth of the dendrites and

Activated neuroblasts

cells

Activating primary jascicle

Fig. 4 The activation of adjacent neuroblasts by an amyelinated (growing)
fascicle. The vertical arrow indicates the direction of growth of the activating
bundle and the direction of its nerve current, which starts at A. The horizontal
arrow indicates the course of the irradiating influence (current) perpendicularly .
to the activating bundle. Notice that the proximal cells are sooner activated
(and have moved further) than the more distant ones. After Bok.

NEUROBIOTAXIS 269

the shifting of the cell body, both of which also only occur later
and which move towards the center, 1.e., poe the current of
the stimulus that proceeds to them.

This observation and Bok’s interpretation of it are very 1m-
portant, and no doubt correct. It is evident, however, that the
final end-point of the growing axis-cylinder can not be deter-
mined by this process alone, as was also realised by Bok, who
came to the conclusion that the final connection was deter-
mined by the principal law of neurobiotaxis, viz., by the stimu-
lative (simultaneous) correlation of the growing axis-cylinder
and its end-point, i.e., the cell or dendrites with which it is
going to be connected.

Bok thought that this could be effected by the fact that if
two centers are in simultaneous stimulation the ideal line be-
tween the two is the path where the plasmodesms undergo the
greatest influence of this relation. He called this the principle
of the ‘doppelte Bahnung,’ and thought that Einstein’s (physi-
cal) law of attraction between synchronic energies also had some
influence on it.

It seems to me, however, that the principle of ‘doppelte Bahn-
ung,’ as laid down in this theory, can not explain from which of
two simultaneously stimulated cells the axis-cylinders grow out,
and that,even the adaptation of the protoplasm to the forma-
tion of the axis-cylinder, eventually a fibrillation of the neuro-
desms, then might begin in the middle between two cells which,
as we know, it never does. Moreover, the expression “‘adapta-
tion of protoplasm to its biological function” is too general an
expression to explain anything.

It has appeared to me that the literature of recent years con-
cerning the microchemistry of the neurones and the phenomena
of tropism and taxis known and experimentally examined in
other organisms, together with Bok’s discovery, concerning the

14 Tf the normal stimulation of the cell body is of little importance or eventually
absent, the cell may also shift in the same direction in which the axon grows out.
(See my paper on the autonomic nervous system. Journal of Physiology, 1908,
vol. 37, p. 139.)

270 Cc. U. ARIENS KAPPERS

stimulogenous outgrowth of the axis-cylinders from the acti-
vated cell by and with the irradiating current from a primary or
activating axis-cylinder in its neighborhood, gives us a key of
exceptional importance to comprehend the phenomena of neuro-
biotaxes in general, and the contrasting behavior in outgrowth
direction between dendrites and axons and allows us to consider,
perhaps to explain, how it is possible that a unit such as the neu-
rone is may exhibit two opposite directions of growth.

EXPERIMENTS CONCERNING PHENOMENA OF TROPISM AND TAXIS
IN PLANTS AND ANIMALS. KATAPHORETIC PHENOMENA

It is evident that, in any attempt to explain the neurobiotac-
tic phenomena, these must be compared with other phenomena
which are better adapted to experimental investigation. As
such we may mention the galvano-tropic phenomena in the growth
of plant-roots and the orientation of animals in the constant
current, about which we have obtained many data during the
last decennia.

As is known, the phenomenon of galvanotropy in plant-roots
was discovered in Hermann’s laboratory by Miiller-Hettlingen,”
who found that, if the sprouting seed of the bean (Vicia faba)
be exposed to a constant current, the tips of the root turn and
grow towards the negative pole (kathode).

An analogy" of this galvano-tropic phenomenon is found in
the galvano-tactie phenomenon described by Bancroft,’ viz.,
that the tentacles and the manubrium cf a medusa, Polyorchis,
during the transmission of a constant current turn towards the
kathode.

In the experiments with the latter this peculiar phenomenon was
observed, viz., that with a long-continued current the side turned
to the anode extended, becoming thinner and weaker; this last phenom-
enon being a symptom of decay, according to this author (vide infra).

16 Miiller-Hettlingen. Ueber galvanische Erscheinungen an Keimenden
Samer. Pfliiger’s Archiv, Bd. 31, 1883, p. 192.

16 Not a homology, probably.

17 Jour. Exp. Zool., vol. 1, 1904, p. 289.

NEUROBIOTAXIS Ze Al

As third example of galvano-taxis the phenomenon discovered
by Verworn'’ in one-celled creatures must be mentioned here,
viz., that these (ameba, for instance) on the transmission of a
constant current through the surrounding medium, send out
enlargements and finally shift in the direction of the kathode.

Verworn at first held the opinion that there were also Pro-
tozoa which shift under normal circumstances to the anode, and
he therefore made a distinction between kathodic and anodic
galvano-taxis. Later investigations revealed that the anodic
galvano-taxis must be considered as being something diffe:ent
from the kathodic, and that the direction and shifting of the
bodies observable in Protozoa under normal circumstances is
invariably a kathodic galvano-taxis.

Also Boruttau (personal communication) holds the opinion
that every real galvano-tropism is a kathodic stimulation phe-
nomenon. ‘This usual kathodic galvano-tropism can be brought
into correlation with Pfliiger’s law, as has been pointed out by
Loeb and Maxwell.”

As far as the anodic tropism is concerned, Loeb and Budgett?’
and after then Coehn and Barratt?! found that when a proto-
zoan, Paramecium, in pure water or in a weak solution of common

18 Verworn. Die polare Erregung durch den galvanischen Strom. Pfliiger’s
Archiv, Bd. 45, 1889. Verworn. Die polare Erregung der Protisten durch den
galvanischen Strom (Fortsetzung). Pfliiger’s Archiv, Bd. 46, 1890. Verworn.
Untersuchingen tiber die polare Erregung der lebendigen Substanz. 3te Mittei-
lung. Pfliger’s Archiv, Bd. 62, 1896, S. 415. Verworn. Die polare Erregung
der lebendigen Substanz durch den Constanten Strom. 4te Mitteilung. Pfli-
ger’s Archiv, Bd. 65, 1897.

18 J. Loeb und 8.8. Maxwell. Zur Theorie des Galvano-tropismus. Pfliiger’s
Archiv, Bd. 63, 1896.

See also J. Loeb und Walter Gerry. Zur Theorie des Galvano-tropismus, IT.
Versuche an Wirbelthieren. Pfliiger’s Archiv, Bd. 65, 1897, S. 41. J. Loeb.
Zur Theorie des Galvano-tropismus, III. Ueber die polare Erregung der Hart-
drusen von Amblystoma durch den Constanten Strom. Pfliiger’s Archiv, Bd. 65,
1897, S. 308.

20 Loeb und Budgett. Zur Theorie des Galvano-tropismus. IV. Mitteilung
iiber die Ausscheidung electropositiver Ionen an der ausseren Anoden flache
protoplasmatischer Gebilde als Ursache der Abweichungen vom Pfliiger’schen
Erregungsgesetz. Pfliiger’s Archiv, Bd. 65, 1897, S. 582.

21 Coehn und Barratt. Ueber Galvano-taxis von Standpunkt der physio-
logischen Chemie. Zeitsch. f. allgemeine Phyziologie, Bd. 5, 7, 1905.

272 Cc. U. ARIENS KAPPERS

salt, was influenced by a constant current, the movement was
in the direction of the kathode, but that this direction of gal-
vano-taxis may be reversed by placing the animal in a stronger
(even physiological) solution of salt. If in the latter case the
constant current was transmitted through it, a movement
towards the anode was observable. This phenomenon of re-
versal, first observed in a galvano-tactic process, was confirmed
shortly after in a galvano-tropic process, in the case of the root-
tips of pease.

Gassner’ found that an increase of salt in the medium influ-
ences the effect of the constant current in those objects also.
When he increased the quantity of salt of the water in which
pea-roots sprouted, the constant current could no longer cause a
kathodic tropism. He was inclined to ascribe this to a dim-
inution in the quantity of electricity running through the tip
of the root, since the greater conductivity of the water (salt
solution) caused a greater quantity of electricity running through
the solution itself.

Schellenberg?* obtained the same result, but went even far-
ther, and on increasing still more the percentage of salt was
able to obtain a reversed tropism, the root then growing to th
anode.

If the percentage of KCl in the water was only 0.074 per cent
the root-tip continued to grow kathodie galvano-tropic; if,
however, the percentage was raised to 1 per cent, a distinct
anodic direction in the growth appeared, and with a fair de-
gree of exactness such a concentration of KCl could be found in
which, after the transmission of the constant current, no trop-
ism was evinced."

* Gassner. Der galvano-tropismus der Wurzeln. Botanische Zeitung, 1906,
Parts 9-11.

23 Schellenberg. Untersuchungen iiber den Einfluss der Salze auf die Wach-
stumsrichtung der Wurzeln, zunichst an der Erbsen Wurzel. Flora, vol. 96,
1906, p. 474.

“Tt may be mentioned that the current strength which caused this tropism
was but slight, and varied from 1/10 to 1/1000 milliampere, with a density of
current of 0.0025 to 0.000025 milliampere per sq. em.

That Elving’s curves (which are also anodic) could be formed under these
circumstances is out of the question, since Brunchorst found the current density
necessary in this case to be about 0.2 milliampere.

NEUROBIOTAXIS 273

Like Gassner, Schellenberg also seems to be inclined to ascribe
the anodic tropism to the greater amount of electricity running
through the water, or rather to the weakness of the current
that runs through the root-tip and which should be too weak to
cause a kathodic tropism, a supposition that seems to be ac-
cepted by Rothert,2> though he admits that it has not been
proved. If only a weaker current were sufficient to cause the
anodo-tropic phenomenon, a smaller amount of electricity
would have to do the same! These authors, moreover, do not
explain biochemically why a weak current should cause an
anodic tropism and a stronger current a kathodic one.

Coehn and Barratt (loc. cit.) tried to explain biochemically
this phenomenon of reversal of the galvano-tropism in the fol-
lowing way. They assumed that on the boundary between the
object used for the experiment and the surrounding medium
(the water) a semipermeable membrane is present that possesses
a different permeability for positive and negative ions. This
assumption is quite legitimate, since the occurrence of such
semipermeable membranes is a very common phenomenon in
nature.

If we now assume that the permeability for negative” ions is
greater in this membrane than that for positive ions, of the
ionized NaCl or KCl (provided the concentration thereof in
the surrounding fluid be greater than in the protoplasm) a
larger quantity of negative ions will be transferred into the cell
than of positive ions, and the cell will then be overcharged with
negative ions and pass to the positive pole on the transmission
of the constant current.

On the other hand, if the concentration of KCI or NaCl in
the medium be less than in the cells, a larger quantity of negative
ions than positive will leave the cell, and the cell-bodies, charged

2> Rothert. Die neuen Untersuchungen iiber den Galvano-tropismus der
Pflanzenwurzeln. Zeitschrift fiir allgemeine Physiologie, Bd. 7, 1907, p. 192.

26 Such a special permeability for negative ions (anions) has been proved to
exist in the case of blood corpuscles by Hamburger (Zeits. f. Biologie, Bd. 28, p.
405, 1891). Compare also Hamburger und Van Lier, Durchlissigkeit der rothen

Blutkérperchen fiir die Anionen von Natriumsalzen. Arch. f. Anat. u. Physiol.,
Physiol. Abt., 1902, p. 492.

274 Cc. U. ARIENS KAPPERS

with a surplus of positive ions, will on the transmission of the
constant current pass to the kathode.

If we accept this theory as correct, we shall have to assume
that in ordinary circumstances—under which the kathodie tro-
pism or taxis predominates—also a greater charge of positive
ions is present in the cell-body of the ameba, or in the proto-
plasm of the tentacles or root-tips, than in the surrounding
extra-protoplasmatic medium. This explanation is not gener-
ally accepted, but that the condition of the extre-protoplasmic
medium is of great importance has also been emphasized by
Loeb and Budgett, who are equally inclined to ascribe the
exceptions to Pfliiger’s law (the anodic migrations) to altera-
tions in the extra-protoplasmic medium. They refer to a
phenomenon which may be exhibited by that side of an ameba
or paramecium that is turned to the anode, viz., the extension
of the protoplasm on that side, eventually followed by lique-
faction. This ancdic extension, first observed by Verworn
(loc. cit.), is the first thing that appears when Protozoa are
exposed to the constant current and precedes the real kathodic
galvano-tropism.

Loeb and Budgett (loc. cit.) have submitted it to a more
detailed examination and also came to the conclusion that this
process is a result of the extra-protoplasmatic medium. Their
explanation of this anodic phenomenon differs from the one given
by Coehn and Barratt. They are, however, equally inclined to
consider this phenomenon as due primarily to changes in the
extra-protoplasmatic medium2? in contrast to the phenomena
of common tropism following Pfliiger’s laws of irritation. It
may be mentioned still that the most favorable strength of cur-
rent in those experiments with ameba was only 0.4 milliampere.

Besides these galvano-tactic and galvano-tropic phenomena of
living protoplasm, we know of polar phenomena in dead organic
substances rendered evident by the direction in which albumen
shifts when subjected to a constant current: viz., the phenome-

27 Perhaps this mode of explanation may be also applicable to the above-

mentioned reversal of the galvano-tropism of root tips and to the anodal phenom-
enon observed by Bancroft (vide supra).

NEUROBIOTAXIS Bae

non of kataphoresis. I refer here to the investigations of
Hardy,?8 which showed that as long as an albuminous solution is
alkaline the particles suspended in it shift towards the anode
on the transmission of a constant current, whereas they shift
towards the kathode when the solution is made slightly acid.

One is apt to look for an explanation of this also in the fact
that on the boundary between a colloid particle and the sur-
rounding fluid, a double layer in the sense of the theory of Helm-
holtz-Quinke is present.

If now the solution is alkaline, a transmission of ions will
take place, in consequence of which the albuminous particle
itself becomes negative and thus shifts to the anode on the
transmission of a constant current, while in the case of an acid
reaction of the surrounding “fluid the contrary takes place.
From the reversibility of the kataphoretic phenomenon (Ham-
burger)?’ the curious fact thus follows, viz., that also proteid
particles have the peculiarity that their electric character is
determined by the reaction of the surrounding medium.

That here too, just as in the above tropism of the root-tips,
an iso-electric condition occurs is clear.

APPLICATION OF THESE EXPERIMENTS TO THE GROWTH OF THE
NEUROBLAST. THE FORMATION OF THE AXON

If, with these facts before us, we consider the phenomena
which appear during the formation of an axis-cylinder*®® in an
activated cell (which precedes the formation of dendrites—see

*8 Hardy. On the coagulation of proteid by electricity. Jour. of Physiol.,
vol. 24, p. 2881, 1899. Proc. Roy. Soc., vol. 68, p. 110, 1900.

29 Hamburger. Osmotischer Druck und Ionenlehre. Wiesbaden, Bergmann,
1904, vol. 3, p. 68. ;

30 Tt is hardly necessary to say that the fact that isolated ganglion cells, as in
Harrison’s experiments, may also send out axis-cylinders proves nothing against
the following text. Harrison (loc. cit., p. 833) remarks that this is a process of
self-differentiation entirely independent of external conditions. This is true to
a certain extent, but we must assume that before it becomes a self-differentiation
its differentiation has been induced to the neuroblast in former generations by
external circumstances and that its doing this by itself is based on hereditary
engrammatic qualities. It is better to see a problem in things than to explain
them by a word which implies a still greater problem.

276 Cc. U. ARIENS KAPPERS

fig. 4), we shall first have to mention the fact that the stimula-
tion center with respect to the surrounding tissue is negative,
forming a kathode with reference to the non-stimulated sur-
roundings, as physiological experiments abundantly prove.

Moreover the strength of the electrolytic potential differences
occurring in the nervous system in consequence of stimulation
appears to be of the same category as those that are applied in
artificial phenomena of galvano-taxis (see above) since it may
vary from 3 millivolt to 0.8 millivolt and lower, so that the forces
developed here are certainly strong enough to influence proc-
esses of formative tropism and functional taxis.

Now, it may be the same whether this stimulated center is
the body surface in or under which nerve cells lie or whether we
start our deductions with a primary growing axis-cylinder which
on its way passes neuroblasts. This negative potential not only
runs along the primary axis-cylinder (fig. 4) but also, we may
assume, as long as the axis-cylinder is not provided with an
insulating medullary sheath, that this negative potential stands
perpendicular to the length of the activating axis-cylinder (or
body surface), irradiating from it.*!

In accordance with this perpendicular irradiation of the
electrolyte nfluence, or current, we see that th neuroblasts
near the primary activat ng bundle send out ax’s-cylinders per-
pendicular to the activating bundle, and that similarly perpen-
dicular collaterals may grow out from the original (activating)
axis-cylinders themselves.

In both cases, in the formation of collaterals as well as in the
outgrowth of the axis-cylinder of the secondary (activated)
neuroblasts, the axis-cylinder substance proceeds in the direc-
tion of the perpendicular irradiation of the stimulated fiber,
i.e., to the anodic pole.

31 The irradiative stimulus of naked axons is very clearly illustrated by the
position of the dendrites of Purkinje’s cells perpendicular upon the parallel
fibers in the molecular layer of the cerebellum and of the dendrites of the motor
cells on the longitudinal (naked) axons in the spinal cord of Petromyzon. See
my paper, Ueber das Rindenproblem, etc., in the Folia Neurobiologica, Bd. 8,
1914, pp. 529-530

NEUROBIOTAXIS 2ae

This first outgrowth which, in the beginning, can be compli-
cated with an anodal katophoretic shifting of the cell body itself
(fig. 4) may be entirely independent of a propagation of the
nervous current itself along the newly formed short axis-cylinder.
As soon, however, as this axis-cylinder is fit for nervous conduc-
tion its rate of outgrowth will be considerably increased, a
much stronger negative current running in the direction of its
growth to the anodal field.

Why does this anodic growth occur before the kathodic trop-
ism of the dendrites and the cell body? I will consider this
question in the light of the above-mentioned experiences.

We know that the neuroblast is embedded in an organic solu-
tion, the pericellular lymph, containing a good deal of potassium
salts.

Macallum has emphasized that the amount of potassium salt
external to the nerve cell is great and that a considerable con-
densation of this element is present on its exterior surface.

Now Verworn has shown that on the transmission of a constant
current the first thing to appear is an anodal expansion oi the
cell body, thus showing that a change of tension may be local-
ised, by electric influences, on the anodal pole.

We may expect that this extension, being under the influence
of a considerable amount of K and Cl, derives certain chemical
and tropic characteristics from it.

That this really occurs in nerve cells is proved by the chemi-
cal constituents of the axon, compared with those of the den-
drites and cell body.

We know from the researches of Macdonald, Macallum, Al-
cock and Lynch that the axis-cylinder is distinguished from the
dendrites and the cell body by a much larger quantity of po-
tassi1um and chlorides*® (which, according to Macdonald, may
also contribute to its conductivity for the nervous current).

32 MacDonald. The injury current of nerves, The key to its physical struc-
ture. Report of Thomson-Yates Laboratory, vol. 4, 1902, p. 213.

MacDonald. The structure and function of nerve-fibers. Proceedings of the
Royal Society, vol. 76, B. 1905, p. 322.

Macallum. On the distribution of potassium in animal and vegetable cells.

Journal of Physiology, vol. 32, 1905.
Macallum. Die Methoden und Ergebnisse der Mikrochemie in der Biologis-

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

278 Cc. U. ARIENS KAPPERS

The large quantity of KCl then present around its colloidal
substance will favor (according to the experiments of Gassner,
Schellenberg, and others) the anodo-tropic character of the axis-
' cylinder.

The phenomenon of the formation of the axis-cylinder and its
collaterals in the direction of the anodic field, may thus be so
expressed that we say that the neuroblast embedded in a solu-
tion containing a good deal of potassium and of chloride ex-
hibits, in harmony with the experiments of Loeb, Budgett,
Coehn and Barratt, a tropism at the anodal side of the neuro-
blast and that the KCl constituents of the neuroblast gathering
on this side thus increase (besides its conductivity) the anodo-
tropic character of its colloidal substance. This anodo-tropic
character of the colloidal substance of the axis-cylinder is, more-
over, in harmony with Hardy’s experiments on the kataphoresis
of albuminoids.

Considering the fact, that the kataphoresis which genuine
albumen and lecithin show is already generally an anodic one
(Hober, loc. cit.) it is clear that the additional composition of the
neurone and its surroundings still favors this, since the colloid
particles of the young axon are embedded in a medium contain-
ing a quantity of KCl, that makes its preponderating reaction
alkaline. Moreover the greater conductivity which KCl gives
it, may cause the greater quantity of electricity to be led
through it.

That the constituents of a peripheral nerve are strongly con-
veyed to the anode is also experimentally shown by Hermann, to
whose experiments I return later (see p. 291).

From every standpoint indeed it seems that the conditions for
the primary outgrowth of the axon along with the kathodic cur-
rent to the anodic field have been realized in the nervous system.
chen Forschung. Ergebnisse der Physiologie von Asher und Spiro, Jahrg. VIT,
1908.

Aleock and Lynch. On the relation between the physical, chemical and
electrical properties of the nerves. PartIV: Potassium, chlorine and potassium
chloride. Journal of Physiology, vol. 42, 1910.

Macallum. Surface tension and vital phenomena. University of Toronto
Studies, No. 8, Physiological Series, 1912.

NEUROBIOTAXIS 279

The outgrowth of the axis-cylinder begins in the chick embryo
about the second day of incubation (Bok).

Not until much later—according to Cajal when the growth
tip of the axis-cylinder has reached, or nearly reached, its end
point (about the 6th day of incubation in the chick embryo,
Bok)—does an outgrowth of the dendrites begin, which make
their way in the direction of the stimulus, that is in the direction
of the kathode.

THE FORMATION AND CONTRACTION OF DENDRITES. THE FINAL
SHIFTING OF THE PERIKARYON

I believe that there is a principal difference biologically as well
as biochemically between the anode elongation of the axon
and the kathodic tropism of the dendrites.

The primary growth of the axon is in the beginning not di-
rected to a certain point, but merely from a certain kathodic
center, the outgrowth of dendrites, however, is much more
influenced also in the beginning by their final end-points.

Their tropism corresponds with the regular appearance of the
law of stimulation of protoplasm and exhibits a kathodic char-
acter, probably related with a more advanced nervous function
for which a further stage of development is necessary.

This kathodic growth direction, as well as the kathodic taxis,
is the usual thing in nature and, as Loeb and Maxwell have
shown, is in harmony with Pfliiger’s law. We only have to
prove that there are no factors which might interfere with it
and change it into an anodal elongation.

This question is the more important since it may be that in
the first phase of outgrowth of dendrites, which is not yet ac-
companied by a secondary shortening of the dendrite and the
shifting of the perikaryon, a kataphoretic process might intro-
duce it or at least be involved in it. Anyhow, the kataphoretic
qualities of the dendrites may never be such that they should
counteract the kathodo-tropic process which certainly is the
chief factor in the shortening (contraction) of the dendrite and
the shifting of the cell.

280 Cc. U. ARIENS KAPPERS

Now we know from the chemical examinations of Grandry*
and Macallum and Menten* that KCl is hardly present in the
cell and the dendrite, so that for us there is no reason here to
expect an anodal kataphoresis.

Looked at from the point of view of Hardy’s investigations,
which according to Greeley* we may apply to intra-protoplas-
matic colloidal granules, there are perhaps more arguments
which favor the shifting of the dendrites, and later of the cell-
body, in the direction of the stimulus. For we know that the
dendrites and the cell-body differ from the axis-cylinder by
the presence of Nissl’s substance which during life is probably
in a more or less fluid condition (see Cowdry’s papers** on the
bio-chemical conditions of nerve cells). This substance is prob-
ably a derivative or compound of nucleic acid*’ and the presence
of acid in it will, according to Hardy’s investigations, promote
the shifting of the colloids which are suspended in them to the
kathode. While, therefore, the absence of a larger quantity of
KCl does not prevent the first outgrowth of the dendrite from
proceeding in the kathodiec direction, the presence of acid nuclein
derivatives would even promote it.

°8 Grandry. Recherches sur le structure du cylindre-axe et des cellules
nerveuses. Bulletin de l’ Academie de Bruxelles, 2 me Series, vol. 28, 1868, p. 304.

Idem. Journal de l’Anatomie et de la Physiologie, vol. 6, 1869, p. 289.

*4 Macallum and Menten. Distribution of chlorides in nerve-cells and fibers,
Proceedings of the Royal Society, vol. 76B, 1905, p. 217.

Macallum. Die Ergebnisse und Methoden der Microchemie in der biologis-
chen Forschung. Ergebnisse der Physiologie von Asher und Spiro, Jahrg. 7,
1908, p. 697.

* Greeley. Experiments on the physical structure of protoplasm of Para-
mecium and its relation to the reactions of the organism to thermal, chemical,
and electrical stimuli. Biological Bulletin, vol. 7, 1904.

8° Cowdry. The development of the cytoplasmatic constituents of the nerve-
cells in the chick. Mitochondria and neurofibrils. Am. Jour. Anat., vol. 15,
1912.

Cowdry. The relations of mitochondria and other protoplasmatic constitu-
ents in spinal ganglion cells of the pigeon. Internationale Monatschrift fiir
Anatomie und Physiologie, Bd. 29, 1913.

Cowdry. The general function and significance of mitochondria. Am.
Jour. Anat., vol. 19, 1916.

‘7M. A. Van Herwerden. Ueber die Nuclear-wirkung auf tiersche Zellen.
Kin Beitrag zur Chromidienfrage. Archiv f. Zellforschung, Bd. 10, 1913.

NEUROBIOTAXIS 281

This substance perhaps also helps to explain the relatively
late formation of the dendrites, since the nuclein substance does
not appear until late in the cell body in the form of Nissl bodies
which, originating from the chromatin of the nucleus (Scott) ,38 pass
through the nuclear membrane in a stage of development when
the axis-cylinder has already completed its growth over a cer-
tain extent, and has even become fairly considerable.

Cajal*® did not find this substance before the time when the
dendrites start to grow out. Consequently when this substance
is present in the protoplasm we observe a tendency in the pro-
toplasm to shift in the direction of the stimulated field (the
kathode) and to be followed by the contraction of dendrites and
finally the shifting of the whole perikaryon in the direction of
the stimulation field, i.e., in the direction of the negative electric
field.

The difference of time between axis-cylinder outgrowth and
dendrite formation thus would be a result of the general anodic
kataphoretic character of genuine albumen and lecithin, the
alkaline reaction of the pericellular lymph and the quantity of
KCl salt present around the young cell and, further, the greater
conductivity that this gives to the axis-cylinder on one hand,
and on the other hand the late appearance of the nucleic acid
derivatives in the protoplasm.

It cannot surprise us in this respect that only small dendrites
are found on those cells whose chromatin is still entirely in the
nucleus (granular cells), and that the smallest quantity of
nuclear chromatin is found in those cells whose dendrites have
developed most (motor cells, reticular cells and others).

Only the question would remain why the alkaline reaction
of the body lymph, which is the same around the axis-cylinder
and the dendrite, does not interfere with a kathodic outgrowth
of the latter and this fact seems to prove the truth of the opinion

38 Scott. On the structure, microchemistry, and development of nerve cells
with special reference to nuclein compounds. Trans. Canadian Institute, vol. 6,
part 142, p. 405, Dec., 1899.

39 Cajal. Textura del sistema nerviosa del hombre y de los vertebrados,
tomo 1, p. 528, Madrid, 1904.

282 Cc. U. ARIENS KAPPERS

of Loeb and Budgett that the kathodie tropism, following the
law of irritation, is chiefly dependent on intra-cellular proto-
plasmatic conditions and that the extra-cellular medium. does
not act such a part here as it does in anodal extensions.

Indeed, it seems more probable that the later outgrowth of
the dendrites as well as their secondary contraction, including
the shifting of the cell body is a process different in principle
from the anodal outgrowth of the axis-cylinder, a process for
which a greater functional completeness of the neurone is neces-
sary, and that we may only say that the character of the chemical
constitution of the dendrite is not such that it would interfere
with it by a disturbing anodal process.

There are still three questions that may be mentioned in this
discussion.

MONOAXONISM AND POLYDENDRITISM

The first question is why only one axis-cylinder leaves the
cell, one which becomes complicated only by collaterals which
proceed perpendicularly from it during its course, while from the
cell-body, a large number of dendrites may and generally do
grow out to several centers of stimulation (monoaxonism and
polydendritism).

To explain the monoaxonism we may first consider what
would happen if two kathodie currents traversed the young
neuroblast at the same time. In a purely polar tropism, as
galvano-tropism preéminently is, it is a familiar feature that the
object under the influence of the current places itself so that the
influence is equally great on both sides of the object. Only
then does the state of equilibrium begin.

J. Loeb“ in particular has shown this repeatedly, for example
in his 7th Lecture, in which he speaks of radiating energy and
heliotropism, and points out that the orientation of a simple
object will continue until all its parts lie at the same angle with
reference to the influence.

40 J, Loeb. Vorlesungen iiber die Dynamic der Lebenserscheinungen. Leip-
zig, Joh. Ambr. Barth, 1906, p. 171.

NEUROBIOTAXIS 283

As long as the influence on right and left, or mdeed on all
sides, be unequal, the object will change its position until the
state of equilibrium is arrived at and the influence is the same
everywhere.

Let us now apply this to a charged field from which a stimulus

irradiates and passes to a cell in the neighborhood.

It will be clear, without anything further, that the outgrowth
of the axis-cylinder in the current from the kathodic field to the
anode has a state of equilibrium only in the course, that is,
lengthways, of the current, i.e., in a collateral growing out
perpendicularly or, where the growth stimulus proceeding from
the irradiating current activated a cell in its. neighborhood, the
latter must send out its axis-cylinder also perpendicularly from
the source along with the current. This explains the peculiar
fact of collaterals of axis-cylinders in the commencement of
their course having invariably a strictly perpendicular position
with regard to the axis-cylinder.*!

The irradiation current which radiates sideways from an acti-
vating axis-cylinder must naturally move in a direction perpen-
dicular to this axis-cylinder. This is a physical fact that is only
changed at the growing point of the activating axone.

It will thus be seen that the presence of one axon, as well as
the perpendicular position of the collaterals on the axis-cylinder,
are but natural consequences of the perfect bipolar character of
the current. Now the same holds good if two or more differ-
ently running tracts, or differently placed centers, activate
one cell simultaneously. We then may also expect only one
axis-cylinder in the resultant line of the two current directions
(two bio-electric fields), since only in this line the equal influence
on both sides of the growing point, the energetic equilibrium,
is realized.

What will be the case if two or more activating centers are
present not acting simultaneously? One of these activating
centers has to be the first and causes the initial outgrowth.

41 At the time when coloration and impregnation methods were not so ad-

vanced as now, the differential diagnosis of collaterals and dendrites was some-
times made on account of the perpendicular position of the latter on the axon.

284 Cc. U. ARIENS KAPPERS

If, however, an axis-cylinder has started to grow, we may
expect that the favorable conditions which it offers for the
current, on account of its greater conductivity, are such that
the obstacle to the formation of a new axon at some other place
is so much greater, that the current will take the present path of
enlarged conductibility, the course of which it may influence
perhaps without, however, causing a new axon to grow out,
the point of application of forces being localized.

The conditions with the dendrites are quite different.

This process is by no means necessarily limited to one part of |
the surface of the cell since its whole body containing Nissl
substance is equally sensitive and any stimulation may cause
protoplasmatic shiftings in their direction, whereby the princi-
pal dendrite and finally the shifting of the cell-body itself will
doubtless take place in the direction of the maximal stimulus.

In other words, if another stimulus than the one which formed
the axis-cylinder reaches the cell, it will form no new way out,
since this would require more energy than a following of the
present path of greatest conductivity, but a new stimulus com-
ing from another center, may produce—or even must produce—
anew dendrite. Since the perikaryon is equally sensitive (except
the axon hillock) to it everywhere and since already existing
dendrites are not in its path, the nearest cellular or dendritic
surface will be the point of application for its influence, 1.e., for
the formation of a new dendritic outgrowth.

THE SELECTIVITY IN THE PROCESS OF NEUROBIOTAXIS IN
HARMONY WITH PSYCHOLOGICAL LAWS

I now come to the second and most important point in the
tract formation, that which determines the selectivity of the
definite connections.

It has escaped the observation of all the earlier investigators
that the selectivity of the tract formation depends upon simul-
taneous, or better, correlative, stimulation. Cajal assumed
chemical secretions coinciding with stages of evolution, also
ascribing an influence to the glia cells in the secretion of such
“substances attractives”’ and without pointing out by which factors

NEUROBIOTAXIS 285

these stages of evolution were defined, which he could not do since
his conclusions were chiefly, if not solely, based on ontogenetic,
that is engrammatic observations. Held speaks of a ‘“‘Prinzip
der Auswahl,” upon the character of which he does not enter,
and with regard to his own researches Harrison* justly remarks:

There is nothing in the present work which throws any light upon
the process by which the final connection between the nerve and its
end-organ js established.

That it must be a sort of specific reaction between each kind of
nerve fiber and the particular structure to be innervated seems clear.

That the relationship for the final connection, which holds
good in the central nervous system for the dendrites and the
cell-shifting as well as for the axis-cylinders exists in the cor-
relative, mostly synchronous stimulation condition of the
elements, I first deduced from the selective character of the cell
shifting, and this could be further clearly demonstrated by the
axonic connections existing in the nervous system. It even
explains a series of peculiarities in the course of the fiber tracts
which otherwise confronted us as constant but inexplicable facts,
especially in the so-called central motor tracts such as the
pyramids.

This fundamental law of neurobiotaxis shows us not merely
that the fundamental law of association in psychology is at the
same time an anatomical law, but also how wonderfully polar
the whole character of tract formation is, an how it therefore
falls within the range of the galvano-tactic and galvano-tropic
phenomena.

In order to explain this phenomenon of selectivity in an
electro-chemical way, I must draw attention to the following
points.

It is presumed that the presence of potassium salts has the
peculiarity that it greatly increases the conductivity of the
axis-cylinder for the electrolytic current.

There is even an inclination to ascribe the strong conductivity
of the axis-cylinder, as compared with the synapse, to the high
percentage of potassium salts in the axon (MacDonald, Macallum).

“” Harrison. The outgrowth of the nerve fiber as a mode of protoplasmic
movement. Jour. Exp. Zodél., vol. 9, 1910, p. 787.

286 Cc. U. ARIENS KAPPERS

We may assume that a state of stimulation once raised at the
beginning of that axis-cylinder will proceed rapidly—it is even
supposed under a gradually increasing force (the axis-cylinder
increases in caliber centrifugally: Johnston,** Tretjakoff‘+)—and
a current of relatively great negative electric potential reaches
the growing point of the axis-cylinder.

If we now assume that in the neighborhood of this growing
point two nerve cells lie, one of which is already in a condition of
stimulation but the other not, on which of these two cells will
this growing point then exercise the greatest influence, and which
cell will exercise the greatest influence on the growing point?

As we know, the cell which has just been stimulated will be
in a state of greater electrolytic dissociation than the cell which
is in rest.

The negative ion current which runs along the axis-cylinder
in its neighborhood, will find its natural selection in this strongly
dissociated field, and not in a cell which is not stimulated and,
being relatively indifferent with respect to this growing axis-
cylinder, does not form a place of selectivity amid all the other
passive (non-stimulated) cells which, so to speak are corpora
aliena for it.

Now we know (see above) that the dendrites of a cell begin
to grow out about the time when the telodendria of an axis-
cylinder reach it or approach very near to it, and this is in strik-
ing agreement with the explanation given here of the neuro-
biotactic processes, because at the moment when the approach-
ing and stimulated axis-cylinder comes into the vicinity of the
cell, the-influence of the approaching kathodic potential differ-
ence will make itself more strongly felt, and a shifting of the
protoplasm into its direction, i.e., a tropism towards the telo-
dendria, is induced, which is a kathodic phenomenon of irrita-
tion like most tropisms under normal circumstances where no
special conditions for a reversal occur.

48 Johnston, J. B. Additional notes on the cranial nerves of Petromyzon.
Jour. Comp. Neur., vol. 18, 1908.

“4 Tretjakoff. Das Nervensystem von Ammocoetes. I. Das Riickenmark.
Archiv f. mikr. Anat. u. Entwick., Bd. 73, 1909, plate 24, fig. 11.

NEUROBIOTAXIS 287

A closer approach of the two neurones, a contiguity, will be
the result. That this will not (or not easily) occur if the grow-
ing axis-cylinder reaches a cell in rest, may result from the fact
that this passive cell, or neurone, is not in that strongly electrolyt-
ically dissociated condition and possesses no considerable electri-
cal potential difference from the surroundings. Or to put it
otherwise, the passive, non-stimulated cell has thus no other
significance for the growing axis-cylinder in its vicinity but that
of a corpus alienum, 1.e., it is fairly indifferent to it.

As far as concerns the fact that axonic endings never com-
municate with axonic endings and dendritic endings never with
dendritic endings, no further explanation is necessary from the
standpoint of polar electrolytic conditions accepted here, which
necessarily implies that homonymic outgrowths do not act on
each other.

FASCICULATION OF AXONS. IMPROVEMENT OF THE NERVOUS PATH

One might ask in this connection why nerve fibers, if homonymic
forces repel each other, tend to group together in fiber-tracts or
bundles, as we always see even if they do not end on the same
level (as the pyramidal tracts).

This process may, however, be analogous with the mono-
axonism, that an axon shall tend to place itself in the way of
the current, and if now such a current reaches a pluricellular
center it is not strange that the chief resultant line for the out-
growth of one cell is also the state of equilibrium for the out-
growth of the other adjacent cells. The orientation of a number
of nerve-fibers (axons) from a cell group into one bundle‘
may be no more than a repetition of the same process concerning
the neurofibrils in one axon, which tend to a state of central
equilibrium in the axis of the neurite.

Perhaps also a sort of magnetic field formed by equally run-
ning currents may exercise an attraction here. Such a magnetic
field is also present around colloidal threads.

45 Perhaps also a sort of magnetic field formed by currents running parallel
exercises an attraction here.

288 Cc. U. ARIENS KAPPERS

Just as we saw that with the dendrites of one cell the question
is different (see above), we also see that dendritic outgrowths
of more cells rarely fasciculate in a bundle. The latter would
be only the case if only one stimulation center attracted them
all, which rarely happens.

As far as concerns the neurites, I will discuss below still another
point in which it seems to be indicated that conditions which
hold good for one neurite may also hold good for a collection of
neurites, for a bundle.

I will not leave, however, the question o' interneuronal con-
nection without emphasizing that the greater conductivity of
the axis-cylinder (based on much more K and Cl) in comparison
with the dendrites, gives a peculiar character to the shifting
of the nerve cell in the direction of the center of stimulation.
This shifting causes a shortening of the dendritic path and a
lengthening of the axonic path for the nervous current and
consequently a diminution of the resistance, or if this expression
be less happy, an improvement in the conductivity.

It seems probable that the retardation which the nerve cur-
rent experiences in the synapse is diminished by this process.
Very interesting in this connection is Mauthner’s cell in fishes,
where the transmission of the afferent current takes place in
part on the axon cap itself (Bartelmez**), and where probably
the least resistant synapse is formed. .

Similar facilitation of the transmission of the current may be
seen in other structures concerned with equilibrium, e.g., in the
basket cells of the cerebellar cortex where, as Oudendal,*? among
others, has shown, fibrillae of the basket are continuous with
the fibrillae in the bodies of Purkinje’s cells.

Since the shortening contraction of the dendrites in such
cases as the descent of the facialis nucleus in mammals is ac-
companied by a lengthening (extension) of the axis-cylinder
(fig. 5), we may ask whether there is not an analogy of this process

‘6 Bartelmez, G. W. Mauthner’s cell and the nucleus motorius tegmenti.
Jour. Comp. Neur., vol. 25, pp. 87-128, 1915.

‘7 QOudendal. Ueber den Zusammenhang der Auslaufer der Korbzellen mit den
Zellen von Purkinje in der Rinde des Kleinhirns. Psychiatrische en Neurologische
Bladen, Amsterdam, 1912.

NEUROBIOTAXIS 289

a

with the process seen in muscles, which at the closure of the
current exhibit, besides the contract'on at the kathode, also
an extension at the anodal pole, the broad analogy between the
law of stimulation for muscles and nerves being known.

Scyllium canicula
(Fish)

Rad. desc. N.V. - i ae oe oe " Varanus salvator
Sse ide z

aS (Reptile)
VII nucleus -—\:

. Ib N ee

Axons -- ee pcaradh be
Mus musculus
ars:
ae un 3 (Mammal)
VII nucleus -- -
Pyramid spies, ole ee eee EEO as

Fig. 5 Migrations of the motor facialis nucleus in the animal series, which

is correlated with a shortening of the dendrites and an elongation of the axis-
cylinders.

290 Cc. U. ARIENS KAPPERS

THE FORMATION OF THE MEDULLARY SHEATH

The third point that might be mentioned in this discussion
is the question as to why most axis-cylinders in the central
nervous system get a medullary sheath, and why this medullary
sheath is not present on the cell body and the dendrites.‘*

If one were content here with a teleological explanation, it
would be sufficient to say that the presence of a myelin sheath
around the axis-cylinder probably has the function of insulating
the current, and that an insulating sheath should not occur in
places where this current proceeds from one neuron to another
(dendrites, cell body, telodendria). And yet that would not
bring us one step nearer to the solution of the question as to the
way in which the process of myelin accumulation is effected by
the axis-cylinder.

Let us endeavor here also to trace the influence which may lead
to the accumulation of myelin around the axon, and why it is
not accumulated sheath-like or otherwise in the cell and the
dendrite.

That the primitive axis-cylinder itself is able to form myelin
is proved most clearly in the central nervous system, where the
cells of Ranvier (i.e., of the neurilemma) which may have to do
with it in the peripheral nervous system, do not occur, and other
adjacent (glia) cells are but seldom found provided with myelin
granules.‘

48T do not refer here to the medullary sheath around the peripheral fiber
of a sensory root, which is a dendrite anatomically and ontogenetically (it
develops later than the central process). In the millions of neurons in the
nervous system this is the only exception, which certainly requires explanation
but at present need not disturb our reasoning concerning the central pathways.
The peripheral nerve fibers—especially the sensory ones—do not seem to be the
most adequate material to elucidate the questions involved here, since they
seem to require more explanation instead of helping to elucidate these questions.
Moreover the fact that spinal ganglion cells belonging to the sensory system of the
skin receive stimuli from other neurones (of the sympathetic system—Dogiel)
proves that nervous currents may also run toward their periphery.

49 Vignal. Le développement des élements du systém nerveux cérébro-spinal.
Masson, Paris, 1889. See also, Ariéns Kappers. Recherches sur le développe-
ment des gaines dans le tube nerveux. Petrus Camper, Amsterdam, vol. 2,
part 2, 1902.

NEUROBIOTAXIS ‘291

We know, from the researches of Ambronn and Held® that
myelin formation is greatly affected by the function of the tracts,
and consequently strongly influenced by the stimuli passing
through it.

I have already referred to the fact that the genuine a ueinons
substance and also the lecithin which forms the chief component
of the myelin sheath generally exhibit, under normal circum-
stances, an anodic kataphoresis.

Concerning the myelin itself this has been experimentally
shown by Hermann, who described its connection to the anode
as ‘“‘eine der gewaltigsten microscopischen Erscheinungen,”’
he ever witnessed.

Putting a part of a peripheral nerve of a frog in a constant
current in the line connecting the electrodes (which, however,
remained at a distance from its ends), he saw a vigorous outflow
of the nerve content—especially the myelin—at the anodal
pole of the nerve, where it collected in a mass.

Reversing the current, this myelin could again be absorbed
by the nerve and the myelin flowed out at the other (then,
anodic) end.

The tendency of the peripheral nerve constituents—chiefly
its myelin—to move in the direction of the anode is clearly
proved by this experiment.*!

If now we apply this phenomenon to the structure of the axon
in the central nervous system we may expect that the nerve
current which has—as pointed out above—an anodal direction,
will convey the lipoid substance, even that which is produced
by the cell itself, chiefly in the axis-cylinder; but, since from this
axis-cylinder an irradiation current of the same character flows
out, the myelin is necessarily conveyed to the periphery of the
nerve fiber.

The difficulty consequently is not why only axis-cylinders
have myelin and why this myelin is conveyed from the center

50 Ambronn und Held. Ueber Entwicklung und Bedeutung des Nervenmarks.
Sitzungsverichte der Kén. Sichsichen Gesellschaft der Wissenschaften, 1895.

51 T am much indebted to Prof. Héber (Kiel) for calling my attention to Her-

mann’s paper, which was unknown to me when | started to write this article.
It is found in Pfliiger’s Archiv, Bd. 67, 1897, p. 240.

292 Cc. U. ARIENS KAPPERS

to the periphery and there gathering sheath-like round it:
but the greater difficulty is why it remains there, and why is it
not conveyed further away from the sheath. Perhaps in the
beginning of sheath formation this really occurs (some glia
cells and lymphocytes are found richly provided with myelin-
like or fatty granules), but when its formation becomes more
abundant it prevents by its nonconducting character the anodal

Ependyma of the
dorsalsac (Parencephalon)

Com. superior
telencephali
- (amyelinated fibers)

r. :
Werte: Seep GSies woes
Baers Fon a a ET Spe

SE 8 cae

Fig. 6 Sagittal section of the habenular ganglion of Scyllium canicula, show-
ing the position of unmyelinated fibers surrounding the myelinated fibers.

current from extending its course and consequently its conveying
influence (kataphoresis) beyond the wall of myelin which thus
thickens more and more.

An induced anodic condition of the direct periphery might
then also cause lecithin substance of surrounding tissue (Ran-
vier cells) to gather on the sheath.

Why we do not find an accumulation of the same substance
at the apex of the axis-cylinder, why the telodendria remain
free from it, is difficult to explain. Perhaps that the conveying
character of the current for this substance is so considerable
there that it does not remain there when formed.

NEUROBIOTAXIS 293

In connection with the accumulation of myelin in the periph-
ery of the axis-cylinder I wish to mention a fact which struck
me repeatedly in the study of the cerebral commissures of ower
animals, where (e.g., in the commissura superior habenulae of
plagiostomes fig. 6) we frequently observe that the medullated
fibers are arranged in the decussating bundle on the periphery of
the non-medullated fibers. The same fact struck me often in
the fasciculus retroflexus, especially in Arius.

Sheldon, too, noticed this in his study of the olfactory tracts
and centers in teleosts, and he makes the same remark with
regard to some thalamic tracts. Whether this is to be explained
as a repetition of the same process—an analogy—of peripheral
accumulation of myelin in the medullary sheath, I do not venture
to say. It seems probable, since we saw that also in another
respect (monoaxonism and fasciculation, see above) the principle
that holds good for an axon seems to hold good also for a col-
lection of axons.

Here, however, we transgress the limits of a scientific hypoth-
esis, which, though not pretending to be more than a mere
hypothesis, must be founded on facts.

I would be perfectly content if this short note might stimulate
others to think about these matters. The dynamic polarization
of the neurone and its biologic character still require a good deal
more light than has as yet been shed upon it, and is worth the
attention of our best physiologists and biochemists.**

RESUME AND CONCLUSION

From the shiftings exhibited (phylogenetically) by the cells
of the motor nuclei it appears that those parts of the neurone
that receive the stimuli (dendrites and cellular body) are formed
and directed to those stimuli trying to approach their center.

® Sheldon, R. E. The olfactory tracts and centers in teleosts. Jour. Comp.
Neur., vol. 22, pp. 177-339, 1912.

°3 T have only one more remark to make. Darwin once said that plants think
with their roots. He did not mean this in a literal sense, of course, but that
there may be some similarities between the sensibility to certain stimuli and the
behavior of the roots of plants (or other centers of growth) and parts of the nerv-
ous system, chiefly the axons, does not seem so very improbable.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

294 Cc. U. ARIENS KAPPERS

Further researches show that this influence is found only on
such nerve cells as have already a certain previous indirect
affinity with those impressions, or with the region where those
impressions accumulate, and it can be proved that this affinity
consists in a simultaneous or successive condition of action
(stimulative correlation); and that consequently, in the material
arrangement in our brains, the law appears which has been
long since acknowledged to be one of the main laws for the
development of our mental capacities, viz., the law of association.

The acknowledgment of correlated function as the fundamental
factor vn the arrangement of the cells and dendrites induced me to
investigate whether the same law could be shown in the final
course and connection of the axons to lower or higher centers
(so-called central-motor paths and higher sensory neurones),
and a careful comparison of the regions where such paths begin
and terminate, showed, that here too such an associative affinity
could be pointed out, that this affinity determines the place
where the axon will end, and explains a number of peculiarities
in the ending of such paths, e.g., throws a light on the singular
fact that the pyramidal tracts do not originally terminate in
motor, but in sensory regions.

Under this fundamental law, that neurobiotactic processes
occur between correlated systems, the tropism of the dendrites and
cell body takes place in an opposite direction to the nerve current,
i.e., towards the center of stimulation: stimulo-petal, whereas
the course of the axon conducting the impression farther is in
the same direction as that current: stimulo-fugal or (more
correctly) stimulo-concurrent.

That, however, also the development of the axon is a con-
sequence of the stimulus has been proved by Bok, who in an
equally convincing and ingenious way showed that at first the
axon does not conduct a stimulus irradiating in the nervous
system, but that on the contrary, this stimulus forms the axon
so that also here a stimulogenous formation occurs, described
by Bok in a very important contribution to our knowledge of
neurobiotactic processes, under the name of stimulogenous
fibrillation.

NEUROBIOTAXIS 295

Taken all in all, we can say that the stimuli which arrive in
the nervous system, especially the relation between those stimuli,
mold the material substratum of the mind; this correlation is
the primary force, and expresses itself in the material arrange-
ments of our nervous system.

This correlation of stimuli thus plays the fundamental part,
in all processes of neurobiotaxis, in which, however, the dendrites
and the cell body grow towards the stimulus center stimulo-
petal, whereas the axon grows away from the stimulus center,
with the influence irradiating from it: stimulo-concurrent.

The question is now: how can we explain these different
tropisms in the nervous system; how can it be, that one nerve
unit, the neurone, shows such a clearly opposite polar difference,
that one part of its protoplasm approaches the source of stimula-
tion (stimulo-petal dendrites and cell body), while the other grows
with the direction of the stimulus-irradiation proceeding from
it (stumulo-concurrent axons)?

In order to find the solution of this problem, we may study
the other tropisms in nature, which are more accessible to experi-
mental research, especially the galvano-tropisms.

In galvano-tropisms we find phenomena which remind us
most forcibly of the manifestations in the nervous system just
described. .

By galvanotaxis we understand the fact that a living being or
part of it, when placed in a constant electric current of certain
strength, is inclined to turn towards a certain pole, in most, or
in nearly all cases towards the electro-negative pole (the kathode).
Thus the root-tips of plants grow towards the electro-negative
pole, monocellular animal organisms move in that direction.

The process 1s however reversible. By putting the object, such as
the root-tips of growing plants or the monocellular animals in a
stronger solution of chloride of potassium or sodium (which at
the same time increases the conductivity of the solution) the
tropism is reversed and goes towards the positive pole (anode).

Albumen also shows a shifting in a galvanic current (kata-
phoresis).

Contrary to the above-mentioned tropism, the shifting of

296 Cc. U. ARIENS KAPPERS

albumen and lecithine takes place under ordinary circumstances
(that is to say in the circumstances in which it usually occurs in
animal bodies) generally towards the positive pole. Addition
of potassium also enhances the anodic character of this process,
and the substance of the axon and myelin sheath of a nerve root,
just cut from the body, shows in a galvanic current even a very
strong displacement toward the positive pole (Hermann).

By acids the removal of the albuminous substance may be
reversed, however, and directed towards the negative pole.

There is much evidence that these galvano-tropic and kata-
phoretic experiments are applicable to the formation of the
nervous system by the stimuli that reach it and act in it.

We know from the negative variation that a part of our nervous
system which is stimulated forms a negative pole, a kathode,
with respect to its surroundings, which in other words form an
anodic field with regard to the center of stimulation.

The nerve-cells which are found in the surroundings of this
electro-negative center of stimulation, will first show an anodic
offshoot going in the same direction as the radiation from that
center of stimulation, on account of the anodotropic character
of their protoplasm. This anodic extension, will derive chemical
and tropic characteristics of the potassium and chlorides in
which it is imbedded. ‘In consequence a larger quantity of
potassium chloride is found in the axis-cylinder than elsewhere
in the neurone (as Macdonald and Macallum and Menten showed
independently of each other and in different ways). This
large quantity of chloride of potassium (conformably to the
above-mentioned experiments with root-tips and ameba) will
again enhance the anodotropic, in casu stimulo-concurrent char-
acter of the axon, and besides it increases its conductivity.

Not until much later do the dendrites appear, and somewhat
later still the cell body begins to move in the direction of the
stimulated electro-negative center.

** Why so much Cl is found in the axonic part of the neurone is unknown.
lt seems possible to me that this is due to the anodotropic character of Cl, this
being ananion. A greater permeability for anions might then be the cause of the

enhanced anodotropic character of the colloid substance of the axon. There is
much in favor of this, that it would be rather the chlorine than the potassium.

NEUROBIOTAXIS 297

This stimulo-petal, kathodic tropism of the dendrites and of the
perinuclear protoplasm is probably a more complicated phenom-
enon, which however is not counteracted by K and Cl, since
this does not occur to any considerable amount in those parts.
On the other hand, it may be favored by a kathodic kataphoresis
since it coincides with the appearance of the nuclear acid de-
rivatives, known as Nissl’s bodies, and does not take place until
the axon has nearly reached its terminus and the neurone is
therefore in a much greater state of perfection (Cajal). This
kathodic tropism, followed by a gradual shortening of the
dendrite and a displacing of the cell itself (as in most kathodic
‘tropisms), is in accordance with the phenomena of kathodic
stimulation, according to Pfliiger’s law (Loeb and Maxwell,
Boruttau), as these become apparent in animal protoplasm
susceptible to stimulation (e.g., also in ameba under normal
circumstances) and causes these parts of the neurone to find their
way to the electro-negative field which is in a state of stimulation.

It is probably accompanied by a facilitation of a stimulus-
transition at that place at the moment when the galvanic cur-
rent which appears in the nervous system makes itself felt (the
enhanced sensitiveness at the kathode well known in neurology).

Thus we find in the first development of stimulo-concurrent
axons a consequence of the enhanced anodotropic character,
experimentally proved, of their substance, strengthened by the
large quantity of K and Cl, while the formation: and contraction
which takes place much later of the dendrites, and the displace-
ment of the perinuclear protoplasm to the kathode is a special
case of Pfliger’s laws, not counteracted by any amount of KCl,
perhaps even favored by nuclear acid-derivatives.

Such may be the explanation of the dynamic polarization of the
neurone. It does not, however, tell us anything about the final
connection of the axis-clyinder.

This final connection is always a territory or cell which has a
correlated activity, that is a simultaneous electrolytic dissociation
with it. Non-stimulated centers are all equally indifferent to
it, i.e., corpora aliena to it. We further saw that monoaxonism
is a result of the effect on the same pole (a resultant line of different

298 Cc. U. ARIENS KAPPERS

forces on the same point of application), while polydendritism is
possible and even usual on account of the fact that their forma-
tion is not a resultant line of different forces on the same pole
(mathematically expressed of different forces on the same point)
of the cell body since the perinuclear and dendritic protoplasm
is equally sensitive everywhere to the kathodic influence and may
respond at several different places to several stimuli of different
origin, each of which may affect it on that part of its wbiquitous
receptive surface that is nearest by.

The ability of the neurone to receive at the same time more
than one stimulus by different dendrites and to lead their com-
pound along one axis-cylinder, may be considered as the material
expression of the formation of a compound impression from
different perceptions, and this compound again acts as a factor
in the formation of higher, more complicated compounds, if the
axis-cylinder runs in the cerebral direction. If it runs in the
aboral direction, the axis-cylinder is the final common path
leading to a somatic effector center.

It seems hardly necessary to emphasize that I do not believe
that nervous life and still less its psychic, conscious realization
are, or even could be, explained by such considerations—pro-
vided they are right. They may, at the best, give us an idea of
some physico-chemic processes that accompany its evolution
and explain the form in which our nervous elements appear.
After all, ‘“‘life’ and its ‘‘données immediates’’ (Bergson)**
remain as self-imposing truths, that are revealed in but not
explained by any phenomenon whatever.

55 Bergson. |’Evolution Creatrice. Felix Alean. Paris, 1907.

FURTHER VERIFICATION OF FUNCTIONAL SIZE
CHANGES IN NERVE CELL BODIES BY THE
USE OF THE POLAR PLANIMETER

DAVID H. DOLLEY
From the Pathological Laboratory of the University of Missouri

THREE FIGURES
INTRODUCTION

Kocher (‘16) has recently published several papers which
make a sweeping denial of any morphological evidence of func-
tional activity in the nerve cell body. He bases this conclusion
chiefly on his failure to find constant differences in the average
size of cells between exercised and undisturbed animals by the
use of the polar planimeter. It became necessary, therefore, for
the writer to test his own positive finding of functional changes
by this method.

The writer has, indeed, used the polar planimeter extensively
to determine the areas of constituent sections of individual cells
in one micron series. This gave part of the data necessary in
calculating individual cell volumes by the prismoid formulas.
Some of these results are published (‘14), some are not. It did
not appear then, on objective as well as theoretical grounds, that
the method by itself would add any essential information re-
garding the functional reaction that was not already supplied
from average of diameter measurements. This the present re-
sults confirm.

It is further my more personal task to reply because the crit-
icism of Kocher is directed entirely at myself, and patently aims
to discredit methods, technic, and conclusions. One only finds
advantage in a censoriousness which overshoots the mark, but I
deprecate being singled out from the large company of workers
who have been convinced that there are morphological evidences

299

300 DAVID H. DOLLEY

of activity. Among these there is noteworthy unanimity when
the phases with which they worked and the parts which they
studied (differentiation) are taken into account, as I have at-
tempted elsewhere to show (711 b). Indeed, from the literature
with which I am familiar, the score stands at present as eighteen
who are willing to admit changes of one sort or another against
two who are skeptical (Eve and Kocher).

Particularly, the credit must go to Hodge, the pioneer. It has
been the writer’s fortune to do no more than confirm every find-
ing of importance which Hodge made. Furthermore, without
these findings of Hodge and his deductions therefrom, and with-
out the contributions of his immediate successors toward filling
in the gaps, the interpretation of the sequence’ of events in the
more highly differentiated cells would have been enormously
difficult for a single investigator, if not impossible, even though
he profited as much as one could by the advance of cytology in
two decades and more. And cytology is just what is meant.

AN ANALYSIS OF THE DENIAL OF A FUNCTIONAL MORPHOLOGY

Were it not for the technical difference of method, repetition
of Kocher’s work would not be necessary, for a critical analysis
of his paper will easily show that certain of his essential con-
clusions do not mean what he appears to think they do, but on
the contrary afford a confirmation of an essential principle of
nerve cell function. This critical discussion will be taken up
first, as it explains why there is no need for further experimental
data than is submitted.

The writer has divided the progress of functional activity
from rest to organic exhaustion into thirteen stages for the Pur-
kinje cell. Kocher states: ‘‘Representatives of practically all
these types of cells were found in my specimens, from the rest-
ing control animal, as well as from those animals exercised for
one, two and a half, and five hours.”’ These stages were so defi-
nite that he counted over three thousand cells in order to deter-
mine the varying distribution (Kocher, table 3).

Our objective findings therefore are the same; the stages exist;
there is no rigid morphology of the cell. He does not explain

SIZE CHANGES IN NERVE CELL BODIES 301

them; he does not appear to think they need explanation; he
does not even consider why he finds them with his technic when
he is so harsh with me about my technic; but he sees them.
Coming as this confirmation does from a professed critic, with
the prestige of a great laboratory behind it, it will doubtless
carry unhoped for weight. The confirmation imposes the greater
debt in that this arbitrary division of a continuous process was
carried to a degree which on its face must have appeared suspi-
cious, though the number was due to the codrdinate inclusion of
intermediate stages, and the division was a practical one for
study.

The effect of varying function between one animal and
another must be either qualitative or quantitative, granting that
there is an effect. For the purpose of further analysis of
Kocher’s findings and conclusions, these possibilities must be con-
sidered separately, and the question of qualitative differences
will be taken first.

His final conclusion which relates to this point is: ‘‘ Further-
more, no qualitative differences in histological characters could be
found between fatigue and resting nerve cells.’’ Or, as it reads
somewhat differently in the text: ‘‘There are neither progres-
sive changes in the morphology of the cells from rest to exhaus-
tion, nor are there any qualitative or quantitative differences in
type of cells from resting and fatigued or even exhausted animals
(italics mine). Qualitative cellular difference between animals
in relative degrees of activity is what he wishes to specify, and
assuredly there is none, if representatives of the thirteen stages,
in orderly relation, are to be found in all, and only those. But
Kocher is artlessly misled because he finds all stages in the ‘con-
trol’ as well as the exercised animal. So his conclusion of lack
of qualitative difference does not mean what he thinks it does,
that nothing has happened. On the contrary, it is a fundamen-
tal conclusion that qualitative differences from function are to
be ruled out. Instead of being destructive to me, this is the
first induction I should wish to be confirmed, since it throws
comparative function on the quantitative principle. It is only

- that our opinions of the significance of an identical conclusion
differ.

302 DAVID H. DOLLEY

It is for qualitative changes for which the main search has
been made, and it is on this point that many interpretations have
foundered. Kocher, so far as he expected qualitative differ-
ences, predicated it on the idea that the cells of an animal pur-
suing its ordinary course are static. That, though possibly not
exactly accessory appendages, still they are unaffected by the to
and fro swing of ordinary existence. He simply neglects the
conception which came in with cellular biology that every. phe-
nomenon of life of the organism is referable to its cells. For he
speaks of the ‘“‘resting cells of the controls” as if all cells in the
undisturbed animal are necessarily static. Only when the extra-
ordinary thing happens then, like being chased around in a tread-
mill, or overdrugged, or cut for appendicitis, should changes be
expected, and these of a peculiar, not to say specific nature, to
fit the assault on the integrity of the cell? When they do not
appear, it is necessary for him to believe that nothing has hap-
pened. But the most ordinary vital phenomenon is a cellular
phenomenon just as well, and must be correlated with the whole
range of extraordinary phenomena. Were nervous phenomena
qualitative, an infinite range and variety would be necessitated.

No animals can be conceived to be static, in one fixed state.
Every reaction of an animal comes from its cells; the outside
environment may disturb those cells. Even the most quiet ani-
mal outwardly might be expected to reflect its own internal
work, and the possible effect of a changed internal environment
on a tissue specialized for irritability has equal possibilities, as
the anatomical facts have proved.

Hence it is that one finds, and would expect to find, varied
evidences of function in different ‘normal’ animals. The only
result of the extraordinary function on this basis is to drive the
cells further along in their phases of reaction, a quantitative differ-
ence in the sum total of reaction. All are in tone, many are
already working,—to this is added more work. The mere exist-
ence of morphological differences within the same animal
would be sufficient clue to something happening which needed
to be correlated and interpreted, when it comes after any technic.
Otherwise all cells would look exactly alike.

SIZE CHANGES IN NERVE CELL BODIES 303

So, can one pick up any animal regardless of its individual
existence, and use it as a control, expecting that it give neces-
sarily a flat level of comparison against other animals of differ-
ent habit, different experience? The true standard exists in the
resting cell, a distinct morphological type, a constant species
type (Dolley, ‘14). The only exact comparison between two
individuals is in terms of the relative distribution of working to
resting cells. Unless one recognize this, he will surely become
involved in a maze of discrepancies.

It now only remains to explain why Kocher failed to find
quantitative differences, as already noted in the citation, to nul-
lify his criticism entirely.

Kocher made differential counts of the distribution of the
stages in four dogs, one the undisturbed control, the others exer-
cised one, two and a half, and five hours respectively. He says:
‘As will be seen in the table, the number of a particular type
of cell varies considerably, but this variation is the same for the
different animals.’’? The understood conclusion is that all were
on the same plane, even granting the existence of morphological
types.

On scrutinizing Kocher’s table 3, one is immediately struck by
what may be a most significant point. Stage 6 stands conspic-
uous by its paucity, if not absence. Taking the counts from
the worm of the cerebellum, he found none in the control ani-
mal, though identifying all succeeding stages. In forced ac-
tivity, the greatest number thus identified was 2 out of 300 cells
surveyed, while in the hardest worked animal none was found.
Nor in the cord counts is he more liberal, three being the maxi-
mum found. Of course, in actual counts of 200 cells in a survey
of 300, this may happen, but from my experience it is not so
uniformly lkely. The average run of stage 6, where all types
are present, has been from 4 per cent to 10 per cent. For ex-
ample, in the first series of counts published (‘09 ¢), there was
a& maximum of 67 out of 600 cells actually counted (11 per cent),
after six hours of exercise, 40 (6.6 per cent) after one hour, and
a& minimum of 25 (4 per cent), in a relatively very resistant dog
in the effect displayed. A failure to identify stage 6 would dis-

304 DAVID H. DOLLEY

turb a count quite considerably. What are the characteristics of
stage 6? Standing at the transition point between the shrunken
hyperchromatic Hodge stages and the following hypochromatism
and upset of the nucleus-plasma relation, it has a more swollen,
vesicular and disproportionate nucleus than the resting type,
though its plasma now comes to show the average distribution
of chromatic substance of that type. I have pointed out several
times that unless its nuclear size and appearance be kept in
mind, it will be mistaken for a resting cell.

A second point: Stage 13 is one of complete basic dechro-
matization. The Nissl substance is gone, likewise the nuclear
chromatin. The rapidity of such dechromatization depends on
the relative differentiation. It may appear within a few hours
in the Purkinje cell, though probably not unless the animal is
advanced in activity to start with. Not only has it never come
under my observation in a lower type of cell within the time
necessary to produce it in the cortex, but the indications have
always been that the lower cells at this time were many stages
removed from exhaustion. It was only marked, though still not
absolute, after two weeks of continuous excitation of the cray-
fish cell.

Yet Kocher is extremely liberal with stage 13. He always
finds it in the cervical and lumbar cord cells, and in two cases
out of the four animals counted there are more than from the
cerebellum. Not only do I regard this as impossible on the basis
of differentiation, but it does not jibe with the text, for he only
mentions grades of plasmic chromatolysis, which obviously is
another thing from nuclear plus plasmic dechromatization.
Nuclear dechromatinization would exact a comment from any one.
In other words, some at least of the stages identified as exhaus-
tion are fairly doubtful, and this carries closely related stages.
One is forced to the same deduction for Kocher’s whole table 3.

It is the sort of rebuttal of a criticism that personally is very
distasteful, for it carries the possible imputation that the orig-
inator of said stages is the only one competent to pass judgment
upon them. This is not true, for eight students who have
worked with me have had no difficulty after several months :

SIZE CHANGES IN NERVE CELL BODIES 305

study in separating them as well as myself. As Kocher denies
‘progressive changes in the morphology of the cells,” it is evi-
dent that he missed the finer points essential to a differentiation.

Outside of these technical points, no denial of the existence of
quantitative differences can be made on the comparison of
four animals. The range of individual variation is too great.
There is no way of telling what the state of activity with which
the experiment begins. Kocher’s control animal may very well
have been two or three times as functionally advanced as the one
exercised the most was to begin with. I have seen several undis-'
turbed animals who showed a degree of activity almost as great
as one subjected to exhausting overstrain. The control com-
parison method, though valuable and frequently the only re-
source, affords no absolute deductions, unless all conditions are
certain. Apparent inconsistencies, of which I have encountered
many, one by one have cleared up as all conditions became known.

Just for one example, age is a factor. Very young animals
usually show a hyperactive state as compared to the adult.
Resting and early active cells may be absent in section after sec-
tion. Very probably this is the reason why Kocher’s three
month old puppies showed ‘‘no discoverable differences in stain-
ing reaction.”

One final rejoinder concerns a matter, which, though even
more distasteful, I refuse to pass over. In April, 1910, I pub-
lished the results of 2200 cell measurements. Even in the pre-
liminary communication of November, 1909, on normal func-
tional activity, which Kocher cites, the results from 1500 of
these measurements were stated, which explicitly did not include
those previously published from the shock and hemorrhage
series. Further, in the same paper the results of differential
counts of 3,600 cells were included. In still earlier communi-
cations, of April and July, 1909, on shock and hemorrhage re-
spectively, which he also cites, it was made sufficiently clear that
preliminary counts of 1300 and 1200 cells had been made, as it
was stated that 100 cells were counted in each experiment.

From this brief survey, it may be imagined with what pained
surprise one reads from Kocher, ‘‘Obviously the observations

306 DAVID H. DOLLEY

were not over a large enough range of sections nor sufficiently
controlled by actual counts of the various types of cells’’ (p. 351).
Kocher’s work was finished in 1912, though the paper was not
published until June, 1916 (see his footnote, p. 341). Before the
end of 1911, I had published six papers, to four of which Kocher
refers specifically in the text, and cites three in his bibliography
—making an error in crediting authorship in that.

He then proceeds to juggle quotations to support his conten-
tion. As in any scientific writings, certain statements of small
numerical amount, treating of finer detail or representing very
preliminary work, are available. For example, he cites from
my second paper (Journal of Medical Research, vol. 21, 104):
‘‘Measurements were made of five cells of each type in two
anemia experiments, one a fatal resuscitation, the other a repeated
hemorrhage.”’ Meagre data surely, and it reads as convine-
ingly as a wilfully isolated text from the Bible. Only my next
sentence, which he does not cite, happens to read: ‘‘Since the
results are the same as for shock, the number is considered suf-
ficient for the present purpose,’ and the context goes on to
enumerate the detailed identity. While not stated in words of
one syllable, it conveys the impression to my mind at least of
a constancy of dimension for each type, even for five cells.
This is not all of the same thing, but it is enough. I leave the
verdict to those disinterested.

EXPERIMENTAL DATA

In imitation of Kocher’s experiment on normal activity, two pup-
pies were chosen. They were females, from the same litter, weighing
2.7 and 2.5 kilograms, and a few days over three months old. One,
the larger, was led on a fast walk over a country road course previously
measured by a Stewart odometer on a Hudson motor car. It was de-
sired to imitate Kocher’s very fast pace of fifteen miles in three and
one-half hours, but my two puppies had never been beyond the con-
fines of the six foot square cage in which they were born and so lacked
training. The animal trotted along willingly enough after it learned
what was wanted of it, but though short rests were allowed, the pace
was too fast, and before two hours it began to show distress. After
two hours and ten minutes it refused absolutely to walk any further.
The actually measured distance in my experiment was a trifle over
six miles. It was then carried to the laboratory, and just as Kocher’s
dog, killed less than one hour after the exercise ceased.

SIZE CHANGES IN NERVE CELL BODIES 307

TECHNIC

The unexercised animal then came into the experiment as the con-
trol, and every precaution was taken subsequently to preserve an exact
identity of treatment. The two were simultaneously anesthetized
with ether through the codperation of an assistant, and killed by simul-
taneous bleeding. Their brains were removed at the same time so far
as possible by duplicate motions, and the specimens from each drop-
ped at the same moment, into the same fixing fluid, in a single con-
tainer as follows: The bottles for each individual fluid used in fixing,
dehydrating and imbedding were divided by a perforated partition
into two parts and the material thus separated was subjected to iden-
tical conditions. Every transfer to the next solution was made by the
simultaneous use of two forceps.

The fixing agent used was

Saturatedumencuntcrehlonidesserar sc oe eee ce eer 95
40 per cent formaldehyde solution. ........¢...200005- 6245 03e22 520°: 5

The material was then run through the graded alcohols,—30 per
cent, 50 per cent, 70 per cent, 80 per cent, being iodized several days
in the 80 per cent to remove the mercury, 95 per cent and absolute. It
was then carried through xylol, xylol-paraffin, and two changes of 52°
M.P. paraffin with the same precautions of identical handling. Fi-
pay the exercised and control tissue were inbedded side by side in one

ock.

The sections were cut by the same stroke of the knife at five micra
in serial, and necessarily subjected to the same conditions of staining.
As customary, the stain used was Held’s erythrosin and toluidin blue.

Yet, save for a certain straining at a finicky precision, the pro-
cedure differed in no respect from previous ones, nor were the
results in any way superior. Still Kocher is very harsh with me
because ‘‘The control and fatigue material was handled entirely
separately. Slight unavoidable variations in the exposure of the
tissue to the various agents and different thickness of the cut
sections would make such material worthless for comparative
study.” Surely not quite so bad as that. Bichloride is bichlo-
ride and alcohol is alcohol, and there are some of us who think
that we get certain cell pictures because of the particular physi-
co-chemical conditions in the cells, for we get them in the same
animal by any fixing and staining reagent—and Kocher admits
that he got them by his method. A microtome that can be de-
pended upon to cut one micron serial sections, and there are

308 DAVID H. DOLLEY

two in this laboratory, will surely cut sections at five micra as
well tomorrow as today. Variations in section thickness are now
and again unavoidable of course, but that is a negligible factor
in median sections of a three dimensional and spheroidal body,
which, excepting the eccentric nucleus, are the ones we use
when plasma, nucleus, and nucleolus come into the same optical
field. For, quoting a mathematical authority (see Dolley, 714)
‘“‘the diameter of the cross section of a nearly spherical body
varies very slowly for plane sections nearly median or diametral.”
Here is the mathematical reason why averages of individual

xw

eens
ng ESR

z ee
B B*
Fig. 1 Diagram of the relation of section frustra to the cell outline in the
case of extra-diametral sections.

stages either of areas or diameters are dependable—they are
from median sections with little variation from that.

The negligible effect of one micron variations in five micra
sections may be illustrated very simply from the diagrams of
figure 1. They represent two cells 20 micra each in diameter,
which is the average for the transverse diameter of the Purkinje
cell. The diameter AB is through the axial or median plane of
each. Each section constitutes a frustrum and it is the edge of
the maximum base of the section frustrum that we outline from
the camera lucida. The frustrum in the left hand figure is a
four micra, and the one to the right a six micra section, both
being unfavorable possibilities outside of the true median section
containing AB. The dotted line in each case marked a coin-

SIZE CHANGES IN NERVE CELL BODIES 309

ciding five micra section. The points one would mark for the
diameter in either case are marked 2, and the slight deviation
from the perfect five micra section indicated by the dotted lines
as well as from the true median section containing AB is
apparent.

THE COMPARISON BY DIFFERENTIAL COUNTS

For the technical interests of this paper only the Purkinje
cell of the cerebellum is considered.

First it will be of interest to discuss the results of the differ-
ential counts which were made for the general comparison of
exercise with the lack of it. The conditions under which both
puppies had lived accounts to me for the striking difference that
resulted from unaccustomed exercise. They were not merely
rested up for a few weeks, for subsidence from activity of any
degree goes most slowly (Dolley, lla), but they had always
lived under general conditions unconducive to wide activity.

TABLE 1
Differential counts of cells

RESTING

han STAGES OF ACTIVITY UNCOUNTED CELLS
© O| 2] oo] o| oD! oO] Oo] o] oO] o| ao] o Hyper- Hypo-
a a|a\a|a|aleala|alalalal|a| chromatic | chromatic
~ »~ a ~ ~ ~ ~ ~ ~ ~ ~ ~ ~
DM NIN|A|N|A|INIA|A|N|D| n\n

Control animal..... 23 10|39|97| 6} 1) 3} 3} 3} 2} 0] 1) 0 105 7

Exercised animal... 0 2| 7/16) 2/13} 9/21)/24)43)12] 2) O 27 _ 122

The data from the counts are set forth in table 1. The counts
were made from paired simultaneously cut sections from the two
cases. In each case a start was made from a corresponding
corner and the line of Purkinje cells followed in continuity until
all in the section were surveyed, and then, as the full quota was
not made up in either case, one jump was made to corresponding
corners of a pair the third removed in the serial to avoid count-
ing any cell twice. Kocher’s prerequisite of a complete nuclear
membrane is as good a working basis as any, only it is to be
remembered that if one admits hypochromatic cells whose karyo-
some is not visible, he can only approximate the exact stage

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

310 DAVID H. DOLLEY

from stages 10 to 13, in which the state of the karyosome is the
criterion. The karyosome is thus most rarely invisible. The
cells which could not be exactly identified because not suffi-
ciently in section were listed as hyperchromatic and hypochro-
matic, as has always been the practice. The differences in the
counts submitted are as striking as in the diagnosed cells. Un-
less the differences are as marked as in the present case I do not
consider a count of less than 200 actually diagnosed cells in imi-
tation of Kocher as adequate, but the survey of 77 paired sec-
tions in this instance showed obviously that the general dis-
tribution was that of the count.

Table 1 may be summarized as follows: The transition to up-
set of the nucleus-plasma relation begins toward the end of the
Hodge stage 5, actually with stage 5”. This is a convenient
point of demarcation for comparison. In the control animal,
173 resting and early type cells, including stage 1-5’, are found,
while in the other only 25 early type cells, with no resting cells
at all, show the effect of exercise in driving cells beyond the
early stages. On the other hand, there are only 19 cells in vari-
ous stages of upset of the nucleus-plasma relation in the con-
trol as contrasted with 126 in the exercised animal. Taking the
undiagnosed cells, the hyperchromatic ones belong between
stages 1 and 5, the hypochromatic ones to the period of upset.
The ratio in the control is 105 to 7, in the exercised animal 27
to 122. The quantitative difference between action and in-
action is everywhere displayed.

It is also worth while to point out that there are no exhausted
cells, namely, dechromatized in nucleus and plasma, in either
case. The exercised puppy was not exhausted in the organic
sense, but had much reserve. Its immediate distress, so far as
it was nervous, came from the other side of fatigue, the waste
product reaction. With rest and elimination, it could have gone
on, as fits experience. Evén the finding of a quota of exhausted
cells does not indicate exhaustion of capacity—so long as there
are other cells.

SIZE CHANGES IN NERVE CELL BODIES ad i

DIFFERENT METHODS OF MEASUREMENT AND THEIR IDENTICAL
RESULT

Since this paper is primarily a comparison of technical methods,
and since the morphological characters of the stages have been
repeatedly summarized and will be again in a further paper to
appear, it does not appear necessary to repeat them here. The
stage numbers are uniform in all publications. The only chance
-of confusion may come in the combination of 4’ and 5’, 4” and 5”
in tables 2 and 3.

TABLE 2

Average diameters of functioning stages in centimeters

CONTROL ANIMAL EXERCISED ANIMAL

NUMBER OF

Diameters of cell

Diameters of
nucleus

STAGE

Diameters of cell

Diameters of

nucleus

6.86 X 3.43 2.38 X 1.59 1 6.91 X 3.44 2.40 X 1.67
7.57 X 4.46 2.65 X 2.16 2 7.42 X 4.35 2.61 X 2.08
6.84 XK 3.80 2.29 X74 3 7.25 X 3.69 2.11 X 1.51
6.43 X 3:31 2.25 X 1.29 4’ and 5 6.50 XK 3.29 197 Daler
6.87 X 3.33 2.37 X 1.50 4’’ and 5” 6.82 X 3.71 2.36 X 1.70
6.96 XK 3.49 2.50 X 1.76 6 CAA X 3279 2.48 X 1.87
7.23 X 4.07 2.49 X 1.93 7 7.81 X 4.13 2.45 X 2.01
8.23 XK 4.32 Pair lla) 8 8.43 XK 4.44 2.47 X 2.14
7.73 X 4.91 2.52 XK 2.22 9 8.47 X 4.98 2.49 X 2.21
8.33 X 4.90 2.51 X 2.28 10 8.34 X 5.09 2.53 X 2.30

It 8.58 X 5.67 2.57 X 2.33

Stages 4 and 5 comprise the Hodge types, which were thus
originally separated because stage 4 represents a more attenu-
ated and smaller cell, and resulted in a somewhat different nu-
cleus-plasma relation. In general, stage 4 now certainly has no
special significance in itself, as a phase of immediate activity,
but merely means a more slender cell to start with. It has,
however, significance in that the slenderness is due either to lack
of development or to functional atrophy. So for uniformity
these stage numbers were kept, though in later publications,
stage 5 has stood for the group. Then, after publishing in sim-
ple numerical order in a preliminary communication, it was
found further that actually there was a transition type between

312 DAVID H. DOLLEY

TABLE 3
Size relations of functioning stages (Areas in square, volumes in cubic
centimeters)
é AREAS BY RELATIVE Laan ie

POLAR NUCLEUS- VOLUMES AS NUCLEUS- | paRaLLELopip- | NUCLEUS-

NUMBER OF PLANIMETER PLASMA CYLINDERS PLASMA EDS PLASMA
STAGE COEFFICI- COEFFICI- COEFFICI-

SS SSS ENTS eee ENTS SESS ENTS

Cell | Nucleus Cell |Nucleus Cell {Nucleus

Control animal

1 16.33} 3.06 4.3 5EROL | 4.87 LORS) | 80 e7ill 602) ear

2 23.32) 4.58 4.1 104.01} .9.89 ONS MSO yA) sia) al

3 19.42) 3.30 4.9 13280, OafAl AS98577 Os O2| see

4’ and 5’ | 14.57) 2.30 5.3 AS E23 |e 22 OT lor oees|eAOL44| Sel lide

_4” and 5” | 15.46) 2.82 ACY FLAS eA | eed et > an ried nme are | sere

6 16.69] 3.42 3.9 Fah GO”, eG) |) bea) cat! 9.9

7 20.25] 3.85 al 33 S2F42 (243 LO OSG) O28 ae eles

8 22.91} 4.10 4.6 OSFOTI Selb) LA 15225911026) e ets 29

9 25.56| 4.44 4.8 |125.50! 9.86} 11.7 |186.33) 12.42) 14.0

10 27.72) 4.61 5.0 135.83] 10.51 11.9 |200.00) 12.86) 14.5
Exercised animal

1 16245) 3.21 4.1 56.59} 5.36 9.6 | 81.77} 6.69 il

2 23.11) 4.31 4.4 |100.53} 8.96) _ 10.0 |140.40) 11.29) 11.4

bb 3 18.66) 2.58 6.2 68.86] 3.90} 16.6 | 98.72) 4.81 19.5

4’ and 5’ | 14.80} 1.88 6.9 48.69] 2.27; 20.4 | 70.36] 2.88) 238.4

Amand 5” 17-16) 3202 4.7 63.66] 5.13) 11.4 | 93.97) 6.82 183 11

6 19.14) 3.77 4.1 ROA eo 973) 102256) S267) 10ks

7 22.02) 3.93 4.6 90.94, 7.90) 10.5 |133.21) 9.90} 12.5

8 25.59) 4.29 5.0 {113.60} 9.18) 11.4 |166.18) 11.31 13370

9 28 .23| 4.35 5.5) 408591 ORG) 12.6 |210.06} 12.16} 16.3

10 30.08) 4.74 doe) MSSelll) LOF90| as Om 216. 07Msrss|i lot

11 34.04! 4.87 6.0 {193.01} 11.35} 16.1 |275.84) 13.95) 18.8

the strict Hodge type, stage 4 and 5, with its dense crenated nu-
cleus and stage 6 with its edematous nucleus, to which nuclear
transition the plasma corresponded with an intermediate con-
dition. Rather than change the numbers, this transition stage
has been indicated since as 4” and 5” to distinguish it from the
strict Hodge type, 4’ and 5’.

Taking up the problem of measurements, it is obvious that
with the cell aggregate distributed as a number of distinct types
or stages, each of a certain significance, one thing to do is to

SIZE CHANGES IN NERVE CELL BODIES 313

measure these types selectively to determine their absolute and
relative sizes. This is what the writer has always done, and it
will be clear enough from the sort of information it affords that
it is the one thing to do. Another thing which might be done,
as Kocher did, and which has always been done by other investi-
gators, is to lump all cells irrespective of type and strike an aver-
age. The uncertainty of this method, which may or may not
give true results, will be very easily shown through the data ob-
tained by stage measurements. The employment thereof is the
one reason for the wide variety of results which has been obtained
by different workers.

The stage measurement is frankly a selective measurement,
but in the sense of the selection of types as they come, not of
picking cells here and there according to their probable suitability
to work out right. It can be made, and has been made, as rigid
in requirement as the aggregate way can be. The cerebellum is
peculiarly suited for this because one can start at a convenient
point and follow the line of Purkinje cells around with no danger
of doubling back until all have been covered. If one then exacts
the requirement to measure and diagnose according its stage
every cell in such a median section that its karyosome and the
trunk of its dendrite are included, he has no more leeway than
by the aggregate method.

The requirement of the trunk of the dendrite is specified for
the following reason in the case of the Purkinje cell. It is
known to be pear-shaped, which I have confirmed by some fifty
reconstructions in wax. I am not going to measure such a cell
unless it is obvious that it is fairly complete in the plane of its
longitudinal axis, for it would be averaging a spheroidal dimen-
sion with elongated ellipsoidal ones.

With a greater or less unequal distribution of stages, the quota
of a uniform number for each stage will be filled up at different
points, one by one all becoming full. One simply proceeds
from section to section, passing over those stages whose quota
is complete, ‘but adding to the incomplete as they come. Of
course this will demand many sections when a stage is scanty,
—for the almost absent resting cells in the exercised puppy it
took 77. One loses the benefit of that ‘‘cut-with-one-stroke-of-

314 DAVID H. DOLLEY

the-knife,”’ but in view of the explanation made and the results
to be shown, this is negligible.

Twenty cells were measured to a stage. ‘There were thus 200
cells (10 stages) measured from the control and 220 (11 stages)
from the exercised animal. Stage 11 was not measured in the
control for the simple reason that none was found.

The procedure was to outline the accepted cells one after
another by means of the camera lucida (Zeiss). The lens system
used was the Zeiss comps. oc. 12, homo. 2 mm. oil immersion,
tube length 153 mm., X 1960.at stage level. The magnifica-
tion was adjusted to conform to some previous measurements,
and it is believed that the area determined from larger magnifi-
cations is less subject to small mechanical errors. At the point
where the dendrite becomes of uniform size the cell outline was
closed. When the full set was outlined, the area in square cen-
timeters was read for each cell from the polar planimeter.. At
the same time, the maximum longitudinal and transverse di-
ameters of cell and nucleus were measured in one-half millimeters,
to compare and test the accuracy of this previous method. The
areas and dimensions were then averaged for each stage set.
The average diameters are set forth in table 2 and the average
areas in table 3 under that head.

From the average diameters the average relative volumes of
cells and nuclei were estimated. The third dimension being un-
known, it can only be approximated on the basis that it will
average the same as the transverse diameter—another thing
that individual cell reconstructions bears out. The term rela-
tive volume is used because the volume was calculated as that
of a parallelopiped, namely, the length by width by depth—the
longitudinal axis multiplied by the square of the transverse.
Mathematically, the formula for such elliptical bodies is 3 [2a.-
x. (b)2], in which a and b represent the major and minor radii.
For the sphere it becomes % (7 r°).

If the ratio between two such bodies whose radii are a and b
and a’ and b’ respectively be expressed as a fraction, it becomes

; a b?
on cancelling out the common factors ——, or the diameters
a

b’?’

SIZE CHANGES IN NERVE CELL BODIES oD

themselves may be used. The relative volumes as parallelo-
pipeds thus obtained are set forth in table 3.

The cell volume less the nuclear volume gives the plasma vol-
ume. The plasma volume divided by the nuclear volume gives
the nucleus-plasma coefficient—the size factor of nucleus to
plasma. The area relations of nucleus and plasma were also com-
puted. The plasma volumes are not stated, but the nucleus-
plasma coefficients appear in table 3.

Between the area method and the diameter method a third
combination is possible. The planimeter gives the area of a
roughly elliptical median plane of the cell. Multiplying this
area in each stage set by the corresponding transverse diameter
of the cell and nucleus gives another set of data of relative vol-
umes. This merely represents the volume as contained within
a cylindrical surface instead of a parallelopiped, and the differ-
ences are in proportion, but there was a curiosity to see how it
would work out. These figures are the middle set in table 3.

A graphic representation of the data of volumes and nucleus-
plasma coefficients gives the most convenient basis for technical
comparison. Instead however of publishing the three sets each
of size (area and volume) and nucleus-plasma curves from each
animal, only the three size curves of the control after the three
methods (fig. 2) and the three nucleus-plasma curves of the exer-
cised one (fig. 3) are presented. A reference to table 3 will show
that the trend of the counterpart figures is identical in the two
cases.

The reduction of the area and volume figures for charting
was made in terms of the ratio of the resting cell body to its nu-
cleus. That is, in the case of the area figures, the ratio of stage
1 is 5.3 : 1; the whole series of cell areas was divided by 5.3, the
nuclear figures by 1. This procedure has the value of making
the curves represent not only absolute size in the ordinates for
each stage, as would be obtained by any convenient divisor, but
also of giving the relative size of each succeeding stage to stage 1.
Further, since this procedure makes the cell and nucleus start
from the same height of ordinate, the shifts of relation between
cell body and nucleus for each stage are shown. It gives in assc- ©

316 DAVID H. DOLLEY

to see 5 OG Pie ere

Fig. 2 Size relations of function compared by the three methods.

SIZE CHANGES IN NERVE CELL BODIES 317

fey)

O

Pw)

po (OD! Seen ene 7 cee Sy! 16k ae

Fig. 3 Nucleus-plasma relations of function compared by the three methods.

318 DAVID H. DOLLEY

ciation with volumes then a graphic representation of the nu-
cleus-plasma relation.

In order to get all three graphs of figure 2 on the same page
the cell and nuclear figures were each further reduced one-fourth.
The lower graph is the area, the middle the relative volume as
a cylinder, the upper the relative volume as a parallelopiped.
The solid lines are cell, the broken lines nuclear sizes in each
case. As the relative sizes to each other by the three methods
have no significance, the graphs are conveniently placed one
above another, and their abscissas omitted for simplicity.

The three nucleus-plasma curves from the exercised animal are
made by plotting the coefficient figures for each set (table 3),
reduced one-half, as ordinates above a base line. They are like-
wise placed one above another in figure 3 for easy comparison,
irrespective of their comparative heights, and their abscissas are
omitted. The comparison is with the resting cell in each case
and not in terms of absolute values. However, the interrupted
scale to the left shows in centimeters the actual height for the
one-half reduction of each curve.

The technical methods may now be compared at a glance.
The planimeter or area method affords results that are abso-
lutely identical in every detail with the diameter method, both
in size and in nucleus-plasma relation. Note the crossings of
cell and nuclear lines in figure 2. Even the slight variation from
the usual steady upward trend from stage 9 to stage 11 in the
nucleus-plasma curve shows up in area and parallelopiped. Pre-
vious results by the diameter method are merely confirmed, no
more, no less.

The planimeter or area method alone, therefore, has no spe-
cial or superior value. ‘True, it gives the exact areas of any sec-
tion through cell and nucleus. It is a valuable check on the diam-
eter method, particularly in the case of irregular cells, but
the irregularity makes consideration of their three dimensions
essential.

On the other hand, exclusive use thereof, in my opinion, would
tend to make one think in terms of two dimensions, as Kocher
did, for his only reference to a third dimension is found in the

SIZE CHANGES IN NERVE CELL BODIES 319

heading of his table 2, ‘‘Volume expressed as square inches,”
a very plane conception of volume.

The area method necessitates the same resource to averaging.
Where relative sizes are all-important, the smaller variations in-
herent in the two dimensional measurements as compared with
the corresponding augmentation of these differences in greater and
ereater degree in three dimensional calculations is quantitatively
misleading and may cause important points to be minimized.
Take 2 x 2, and 3 x 3; one is 4, the other 9; but 2x 2x 2 is 8, and
3x 3x3 is 27. Here is another fault in all size comparisons in
the literature which makes them less productive—the diameters
alone were used (compare tables 2 and 3). Mathematically
stated, in a series of increasing squares the first differences are in
arithmetical progression, in one of cubes the second differences
are in arithmetical progression.

Along with the confirmation of previous technical methods
certain important conclusions are corroborated by the added
data.

In the first place, the nucleus-plasma coefficients of the resting
cell (stage 1) are 12.4 for the control, and 11.2 for the exercised
animal by the diameter method, and 4.3 and 4.1 respectively by
the area method. The average resting cell coefficient so far is
11.7 by the diameter method, and the range of deviation is 11 to
12.4. The two figures above, 12.4 and 11.2, fall therefore within
this range. Two additional individuals conform to the law, of
species identity of the nucleus-plasma norm (Dolley, ‘14).

In the second place, it may be noted that the coefficient figures
of stage 2 do not vary more than those of stage 1 just discussed
—12.4 to 11.2 and 11.2 to 11.4. In short, as has always occurred
in stage 2, though the size undergoes a 50 per cent or greater
increase, the nucleus-plasma relation remains constant. This
is most important for the deduction of an exact proportionate
increase of nuclear and plasmic materials in the beginning of
activity, a purely quantitative increase of the same materials in
each element.

The third point is the close identity of area and volume for
the resting cells in the two cases—16.33 and 80.71 with 16.45
and 81.77. What does it probably mean? The deduction has

320 DAVID H. DOLLEY

been made in previous work (’14, p. 494), and more strongly sup-
ported in some unpublished work, that function is the sole deter-
minant of absolute size. Non-divisional growth in mass is a fune-
tional growth. Here are two animals born together and living
under functional conditions as identical as may be. Their cells
show the same absolute size. It is a noteworthy verification of
the deduction.

It fits in with this relation of function to size that the evi-
dence is accumulating of a tendency to a ‘uniformity of absolute
size among corresponding nerve cells of animals of the same spe-
cies. When sufficiently demonstrated it would be understandable
on the basis of average general functional usage. The excep-
tions thereto so far in the dog, the unusually large cell, have
been associated with a known history of unusual training and
activity. It makes the nerve cell agree with Conklin’s conclu-
sion that within the same species cell size is approximately con-
stant. Making simply a statement here of the probable prin-
ciple, it is to be noted that these two dogs, being not yet grown,
offer no evidence for or against species uniformity of absolute
size, save that they are progressing together under identical
conditions.

It might be expected and to some extent it is true that all
stages succeeding stage 1, being based quantitatively upon it,
might show this same correspondence of absolute size. How-
ever, in all stages except stage 1, one encounters a shifting range
of size throughout the stage. The results will vary according as
the majority of cells are at one end or the other, or well distrib-
uted in the chance of a section. Stage 1, though there are inter-
mediate grades to stage 2, was frankly selected in both animals
as the nearly flat type, with this very point in view, and inter-
mediate stages were thrown to stage 2.

THE INCONSTANCY OF COLLECTIVE AVERAGES OF FUNCTIONING
CELLS

It only remains to demonstrate from the data in table 3 the
inconstancies which may result from averaging all cells irrespec-
tive of their functional state, and to expose the fallacy of deny-

SIZE CHANGES IN NERVE CELL BODIES oul

ing on such a basis the existence of functional size changes.
For the inconstancies, take the control data: The average area
of thes mallest cell, stage 5’—and the average covers its own varia-
tions—is 14.57, that of the largest is 27.72 or nearly double;
the average volume of the smallest cell is 70.44, that of the
largest 200 or nearly triple the size. In the exercised animal
with the still larger stage 11, the largest volume is nearly quad-
ruple the smallest, and its area again more than double. Is it
not apparent to any one that if such widely variant sizes or areas
are averaged, the result depends upon the particular distribu-
tion of types and that a wide range of results is possible? I,
out of 20 cells, even in area computation, 5 measure 14 sq. cm.
and 15 measure 27, the average is 24, whereas if 15 measure 14,
and 5 measure 27, the average is 17 sq. em. The results may
or may not prove anything about the immediate functional state.

One can then with fair probability explain what did happen in
Kocher’s case. He finds only small variations in average area
size between control and exercised animals and these not con-
stant. So far as different functioning stages appear, they tend
to be distributed rather than bunched. <A general average, taking
into account the smaller range of variation of area figures, would
tend to equalize the differences due to unequal distribution of
various-sized types.

So Kocher, having smoothed out individual cell variations by
averaging, found no great difference between an animal and its
control. His results are just what might be expected in prob-
ably the majority of cases, and instead of confounding the writer
in respect to functional size changes, tend only to support the
induction previously stated of a uniformity of cell size as a gen-
eral rule for a species. Were it not for this tendency to equality
of size of corresponding cells, collective averaging would not have
afforded so many positive results as it has.

Since the method of collective averaging is the one which has
been always used, how about such results as those of Hodge?
Are they discredited? No, but they must be qualified. Hodge
found a smaller size in the stimulated spinal ganglion as com-
pared with the unstimulated simply because there were enough

Bee DAVID H. DOLLEY

smaller cells to bring the average down. In such a slowly re-
acting type morphologically, it would be the usual result for a
certain period, but not indefinitely. Prolonged stimulation
would be driving the cells after they reach this point of shrink-
age to enlargement—and enough of these larger cells would first
equalize the comparison and then bring a larger size in the stimu-
lated cells. So Kocher did not with entire consistency obtain
Hodge’s results. Yet as a matter of fact, accepting the smallest
differences, he did duplicate Hodge’s results in eleven out of
fifteen series from the spinal cord and associated ganglia. Fur-
ther, his exceptions and the trend of his figures follow to a con-
siderable degree the explanation given.

To sum up for collective averages, variations may indicate
first the predominance of related types of present function.
When they do, the variations may be above or below the mean,
depending on what sized types predominate. No variations will
show up in certain distributions of types. Second, variations
may indicate an acquired state of functional hypertrophy, which
has nothing to do with the immediate function, but which, as
every one should know, may enter as a condition and not a the-
ory for the specialized cell. When this complication is introduced
it may combine to lessen, equalize or exaggerate the other possi-
bilities of variations from the first group of immediate function.
The functional hypertrophy has an opposite possibility, the func-
tional atrophy. Why waste any more time over this method?
It gave of what it has to the pioneers. It is a scientific solecism
that function, the one faculty which results from the differen-
tiated state, is the one and only factor which has been neglected
in making cell measurements on differentiated cells.

If any one wishes to investigate the size changes of function,
he must identify and measure functioning stages and the rest-
ing type from which they spring. This will give him function
in itself. The resting cell and that alone will give him species
size, its mean and its variations, species relativity of plasma to
nucleus, and a basis of comparison between individuals uncom-
plicated by the degree of present function. For species size,
once the mean is determined, the variations may be analyzed,

SIZE CHANGES IN NERVE CELL BODIES 323

and all complications of comparison sufficiently discounted.
For species relativity of plasma to nucleus, a constancy can not
be denied until it is analyzed apart from immediate function.
This applies not only to the nerve cell, but to all specialized cells
in their proper measure. Until that be done, criticisms of Rich-
ard Hertwig’s nucleus-plasma relation, of which the species con-
stancy is an extension for the nerve cell, are not worth the paper
on which they are written. When it is done, there is still no
reason to this investigator to doubt that the nucleus-plasma
relation will be a fact and no longer a theory.

CONCLUSIONS

The planimeter or area method applied to the stages of func-
tion affords results which are identical with those from the dia-
metral method of measurement, both in size and in nucleus-
plasma relations. Previous conclusions for functional size
changes and species size relations of nerve cell bodies are verified.

The value of the planimeter method is as a mutual check on
the diametral method, particularly in irregular cells. Alone it
has no special or superior value, while it gives only two dimen-
sions, with smaller variations than in volume calculation, and is
quantitatively misleading.

Collective averages of cells irrespective of their functional
state, which has been the usual basis of comparison between in-
dividuals, afford inconstant results and should be discarded.
It is a scientific solecism that function, the one faculty which
results from the differentiated state, is the one factor which has
been neglected in the measurement of differentiated cells. To
deny functional size changes on this basis because of small and
inconstant variations between one animal and another, as Kocher
has done, is a fallacy, and such results indicate only the tend-
ency to a uniformity of absolute species size for corresponding
nerve cell bodies.

324

DAVID H. DOLLEY

BIBLIOGRAPHY

Douiey, D. H. 1909a The pathological cytology of surgical shock. 1. Jour.

Med. Research, vol. 20, 275.

1909 b The morphological changes in nerve cells resulting from over-
work in relation with experimental anemia and shock. Jour. Med.
Research, vol. 21, 95.

1909 ¢ The neurocytological reaction in muscular exertion. Amer.
Jour. Phys., vol. 25, 151.

1910 The pathological cytology of surgical shock. Il. Jour. Med.
Research, vol. 22, 331.

1911 a Studies on the recuperation of nerve cells after functional
activity from youth to senility. Jour. Med. Research, vol. 24, 309.
1911 b The identity in dog and man of the sequence of changes pro-
duced by functional activity in the Purkinje cell of the cerebellum.
Jour. Med. Research, vol. 25, 285.

1914 Ona law of species identity of the nucleus-plasma norm for nerve
cell bodies of corresponding type. Jour. Comp. Neur., vol. 24, 445.

Kocuer, R. A. 1916 The effect of activity on the histological structure of

nerve cells. Jour. comp. Neur., vol. 26, 341; Jour. A. M. A., vol. 67, 278.

THE FOREBRAIN OF ALLIGATOR MISSISSIPPIENSIS

ELIZABETH CAROLINE CROSBY
From the Anatomical Laboratory of the University of Chicago

FORTY-SIX FIGURES

CONTENTS
Materialsramcdem ech GS nthe ceteris. 0.2 a1: ual lee ene esos std eons eee 327
HAN SCORUC ARM OES hee eed Mee cere is: cso, od ee on ah ete omigetite ace 328
GellGstrrcG tunes aes We Sey Pa RS TN Se co-chair ephee S R O 329
OTA ctor yl cd sca A perceavaets ee A Le 5h sop aR eae occa) PN eas ac St 329
DY Gree, le ial ee ee a ee ee 330
GramulexcellMlayieieccir < s.)% e-<.6,iac,5 1 Re a etree ea. S - 331
Olfactonyacnus neti teeter end acre. 508.555 tue SR a Seo apo ne air Anh sed
Centersiombne mens pMere: 20 966s: «li s2 Ra eae ace ook EOD:
Niuclensroliactoniustnterlors +... 4. seen eee eee eee ane 335
INTCASD ALO UMC OVI raya carers: : Ss 5-81 Oe Oe REE Dialect. heen, 335
ItMloeieul lan Olbitveuorbbnanl sso5e conc amenococecéusnccouoseuhsnooaenboe 338
INuclewsiconmmissurac hippocampi... 52 ste aeeeeien tes: . “ae ee 339
INucleusicommissunaeanteniOnis: ce oee eee eee eee aero oo
INCleUSh Dre Op LICMS Stes taee as 4 cree. Se os) oe EC ae See 340
Iinibenstitilallemucleuists.2e8 sects ties ote wees «optic een mee oe 340
: Nucleusiof thesdiagonal band df: Broca-sepsiecry.0e sci aie ok 341
Basdenuclewoimunenlatenalewallllaaess 2 see ee rere een paren ns rca 341
Functional complexes formed by the basal nuclei of the lateral wall 349
@ornpicalcenters:of theshemispheres....:. uae saree tee ci boas! 351
Wentersioithecdiencephalon: 22.4 sate ccc doo Sgeilvte «3 opts boldes Shoe n seu 359
1 ERySNT HL 73 LENS a UIST fo hereon types ee a ie Re 48 ch ea AN el ne 359
Raul pants. bial s Ae Moe eRe Ree ed eS Ue Uk eek Se Ed a eat OS 359
dB NOOR RL) Ova 7 FISK ee Oe a OO OR Oe tee, So aes, § O89 Pe ae eee oe 361
Mibericonne CHONS per er Ee ven) eee a ee STON ay Ae Re es ee 361
ETSctusOlLACKORI SH eT eee S cote Cntr Aer eee oe ee oe cat ee BOE
Tractus olfactonimeumed (align. i04550.02 0k st oleae. Dee leme VEN 362
Tractus oliactoruerimtenmed wis). aceise Getic. orale ela et. 362
Practusrolfactonitismaterslitsupe te. ye xictecee fie. oisse lovee tes «ele eve ented 363
SErAchusiuMbenculo-cOnticallis haan a Teeter ree ke cr ii eee 363
Baroliacto=cortical tracts ean eee eae a eens 364
ractus:parolfaeto-corpicalise sagen) ss 5 Sed kek: eu one 364
cRrachustcorieo-parolbactoOritls oar See eee 364

3250

THD JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

326 ELIZABETH CAROLINE CROSBY

Commissures oluhestorebraine-s)- eee ee eee eee ee eee ee 365
Commissura hippocampl............... ected hin i, od A AE ie ap eA Ee aa 365
Gommissuraranterior..4c sae eee: Oe ee eee 366

Tractiolsae diagonal bandiof:Brocatan... 0s: eee aes ce eee er 366

SErtacbemmarmaligeys «sic. sewer aot Rees Mee ec Re ee OT 367

‘ ‘Lheycommissural portionis..9-62 6-6 eee ee Ee eee eee 367
aRHEypreo pele, POLblOM waste eco re tn Eo eee 367

PANS CUS soiree ace else be eee oe eee EEE oe ee 368

EY Coc Ove) ts pena I Anon ARP ub i 06 Ton am ule in ine Melgar eer Cie be 368

Hibraetangentialess-<.ccnd cise eee ee ee ee ae ne 368

1 OY0Y i 01S, Cee Cera es bea i), ER RE eI a CNR IR Wee Rm: ORE Very ate ra) Bs | 369
Colummayforniciss2 cease Ce ee ee ras en ee ee 369
POrDIXA ON GUS scr. rp.< se oor ho eo Ne os en ee 369

Stria ime dulllanis: 3c. sh cs eee oo eee ee eee ae Se ee 370
‘iraActusicortico-nabenularis medialis: 4b eee ALE ee eee 370
Tractus cortico-habenularis lateralis anterior....................... 370
Fractus cortico-habenularis lateralis posterior...................... 372
tractus olfacto-habenulanigimedialistes. ss 54-0 pees nee nee 372
iractusomacto-habeniulanisilatenclisues sere eee eee eee 372
fractus olfacto-habenularis posterior... 2.00240... sees) cece oe 373

Olfactory proyection+tracts, 2. tc a eee 373
Ventral olfactory projection tract. fossa) hse oe a eee 373
@lisetory prorection tractiol Cajal sto es Sate ee ee ee 373

Basalkforebrain: bundles rs. 4 1h een eR ee pe tae Ae a 374
Miediralitionebrainybundlless se eee eae ene ee eee 374
wateralvforebrainibundlev sm an.ceeetis® stone a eee 375

General-dis cussion 4s) )o ke tee ee oe eee te ied te ee ee 375
Sinmniivary: 504 Aer Ne. Pee he OT eae cA et Pet ie eRe eee 384

The olfactory system is highly developed in the reptilian fore-
brain. Not only are large basal nuclei present, but there is
clearly differentiated cortex and all the important olfactory
tracts of higher forms are represented. The larger part of the
forebrain, then, is concerned in the reception of olfactory im-
pulses and their correlation with incoming diencephalic impulses
of various sorts. In fact, the diverse distribution of the incom-
ing diencephalic tracts, each of which has its own characteris-
tic functional significance, has been one of the prime factors
in the differentiation of the forebrain into its various cortical
and basal centers. Although the greater part of the reptilian
forebrain is under the influence of the olfactory fibers, there is
a considerable portion which receives a much smaller number
of these fibers and is dominated by the ascending fibers from
the somatic nuclei of the thalamus. Through these somatic

THE FOREBRAIN OF THE ALLIGATOR ah

diencephalic fibers, the functional motive for the formation
of the corpus striatum and the neopallum has been introduced
into the hemispheres and in the alligator forebrain these somatic
centers are already beginning to take form and to establish cer-
tain characteristic and fundamental relationships which will be
discussed later.

The specific distribution of the olfactory and the non-olfac-
tory fibers, the positions and the relations of the various cen-
ters and, finally, an analysis of these data in terms of their
functional significance, these are all essential to the adequate
understanding of the morphology and the evolution of the
forebrain. The present report is concerned with a descrip-
tion of these centers in the alligator forebrain and of the fiber
tracts which put these centers into relation with each other
and with the diencephalon. Finally, an attempt has been
made to effect a partial correlation and interpretation of the
factual data obtained.

The advice and assistance of Dr. C. Judson Herrick have
made possible whatever there may be of value in these notes.
For these things and for the opportunities accorded me in
his laboratory, I wish to thank him most sincerely. I am
indebted to Miss Jeannette B. Obenchain for assistance in
technique and to other members of the Department of Anat-
omy of the University of Chicago who have given helpful sug-
gestions. Mr. Streedain has very kindly made the drawings
of the gross material and has lettered the drawings of the micro-
scopic material and otherwise aided in preparing them.

MATERIALS AND METHODS

The animal chosen for study was Alligator mississippiensis.
The individuals were small, varying in length from 30 to 55 em.
The drawings of the surface anatomy (figs. 1 and 2) were made
from the brain of a 55 em. alligator. |

The silver impregnation methods of Golgi and Cajal and the
toluidin blue method were the chief ones employed. Two
rather imperfect series, one stained with Ehrlich’s haematoxylin

328 ELIZABETH CAROLINE CROSBY

and the other by the Leuden van Heumen method, were used
to check certain fiber tracts, but the paths described in this
paper are almost entirely those brought out by the method of
Cajal. These series were further supplemented by a trans-
verse series stained with carmine and a second such series stained
with haematoxylin (both the property of Dr. C. J. Herrick).
One of the Cajal series was very kindly loaned by Dr. P. 8.
MeKibben.

Several specimens were stained by various modifications of
the Weigert method. While the results were very satisfactory
for the study of parts of the brain below the thalamus, these
preparations contributed little of value in the study of the con-
nections of the cerebral hemisphere because practically none of
the fiber tracts in this part of the brain at the ages here inves-
tigated have become myelinated.

HISTORICAL NOTES

Rabl-Riickhard (’78) gave an-excellent description and some
very clear pictures of the gross appearance of the brain of the
adult Alligator mississippiensis. A brief description of the ex-
ternal form and some details of the microscopic anatomy of
the same species were given by C. L. Herrick (90), based upon
young specimens under 45 em. long. Figures of the alligator
brain are given in Wiedersheim’s Comparative Anatomy and
other figures and descriptions of the external form are scat-
tered throughout the literature and it is unnecessary to enter
into a detailed account of the gross relations, the essential fea-
tures of which are shown in figures 1 and 2. DeLange (711)
presents a series of photographs of surface views of reptilian
brains, among which are those of Alligator sklerops, and in a
later paper (713) the same author publishes a series of twenty
sketches of cross sections through the thalamus and the mid-
brain of this species. Unger (’11) has given a brief account of
structure and fiber tracts of the forebrain of young specimens
of Alligator lucius and Crocodilus niloticus which is preceded
by an excellent summary of previous work on the forebrain of
the Crocodilia.

THE FOREBRAIN OF THE ALLIGATOR 329

Many workers have indicated the presence of an epiphysis
in the alligator brain. The work of Albert Reese (10) has shown
the structure so named is a paraphysis and that no epiphysis
is present in the alligator, even in the embryo. In 1908, Reese
published an account of the general embryological development
of Alligator mississippiensis.

No attempt will be made to review systematically the ex-
tensive literature on the brains of reptiles in general, though
references to this literature will be made as occasion may
arise. Among the classical descriptions of the rept lian brain
especial mention should be made of the valuable descrip-
tion and figures of the turtle brain published in 1895 by Mrs.
Susanna Phelps Gage, in commemoration of whose important
contributions to comparative neurology the current volume of
The Journal of Comparative Neurology is dedicated.

CELL STRUCTURES

The positions of the nuclei of the telencephalon, with such
details of their cell arrangement and cell structure as have been
observed, will first be described, together with some more frag-
mentary observations on the diencephalic nuclei. Then using
these facts for orientation, the courses and connections of the
fiber tracts will be considered.

Olfactory Bulb

Johnston (’13) described the presence of a nervus terminalis
in the reptiles. The preparations available are not suitable
for the identification of this nerve in the alligator.

The cell bodies of the peripheral olfactory neurones lie in the
olfactory epithelium of the nasal cavity. Their axones pass
back as fibers of the olfactory nerve or ‘fila olfactoria.’ These
fibers are unmyelinated and, after a very short course, enter
the olfactory bulb. In its outer portion they break up into ter-
minal arborizations which form synapses with dendrites of the
mitral cells (figs. 23 and 24) and with other receptive cells of
the olfactory bulb.

330 ELIZABETH CAROLINE CROSBY

These places of synapse are called glomeruli and, scattered
among these glomeruli, are a number of small cells which send
their dendrites and, probably, their axones (though there is no
proof in the material used for this statement), into the various
glomeruli and so serve for the correlation of impulses. These
are the type that Cajal calls intraglomerular cells.

Mitral cells (figs. 23 and 24). In transverse sections, the
mitral cells have a ring-like arrangement around the granule
cells as a center (fig. 13). Near the anterior end of the bulb
they form a somewhat diffuse mass but soon take on their char-
acteristic arrangements. They are replaced by other cell groups
in the olfactory crus.

In the ventro-medial portion of the bulb, near its anterior
end, the mitral cells form a curious depression or ‘fossa.’ This
‘fossa’ was first described by C. L. Herrick (’90), who said that a
separate slip of the olfactory tract arises from it. The ‘fossa’
is very evident in both the toluidin blue series and those series
prepared by the Cajal method. In the latter series, the fibers
can be seen passing caudad from it and forming a part of the
tractus olfactorius. There is a special thickening of the glomer-
ular layer in that region, which pushes. the mitral cells inward
and causes the depression. Beyond being a point of entrance
for a particularly large number of olfactory fibers, it does not
appear to have any special significance.

A study of the toluidin blue preparations shows a considerable
variation in the shape and size of the different mitral cells.
On the whole, the nuclei tend to be rather large and are usually
placed nearer the ventricular border of the cell. An abundance
of Nissl substance is present in the cytoplasm.

The variations in size among the mitral cells are brought out
most clearly in the Golgi preparations. Some of the different
types observed there are illustrated in figures 23 and 24. Round,
stellate, and large pyramidal forms are seen. A mitral cell
usually has two main dendrites and several smaller dendritic
branches. The larger dendrites are thick and thorny and enter
into the formation of glomeruli with the incoming olfactory
fibers. The smaller dendrites extend as far outward as the

THE FOREBRAIN OF THE ALLIGATOR So!

glomeruli but have never been observed entering into the for-
mation of a glomerulus. They intermingle with the dendrites
of other mitral cells and granule cells and so make one of the
important elements of the plexiform layer. This plexiform
layer, then, provides an additional mechanism for the increasing
and the summating of stimuli.

The axones of the mitral cells arise from their ventricular
border. A short distance from the cell body, the axones of the
larger cells divide into two main branches of approximately
equal size. One branch enters the granule cell layer and comes
into synaptic relations with its neurones. The other branch
runs caudad in the tractus olfactorius, giving off, at various
levels, numerous fine collaterals into both the granule cell and
the plexiform layers. The first branch and fine collaterals of
the second branch are chiefly (although not entirely) to pro-
vide a mechanism for the summation and strengthening of stim-
uli. The main part of the second branch provides for the con-
duction of the impulse to the secondary centers. In some of
the mitral cells apparently only one of the branches may be
present. When this is the case, it is usually the one into the
tractus olfactorius which is represented.

Granule cell layer (figs. 138 and 25 to 28)* The inner granule
cells occupy the inner portion of the bulb about the ventricle,
next to its ependymal lining. Extending caudad, at about
the beginning of the olfactory crus, this layer is replaced by the
cells of the nucleus olfactorius anterior, although the material
at hand has not permitted the drawing of so precise a line of
demarcation as Johnston (’15) is able to do in Cistudo carolina.

In teleosts, Sheldon (’12) has called the whole mass a part of
the nucleus olfactorius anterior and then explained that certain
neurones function as granule cells. The conditions found in
the alligator represent an advance in differentiation over those
described for teleosts; yet even here it does not appear that the
granule cell layer is so physiologically distinct from the nucleus
olfactorius anterior since, in part, its cells still serve as second-
ary olfactory neurones.

aoe ELIZABETH CAROLINE CROSBY

The granule cell layer shows a wide range of types among its
neurones. The following types, based on a study of Golgi prep-
arations, have been distingushed among the granule cells of
the bulb.

1. Intrinsic or type II cells. These neurones have small
cell bodies, with dendrites that are short, thorny, and branching,
and which pass out in every direction from the cell body. No
axones can be distinguished. These cells are intrinsic neu-
rones, serving for the correlation of impulses within the layer.
Some of the smaller stellate cells appear to serve as intrinsic
neurones, at least so far as can be judged from the material
studied.

2. Stellate cells (figs. 27, 28).. These are similar in appear-
ance to the cells so named by Sheldon in the teleostean olfactory
bulb. The cell bodies are angular or somewhat star-shaped
as the name indicates. The dendrites are thick and thorny
and many branched and extend out towards the periphery of
the bulb. In the plexiform layer they interlace with the den-
drites of the mitral cells and of the goblet cells. Some of the
dendrites extend outward into the glomerular layer but it was
not determined whether these dendrites actually entered into
the formation of glémeruli, as Sheldon (’12) found to be the
case in the teleosts. The axones in many cases form synapses
with branches of the mitral cell dendrites. Sometimes they
enter the tractus olfactorius, although they have been followed
no great distance in it. Some of the smaller stellate cells do not
send their dendrites outward beyond the cell bodies of the mitral
cells. Furthermore the axones of such cells often end about
other cells of the bulb and so serve as intrinsic neurones.

3. Goblet cells (figs. 25 to 27). These are large, oval cells
whose dendrites are similar in appearance to those of the stellate
cells. Sometimes the dendrites of the goblet cells reach the glo-
merular layer, and have been seen entering into the formation
of a glomerulus. In other cases, the dendrites of the goblet
cells do not enter the glomerular layer but are dependent upon
the mitral cells for their stimulation. The axones of the gob-
let cells enter the tractus olfactorius, at least in some cases.

THE FOREBRAIN OF THE ALLIGATOR ooo

From the.standpoint of their types of synaptic connection ap-
parently three functions are served by the neurones of the granule
cell layer. ‘The first of these is that of diffusing and summating
the incoming olfactory impulses and so strengthening the dis-
charge into the hemispheres. This purpose is served by the
type II cells, the stellate cells, and, in part, by the goblet cells
(particularly those found in the anterior part of the bulb). All
these cells receive their impulses by way of the mitral cells and
do not send their axones into the tractus olfactorius.

A second group of these stellate and goblet cells send their
dendrites into the glomeruli and their axones into the tractus
olfactorius and so, from a functional standpoint, are practically
mitral cells.

The third function served by neurones of the granule cell
layer is that of acting as the cells of secondary olfactory nuclei.
Such cells receive impulses from the mitral cells and send their
axones into the tractus olfactorius. The goblet and stellate
cells offer examples of this type of neurone.

Judging from what is known of the development and spe-
cialization of the centers of the central nervous system, it seems
but fair to suppose that, in phylogeny, the centers of the olfac-
tory bulb arose from undifferentiated central gray. Johnston
(98) has shown that in Petromyzon and in Acipenser, neurones
of this mitral cell type are found all through the central gray.
The same author (15) has described, in Cistudo carolina, a
granule cell layer in which are cells functioning as mitral cells.

Certain cells of the central gray (on the whole those nearer
the periphery) will receive a larger number of the incoming
olfactory impulses. Under the operation of neurobiotaxis
(Kappers, ’14) such cells will be drawn toward the periphery
and, in this way, a mitral cell layer will be formed. Accom-
panying such a migration toward the surface and the conse-
quent higher specialization, there will be a differentiation in
form and in size to meet the greater demands.

Not all the cells left in the central gray will lose their connec-
tion with the fila olfactoria and so certain goblet cells and prob-
ably some of the stellate cells (although the proof for this is not

334 ELIZABETH CAROLINE CROSBY

absolutely clear) found in the olfactory bulb of the alligator
illustrate this condition, for they lie in the granule cell layer—
the position of the primitive central gray—yet aid in the for-
mation of the glomeruli and send their axones into the tractus
olfactorius. Sheldon (’12) has shown a similar condition in his
description of certain stellate and goblet cells in the olfactory
bulb of teleosts.

Other stellate cells and many of the larger goblet cells of the
reptilian granule cell layer have lost their connection with the
fila olfactoria and receive their impulses by way of the axones
of the mitral cells. In turn, they send their axones through
the tractus olfactorius and, from a functional standpoint, are
secondary olfactory neurones. A similar state of affairs has
been described by Sheldon (’12) for teleosts.

A smaller number of small stellate cells and goblet cells have
lost their connection with the tractus olfactorius and discharge
into the plexiform layer, serving apparently the usual correlat-
ing and summating function of granule cells. Are these the
forerunners of the most highly specialized granule cells of higher
forms?

Olfactory crus

The anterior continuation of three of the centers of the hem-
isphere are to be found in the crus (fig. 14). These are the nu-
cleus olfactorius anterior, the pyriform lobe complex, and the
hippocampus. Mitral cells are found in the olfactory bulb
back to the point where it passes over into the crus. There
they are replaced on the lateral side by cells of the pyriform
lobe complex and on the medial side by the anterior continua-
tion of the hippocampus. In the anterior end of the crus, the
granule cell layer is replaced by the cells of the nucleus olfac-
torius anterior, which takes its characteristic ventro-medial posi-
tion. The position, cell structure, and significance of these
centers will be discussed immediately under the head of centers
of the hemisphere.

THE FOREBRAIN OF THE ALLIGATOR 335

Centers of the hemisphere

Both basal and cortical centers are found in this region in the
alligator. The former will be described first. The term olfac-
tory lobe will be used a number of times in the following de-
scription. By that term is meant the anterior portion of the
hemisphere, including the secondary olfactory nuclei from the
posterior end of the crus to the beginning of the primordial
general cortex.

Nucleus olfactorius anterior (figs. 3 to5 and 14). In the crus
the nucleus olfactorius anterior occupies a ventro-medial po-
sition. It extends back into the hemisphere and runs caudad
for some distance.. It is gradually pushed away from the sur-
face by the cortical layer of the tuberculum olfactorium and
is directly continuous with the non-cortical part of that nu-
cleus. Throughout most of its extent in the hemisphere the
boundary between it and the anterior end of the caudate nucleus
ean not be defined. Johnston (’15) has described this nucleus
in the turtle. He considers, however, that it makes up, for
the most part, just the head of the caudate nucleus.

So far as has been observed in the preparations made after the
Golgi method, the cells of the nucleus olfactorius anterior are
round or goblet shaped and are comparable in form and general
appearance with the goblet cells of the granule layer of the olfac-
tory bulb. Figure 29 shows a goblet cell of this nucleus.

This nucleus is a secondary olfactory center, receiving im-
pulses by way of the tractus olfactorius and discharging, through
the axones of its cells, into the tuberculum olfactorium, the parol-
factory region, and the hippocampus.

Area parolfactoria (figs. 7 to 9, 16, 17). The cell groupings
found in the ventro-medial wall of the hemisphere in lower ver-
tebrates have caused much discussion. Meyer (’95), Unger
(06), C. J. Herrick (10), Johnston (’13 and 715), and a number
of other observers have mapped out the cell groups in this region.
They have not all agreed in regard to the embryological and
phylogenetic significance of these cell masses and as a result
a somewhat confusing nomenclature has appeared.

336 ELIZABETH CAROLINE CROSBY

In his 1913 paper Johnston has discussed fully the relative
positions and extents of the cell masses in this ventro-medial
region of the hemisphere in cyclostomes, selachians, reptiles, and
monotremes. Among the reptiles, he has described particu-
larly the conditions found in the turtle. In regard to the alli-
gator he says (13, p. 387), “In Alligator mississippiensis the
features described above (that is the relations of the primor-
dium hippocampi and the parolfactory area in the turtle) are
repeated so exactly that it is unnecessary to present separate
drawings. There are differences in general form, and the area
parolfactoria is relatively smaller than in the turtle.”

In the turtle Johnston has pointed out the presence of a pri-
mordium hippocampi, ventral to the hippocampal formation.
A similar primordium is present in the alligator and below this
primordium and separated from it by a cell free zone and, in
turtles, by a sulcus limitans hippocampi (Johnston, 713) is the
area parolfactoria (in part, Herrick’s septal area). This parol-
factory area is divided into medial and lateral portions which,
following Johnston (715), have been termed in this account the
medial and lateral parolfactory nuclei respectively. This area
parolfactoria is not the lobus parolfactorius of Edinger.

Medial parolfactory nucleus (figs. 7 to 9, 16, 17). This nu-
cleus consists of cells of the more medial part of the parolfac-
tory area. In the anterior end of the hemisphere it cannot be
sharply separated from the lateral part of the area but farther
caudad it is separated off by the fibers of the medial forebrain
bundle. This nucleus as it is here described is practically the
same as the medial parolfactory nucleus described for Cistudo
carolina by Johnston (715). Medialward it is bounded by the
nucleus of the diagonal band of Broca.

Lateral parolfactory nucleus (figs. 7 to 9, 16,17). The lateral
parolfactory nucleus bulges out‘into the ventricle. Ventralward
it follows the ventricle and cannot be sharply separated from the
nucleus accumbens, so that by some writers the whole cell mass
has been called nucleus accumbens septi. Dorsalward this lateral
parolfactory nucleus, in the more anterior region of the forebrain,
is clearly marked off from the primordium hippocampi by a cell-

THE FOREBRAIN OF THE ALLIGATOR 337

free zone and, in the turtle, by the sulcus limitans hippocampi.
Farther caudad, however, the line of separation between the two
becomes indistinct. In the region just anterior to-the commis-
sure, if the writer has understood Johnston correctly, the sulcus
disappears. Certainly in both the alligator and the turtle (Cis-
tudo carolina) the cell-free zone disappears and there is no line
of demarcation, so far as could be determined from the material
available, between the primordium hippocampi and the more
posterior portion of the nucleus parolfactorius lateralis. A cell
mass is thus formed which has cells apparently of the type both
of the primordium hippocampi and of the lateral parolfactory
nucleus, although the latter type appears to predominate. Con-
sequently, Herrick (10) after a study of amphibian and reptil-
ian material (including Lacerta, Cistudo carolina, and Alligator
mississippiensis) and after an examination of embryonic reptilian
brains from the Harvard collection, reached the conclusion that
this nucleus was a part of his septal nucleus, consisting of cells
of the basal region which had invaded this region, migrating
upward along the descending hippocampal fibers.

Johnston (713), on the other hand, has considered this inter-
mediate cell group a part of the primordium hippocampi, basing
his conclusion partly on the presence of the fornix fibers and the
fibers of the hippocampal commissure, and especially on its
position, as he believes from a study of embryonic material,
above the neuroporic recess in a thickened portion of the lamina
supra-neuroporica.

In the more anterior part of the brain, the ventro-lateral, small
celled area (Johnston’s caudate nucleus) is apparently continued
around the corner of the ventricle to the medial surface (figs.
7 to 9, 16, 17). This continuation of the caudate has been
termed nucleus accumbens by many observers. Johnston in-
cludes this nucleus accumbens in his nucleus parolfactorius
lateralis.

It will be seen from the above discussion that the terms lat-
eral septal nucleus (Herrick 710) and lateral parolfactory nu-
cleus (Johnston *13 and 715) are not synonymous. If the writer
has understood the authors correctly, the two masses compare

338 ELIZABETH CAROLINE CROSBY

as follows. The lateral septal nucleus (Herrick ’10) equals
the lateral parolfactory nucleus (Johnston ’13 and ’15) plus a
part of the posterior portion of the primordium hippocampi
(Johnston 713 and ’15) minus the nucleus accumbens (Herrick
eb)

Having had no opportunity to study reptilian embryological
material, the writer is in no position to decide which nomen-
clature is the better of the two. A most thorough study of the
nuclei in the developing brain will be necessary before any-
thing definite along that line can be determined. For con-
venience the terminology of Johnston has been adopted except
that the name nucleus accumbens has been retained.

The essential point is that in the alligator, in the region of the
fornix fibers and other descending hippocampal systems, there
is a cell mass which serves as a place of synapse for many of the
descending fibers. This cell group has been identified in a num-
ber of reptiles besides Alligator mississippiensis. Further-
more, so far as is now known, there are two possible sources of
origin for this cell mass. The first theory is that it is a special-
ization of a portion of the primordium hippocampi as a place of
synapse between the hippocampus and the basal centers. The
second theory implies that cells, situated in the basal region
and serving as places of synapse for descending fibers, moved
upward toward their source of stimulation according to the
principle of neurobiotaxis (Kappers, 714) and invaded the re-
gion of the primordiun hippocampi.

Tuberculum olfactorium (figs. 5, 6, 7, 15). This nucleus be-
gins in the hemisphere a short distance behind the olfactory
crus. It is ventro-medial in position and is continuous anteri-
orly with the nucleus olfactorius anterior, from which it can be
distinguished by the cortex-like arrangement of its outer layer
of cells. Its inner portion is made up of small groups of cells
which show, though not so clearly as in some forms, the arrange-
ment into islands so characteristic of the highly developed
tuberculum olfactorium. The cortical and non-cortical layers
of the tuberculum olfactorium are shown in the drawings of the
toluidin blue sections from this region (figs. 5 to 7).

THE FOREBRAIN OF THE ALLIGATOR 339

Laterally the tuberculum olfactorium lies in close relation to
the pyriform lobe, although its cortical layer is separated from
the latter by an area of scattered cells which belong apparently
with the nucleus of the lateral olfactory tract (see discussion
of that nucleus). Medially the tuberculum olfactorium is in
close relation with the parolfactory region.

The relations described here are in all essential points the same
as those described for the tuberculum olfactorium of turtles
(Johnston, 715). The differences in the two forms are to be found
in the somewhat higher development of the islands of Calleja
in the turtle and in the absence in the turtle of so well developed
a cortical layer as is found in the alligator. The tuberculum
olfactorium does not appear as a clearly defined nucleus in
Amphibia. In certain Dipnoi (Lepidosiren) described by Elliot
Smith (08) it appears in an exaggerated form. The Golgi
material available does not show the cell forms in this region.

Nucleus commissurae hippocampi (figs. 18 and 19). This
nucleus is really only a specialized portion of the primorium
hippocampi, consisting of clusters of cells of that primordium
which are mingled with the descending hippocampal fibers and
which collect particularly about the point of decussation of the
fibers of the hippocampal commissure. The cells of this nucleus
serve as a place of synapse for commissural fibers and as cells
of origin for some of the fibers of the medial cortico-habenular
tract. In fact cells of this nucleus accompany this latter tract
until it enters the stria medullaris and are probably a remnant
of the broad gray connection found in lower forms between the
hippocampal and the habenular regions.

Nucleus commissurae anterioris (fig. 18). This name has
been given to the cells forming the bed nucleus of the anterior
commissure. They resemble in general character the cells of
nucleus preopticus but are quite distinct in type from those
forming nucleus commissurae hippocampi. The cells of the
nucleus commissurae anterioris afford a place of synapse for
some of the fibers of the commissural division of the stria
terminalis.

340 ELIZABETH CAROLINE CROSBY

Nucleus preopticus (figs. 9, 10, 18 to 21). This term has been
applied to the cell mass which appears in the region of the pre-
optic recess just in front of the level of the commissures and which
extends caudad still occupying this position. It passes over
into the hypothalamic region with no definite line of separation
between the two areas. The nucleus preopticus receives im-
pulses from the stria terminalis, from fibers of the medial olfac-
tory tract which have decussated by way of the anterior com-
missure, and, at its anterior end, from some few fibers of the
tract of the diagonal band of Broca.

Interstitial nucleus (figs. 10, 19, 20). Cajal (11, vol. 2; p.
723) described this nucleus and figured it in the mouse, calling
it ‘“‘noyau interstiel de la voie de projection de |’écorce tempo-
rale.’ He says further ‘‘Malheureusement, il ne nous a pas
été possible de déterminer de facon précise les relations qui
existent éntre la bandelette semi-circulare et ce noyau, et cela
& cause de la rareté des bonnes imprégnations. Ajoutans que
cet amas de la région sousthalamique pourrait fort bien étre
encore un ganglion moteur.”

Johnston (’15) has described the olfactory projection tract
of Cajal for the turtle but has said nothing of the interstitial
nucleus. In the alligator the nucleus appears in the preoptic
region near the posterior end of the hemisphere as a ridge of
cells extending lateralward in close relation with the nucleus
ventro-medialis, arching dorso-medialward above the forebrain
bundles and extending medialward into relation with the more
dorsal part of the mass of the preoptic nucleus. It extends
caudad throughout the preoptic region but in the hypothalamic
region is gradually replaced by the hypothalamic nuclear ridge.

Part of the fibers of the olfactory projection tract of Cajal
arise from the ventro-medial nucleus. Cajal (11, vol. 2, p.
725, fig. 463) has shown some of the neurones of that nucleus
giving rise to these fibers. Many of the fibers having such an
origin quite probably send off collaterals among the cells of the
interstitial nucleus. Part of the fibers of this olfactory projec-
tion tract arises from the interstitial nucleus. The tract passes
caudad with the fornix into the ventral part of the hypothalamus.

THE FOREBRAIN OF THE ALLIGATOR 341

Nucleus of the diagonal band of Broca (figs. 8, 9, 17). This
nucleus was first described for reptilian brains by Johnston (’15)
in the turtle, Cistudo carolina. It is present in the alligator
in practically the same relations as in the turtle. It appears
behind the level of the tuberculum olfactorium as a dense col-
lection of cells arranged in a cortex-like layer in the ventro-me-
dial angle of the hemisphere. It extends dorsalward along the
medial surface as a somewhat less dense, cortex-like layer which
comes into relation with the medial parolfactory area and can-
not be sharply distinguished dorsalward from the cell mass of
the primordial hippocampus. It extends from the ventro-me-
dial region lateralward, as scattered clusters of cells, into rela-
tionship with the nucleus of the lateral olfactory tract. The
nucleus of the diagonal band extends posteriorly just outside
of the medial forebrain bundle into the region of the preoptic
nucleus. It is accompanied, as in the turtle, by bundles of
fibers which serve for connecting the lateral and medial olfac-
tory areas. The writer is particularly indebted to Dr. C. J.
Herrick for aid in identifying this nucleus in the alligator.

Basal nuclei of the lateral wall. Students of the reptilian brain -
have generally recognized two basal centers in the lateral wall
of the cerebral hemisphere, the corpus striatum and the epi-
striatum, and some have recognized a third region distinct from
both of these, comparable with the mammalian nucleus amygda-
lae. According to these observers, the epistriatum is the more
dorsal member of the complex and is in continuity with the cor-
tical lamina. The extent of this continuity varies in different
reptiles, depending upon the species and the general form relations
of the hemisphere, particularly upon the ventro-lateral extent
of the ventricle. In Testudo graeca, DeLange (13a, p. 113,
fig. 8.) has shown that the epistriatum is continuous with the
lateral or pyriform lobe cortex throughout its whole extent.
Kappers and DeLange consider the epistriatum to be striatal
in origin and to have acquired secondarily a connection with the
cortical lamina. They consider the epistriatum an olfactory
nucleus of the second order and the entire epistriatum complex
the homologue of the mammalian nucleus amygdalae.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

342 ELIZABETH CAROLINE CROSBY

On the other hand, Elliot Smith (10) has considered the
epistriatum to be of cortical origin. He believes that the effect-
ing of olfacto-somatic correlations in the reptilian hemisphere
and, particularly, the entrance of tactual fibers into the dorsal
part of the hemisphere lead to the disturbance of the morpho-
logical relations of the centers of the forebrain. He says ‘‘One
curious manifestation of these disturbing influences is seen in
the ingrowth (toward the lateral ventricle) of part of the over-
growing pallium, forming a structure to which Edinger gave the
name ‘epistriatum.’ The epistriatum is not a part of the striate
body but is cortical in nature. Moreover it is not a morpholog-
ical subdivision of the hemisphere which can be identified in
other groups of vertebrates, as many anatomists believe. It
is merely a peculiar adaptation of structure to meet the con-
ditions favorable to the reptile;—namely the disturbing influ-
ence of the recent admission of tactile impressions into the
hemisphere.”

In his 1915 paper, Johnston, following Edinger and Kappers
(Kappers, 06, p. 9), suggests that the term ‘epistriatum’ be
dropped, basing his suggestion on the facts that “the structure
to which the term was first applied, does not appear as a special
body or ridge in the turtle brain” and that “the author
(Edinger) of the term uses it for at least three different bodies
in the reptilian brain.”’ The conditions found in the forebrain of
Alligator mississippiensis certainly support Johnston’s suggestion.

In the anterior end of the hemisphere of the alligator several
large cell masses are found in the basal portion of the lateral wall.
1. There is a dorso-lateral area which includes a part or all
of the ‘epistriatum’ as that term is used by some recent writers
on the reptilian brain and is comparable with the dorsal ventric-
ular ridge (Johnston, ’15) in turtles, though perhaps not exactly
homologous with that area. 2. Below the dorso-lateral area, in
a ventro-medial position, are two nuclei which belong to the cor-
pus striatum of Johnston. The more dorsal large celled mass
is the ventro-lateral, large celled nucleus of this description,
comparable with Johnston’s nucleus lentiformis in the turtle.
3. The more ventral small celled mass is the ventro-lateral,

THE FOREBRAIN OF THE ALLIGATOR 343

small celled area of this description and is comparable with the
area termed nucleus caudatus in turtles. 4. Between the
dorso-lateral area and the ventro-lateral, large celled area, in
the anterior end of the brain, there is an intermedio-lateral area
which at first is closely tied up with the anterior part of the nu-
cleus of the lateral olfactory tract, but later becomes continuous
with the dorso-lateral area and probably is a part of that area. 5.
The nucleus of the lateral olfactory tract is situated in the later-
al part of the hemisphere, ventral to the dorso-lateral area and
dorso-lateral and lateral to the ventro-lateral areas (the corpus
striatum of Johnston’s description). It lies in intimate relation
with the intermedio-lateral area and is apparently continuous
with it. Dorsalward it is at first clearly distinct from the dorso-
lateral area but finally merges with it. Behind the level of the
tuberculum olfactorium the more ventral part of the nucleus of
the lateral olfactory tract becomes continuous with the nucleus
of the diagonal band of Broca and then swings farther ventral-
ward until it occupies the greater portion of the ventral region
of the hemisphere internal to the cortex of the pyriform lobe
and in close relationship with it. The nucleus of the lateral
olfactory tract of the alligator, as the name is used in this paper,
includes both the nucleus of that name and a small celled, ven-
tral portion of the pyriform lobe as described in turtles. 6. In
close relation with the nucleus of the lateral olfactory tract in
the ventro-medial angle of the posterior half of the hemisphere
is a nucleus to which the name of the ventro-medial nucleus
has been given. This nucleus (figs. 18 to 21) gives rise to the
projection tract of Cajal, and corresponds to the medial large
celled nucleus described in turtles (Johnston,’15). 7. The outer
ventro-lateral and ventral portions of this lateral wall are occu-
pied farther cephalad by the cells of the tuberculum olfactorium
and behind the level of that cell mass (8) by a part of the nu-
cleus of the diagonal band of Broca. These two centers have
been described previously. (For a more complete discussion
of the extent, relations, and fiber connections of these nuclei
of the lateral wall see the special headings.)

344 ELIZABETH CAROLINE CROSBY

Dorso-lateral area (figs. 7 to 10, 12, 15 to 19). This area
forms most of the large eminence which projects from the ven-
tro-lateral wall of the hemisphere into the lateral ventricle and
nearly fills that cavity. Its lateral aspect is exposed in the dis-
section illustrated in figure 2. The anterior end of the dorsal
area is marked by the previously mentioned inward fold of the
dorso-lateral cortex (figs. 5, 6, 7) which may be considered pri-
mordial general pallium. In the turtle, as many writers have
shown, there is a pallium-like infolding throughout the whole
extent of a somewhat similar area, the dorsal ventricular ridge
of Johnston (15). In all but its most anterior portion, the dorso-
lateral area in the alligator is cut off from the pallial areas by
_ the outward and downward growth of the lateral ventricle, so
that the infolding can be plainly seen only in the anterior end
of the hemisphere. The dorso-lateral area is bounded ven-
trally by the intermedio-lateral area and then by the ventro-
lateral large celled area and ventro-laterally by the anterior
end of the nucleus of the lateral olfactory tract (figs. 7, 8). Be-
hind the ventro-lateral areas (the lentiform and caudate nuclei
of Johnston) it is bounded ventrally by the posterior portion of
the nucleus of the lateral olfactory tract (fig. 12). Olfactory
fibers from the lateral olfactory tract distribute to the dorso-
lateral area from behind the level of the general cortex infolding
to the posterior end of the area. Ascending somatic sensory
fibers from the thalamus also distribute throughout practically
all parts of this region and, in some parts of the area, these
somatic connections alone are present without admixture with
olfactory fibers. This purely somatic region includes practi-
cally all the dorso-lateral area at the anterior end of the brain.
Farther caudad an increasingly large amount of the lateral and
dorso-lateral portions of this area receives olfactory fibers and
only the dorso-medial portion is relatively pure somatic in type
of correlation. The ridge of primordial general cortex implies
a very close relation between the somatic dorso-lateral area and
the general cortex.

The ventro-lateral areas as they have been termed in this de-
scription of the alligator brain are apparently directly compara-

THE FOREBRAIN OF THE ALLIGATOR 345

ble with the corpus striatum which Johnston has described in
turtles. If that author has been correctly interpretated, the
ventro-lateral small celled nucleus of this description is nucleus
caudatus, while the ventro-lateral large celled nucleus. is the
nucleus lentiformis of turtles. The writer has avoided the
specific terms employed by Johnston because she does not have
sufficient knowledge of the development of the striatum through-
out the vertebrate series to be certain of the homologies.

Ventro-lateral small celled area (Johnston’s nucleus cauda-
tus). This nucleus (figs 7 to 9) begins a short distance behind
the olfactory crus and, increasing in size, extends backward to
the level of the anterior nucleus of the thalamus with which
its posterio-medial portion lies in close relation. Posteriorly
its ventral and lateral portions lie in close relation with the
posterior part of the nucleus of the lateral olfactory tract. An-
teriorly it cannot be sharply delimited from the nucleus ol-
factorius anterior. Johnston (715) in turtles, where the rela-
tionships of the caudate nucleus are practically the same as the
relationships of this area in the alligator, has considered the nu-
cleus olfactorius anterior and some associated gray as giving
rise to the head of the caudate nucleus in mammals. The great
increase in the neopallial area in higher forms is accompanied
by an increase in the number of fibers (internal capsule fibers)
distributing to that cortical area. These fibers are imbedded
in the caudate nucleus and more posteriorly are ventral to it
and so push this cell mass dorsalward and caudalward as they
increase in number during phylogeny. The upward, backward
and downward growth of the general cortex and the downward
growth of the pyriform lobe have produced the typical curve of
the caudate nucleus of higher forms.

The ventro-lateral small celled area is continuous around
the ventral border of the ventricle and onto its medial surface and
this continuation represents the nucleus accumbens of higher
forms. This apparently belongs to the striatum complex, al-
though Johnston (’13, p. 421) has joined it to the nucleus later-
alis septi of previous authors under the name of nucleus parol-
factorius lateralis (see the discussion of the parolfactory nuclei).

346 ELIZABETH CAROLINE CROSBY

Ventro-lateral large celled area (Johnston’s nucleus lentifor-
mis) (figs. 7 to 10, 17 to 19). This nucleus appears as a group of
cells just dorsal to the small celled area at the anterior of the
hemisphere and laterally close to the nucleus of the lateral ol-
factory tract. Farther caudad it is partially separated from the
ventro-lateral small celled area (Johnston’s caudate) by a spe-
cial fascicle of the lateral forebrain bundle. In the posterior
part it forms a heavy ridge of cells over the dorsal and dorso-
lateral portions of the small celled area. It disappears in front
of the posterior end of the latter area at about the level of the
foramen of Monro. The larger size of the cells of the ventro-
lateral large celled area makes it easy to distinguish this nucleus
from the ventro-lateral small celled area.

Intermedio-lateral area (figs. 7, 8). This area is found at
the level of the posterior part of the infolding of the general cor-
tex and in the region just caudad to that infolding. The inter-
medio-lateral area is ventral to the dorso-lateral area, dorsal
to the ventro-lateral areas and medial to the nucleus of the
lateral olfactory tract. It lies in so close relation with this last
nucleus especially in its more anterior extent that it is not prac-
ticable to attempt to draw any sharp boundary line between
them. The intermedio-lateral area is separated from the ven-
tro-lateral areas by a cell free zone in which are fibers which
belong in part at least to the lateral forebrain system. A sulcus
in the ventricular wall indicates the position of the boundary
line between the intermedio-lateral and ventro-lateral area.
The anterior portion of the former area is separated from the
dorso-lateral area by a cell free zone but the two areas fuse into
one, behind the level of the infolding of the general cortex. So
far as the evidence goes, the intermedio-lateral area appears to
belong with the dorso-lateral area. Possibly it is a representa-
tive of some part of the striatum complex of higher forms.

Nucleus of the lateral olfactory tract (figs. 5 to 10, 12, 16 to 19).
This nucleus can be distinguished from the other cell masses in
the lateral wall of the hemisphere by the smallness of its cells.
It begins as a small cluster of cells scattered along the inner
border of the cortex of the pyriform lobe and between that cor-

THE FOREBRAIN OF THE ALLIGATOR 347

tex and the cortex-like superficial layer of the tuberculum ol-
factorium. Close to its anterior end the cells of the upper or
more dorsal portion of the nucleus group themselves more close-
ly together and a clearly defined nucleus is formed which is ven-
tral to the dorsal-lateral area and lateral to the ventro-lateral
large celled area. At first this upper portion of the nucleus of
the lateral olfactory tract remains distinct from the surrounding
cell masses of the hemisphere wall; but as it is followed caudad
it gradually comes into close relation with the cell mass of the
dorso-lateral area and finally merges with it with no sharp de-
limiting line between the two, a greater and greater number of
large cells appearing among the small cells in that region until
apparently the mass has become a part of the dorso-lateral area.
The ventral portion of the nucleus, at the anterior end of the
brain, consists of diffuse clusters of cells lying in close relation
with the pyriform lobe, the cortex of the tuberculum olfactorium,
and, farther caudad, the nucleus of the diagonal band of Broca.
At approximately the level of the fusion of the anterior dorsal
portion of the nucleus of the lateral olfactory tract with the
dorso-lateral area, the ventral portion of the former nucleus
broadens out and extends to the posterior end of the hemisphere,
occupying first a ventro-lateral and then a ventral position.
A part (probably the more ventral portion) of the anterior
dorsal portion of this nucleus of the lateral olfactory tract, as it
has been described for the alligator, is quite probably compar-
able with the small celled, ventral part of the pyriform lobe
observed by Johnston (715) in the turtle. In describing this small
celled part Johnston says that in the rostral part of the brain of
Cistudo carolina it may be sharply distinguished from the large-
celled portion both by the difference in cell character between
the two regions and by the more ventral position of the small
celled portion, which extends below the sulcus endorhinalis and
expands behind the posterior part of the striatal area into
the nucleus of the lateral olfactory tract. In the alligator the
continuance of the pyriform lobe cortex (Johnston’s large celled
medial portion) farther ventralward, has pushed this small celled
portion inward and crowded it somewhat dorsalward so that

348 ELIZABETH CAROLINE CROSBY

it lies for:'the most part, medial to the pyriform lobe cortex in-
stead of ventral to it as in turtles. Furthermore the anterior,
dorsal end of the nucleus of the lateral olfactory tract (Johnston’s
small celled, ventral portion of the pyriform lobe) is much larger
in the alligator than in the turtle and this increase in size has
probably been another important factor in bringing about the
change in the relative positions of the two cell masses.

The more ventral and posterior portions of the nucleus of the
lateral olfactory tract as that nucleus has been described for
the alligator are comparable to nearly all of the nucleus of that
name described for the turtle (Johnston, ’15). Here again,
however, there is one point of difference, for, while in the turtle
the nucleus occupies the outer portion of the hemisphere in the
posterior part of the forebrain, in the alligator the cortex of the
pyriform lobe extends downward and occupies the outer portion
of the ventral wall, the nucleus of the olfactory tract lying in-
ternal to it and in close relation with it. (For a further dis-
cussion of these relations and their significance see the account
of the pyriform lobe.)

To summarize, the nucleus of the lateral olfactory tract as it
is present in the alligator is practically the equivalent of the
nucleus of that name and the small celled ventral part of the
pyriform lobe in turtles, except that the last named area has
been greatly elaborated in the alligator and its more anterio-
dorsal portion may very well have taken on an added significance
from its intimate relation with the somatic dorso-lateral, area.

Ventro-medial nucleus (figs. 10, 12, 18, to 21). This nucleus
occupies the extreme ventro-medial portion of the hemisphere,
extending throughout about the posterior half of the hemi-
sphere. It has broad connections with the habenula by way
of the stria medullaris and it gives rise to the great olfactory
projection tract of Cajal.

This quite evidently is the medial, large-celled nucleus de-
scribed by Johnston (715) for Cistudo carolina, although the cells
of this nucleus, in the alligator, resemble in size and in cell char-
acteristics the cells of the nucleus of the lateral olfactory tract,
except that they are massed somewhat more closely together.

THE FOREBRAIN OF THE ALLIGATOR 349

Functional complexes formed by the basal nuclei of the lateral
wall. In the foregoing paragraphs, an account has been given
of the relative positions and the extents of the various nuclei
found in the lateral wall of the hemisphere, and something has -
been said of their fiber connections. Two problems then arise,
the first regarding the way in which these centers interact in
the functioning brain of the alligator; the second regarding their
phylogenetic significance as forerunners of centers found in mam-
malian brains.

Two types of nervous impulses enter the lateral wall of the
cerebral hemisphere, (1) descending mpulses from the olfactory
area, and (2) ascending somatic sensory impulses from the cen-
ters of the thalamus. The nuclear pattern of this basa area
of the forebrain has been determined in large measure by the
distribution and mutual interconnections of the incoming fibers
of these two systems.

The first type of nervous impulse includes the secondary and
tertiary fibers of the lateral olfactory tract, which, entering from
in front, distribute to the nucleus of that tract throughout its
entire extent and, turning gradually dorsalward, in the posterior
half of the hemisphere distribute to the lateral portions of the
dorso-lateral area. The lateral part of this dorso-lateral area,
the nucleus of the lateral olfactory tract, and the ventro-medial
nucleus all give rise to fibers of the stria medullaris and the
first two masses (and in turtles the ventro-medial nucleus also)
discharge through the stria terminalis. The ventro-medial nu-
cleus in both the turtle and the alligator discharges into the
diencephalon through the great olfactory projection tract of
Cajal. .

The identification of the amygdaloid complex of higher forms
is based on the following features: 1) upon its reception of fibers
from the lateral olfactory tract (figs. 16, 18); 2) upon its relation
to the pyriform lobe cortex (figs. 7 to 10); 3) upon its giving rise
to fibers of the stria terminalis (figs. 19 to 21—in this is included
its connection with the opposite side of the brain by way of the
anterior commissure) ; 4) upon its giving rise to fibers of the stria
medullaris (figs. 16 to 21). It is evident that the group of centers

350 ELIZABETH CAROLINE CROSBY

just discussed, i.e., the nucleus of the lateral olfactory tract, the
ventro-medial nucleus, and the more lateral part of the dorso-
lateral area, make up such an amygdaloid complex.

The second type of impulse which enters the lateral wall of
the hemisphere is somatic, being transmitted by the somatic
sensory radiations from the lateral and medial nuclei of the thala-
mus to the ventro-lateral areas (caudate and lentiform nuclei).
These areas, then, are centers for the correlation of somatic
sensory impulses in the hemisphere and are, therefore, the fore-
runners of the mammalian corpus striatum. They discharge
into the lower brain centers through the lateral forebrain bundle.

A part of the somatic sensory fibers pass beyond the ventro-
lateral large celled area (Johnston’s nucleus lentiformis) into the
dorsal area, so that at the level of the primordial general cortex
(figs. 5, 6, 15), this dorsal area is entered almost exclusively by
the somatic correlation fibers and hence is a somatic correlation
center of striatal type. This area at its anterior end probably
exhibits the highest type of somatic correlation tissue found in
the brain of the alligator, and the entrance of association fibers
from the adjacent cortical centers into its dorsal part has given
the conditions favorable for the differentiation of primordial
general cortex (i.e., cortex approaching the neopallial in type).

It will be remembered that behind the level of this thicken-
ing representing primordial or transitional general cortex the
lateral part of the dorso-lateral area receives olfactory fibers and
probably some somatic fibers, and belongs to the amygdaloid
complex. Whether this portion becomes a somatic part of the
amygdaloid complex of higher forms, as Johnston (715) believes
is the case with the dorsal ventricular ridge in turtles, or whether
it is a step toward the enormous striatum complex found in birds,
cannot be decided without a much greater knowledge of other
vertebrate forms than the writer possesses. In any case, it is
very evident that the dorso-lateral area must be regarded as a
structure of intermediate or transitional type, containing pri-
mordia related to three diverse structures in the mammalian
brain, viz., corpus striatum, amygdaloid complex, and . the
general cortex.

THE FOREBRAIN OF THE ALLIGATOR 351

Cortical centers of the hemisphere. Within the pallium three
types of cortical centers may be distinguished. One of these,
the hippocampus, is concerned primarily with olfacto-visceral
correlations. The pyriform lobe cortex is concerned chiefly
with olfacto-somatic correlations, with some involvement of the
general visceral centers of the hypothalamus. The third type,
the general cortex, is largely concerned with somatic correla-
tions, and is differentiating toward true neopallium.

Hippocampus (figs. 3 to 10, 12, 14 to 21). Spitzka was the
first to suggest that the dorso-medial wall of the hemisphere was
hippocampus, although he still called the hippocampal com-
missure the corpus callosum. From that time the homology of
this medial cortex has been recognized by most observers, includ-
ing Edinger, Brill, Meyer and Elliot Smith. Very good sum-
maries of the earlier studies on the hippocampus and its com-
missure are given in Elliot Smith’s article (03) dealing with
the morphology of the cerebral commissures in vertebrates and
in the Arris and Gale lectures (10). In this connection it is
interesting to note that Johnston (15) has reopened the ques-
tion of the presence of true callosal fibers in the dorsal or hip-
pocampal commissure of both marsupials and reptiles. His evi-
dence for their presence is based on expérimental work. In
regard to reptiles he says, page 404, ‘‘in the turtles the lack of
medullation in the dorsal commissure has made it impossible
thus far to secure positive evidence as to the presence of callosal
fibers.’”’ He argues that they should be present because of the
great number of ascending fibers carrying sensory impulses from
the thalamus to the telencephalon in reptiles. Others, as Ra-
mon y Cajal, Unger, and Pedro Ramon, have claimed that various
reptiles have true callosal fibers.

Adolf Meyer (92) was the first person to distinguish between
the dorsal and the dorso-medial portions of the hippocampus.
The dorso-medial portion arises rostrad in the narrow part of the
olfactory crus and there occupies a somewhat dorsal as well as
a dorso-medial position (fig. 3). At this level it lies in close
relation dorso-lateralward with the cortex of the pyriform lobe.
As it extends caudad into the hemisphere, the dorso-medial

352 ELIZABETH CAROLINE CROSBY

cortex takes its characteristic position and dorsal to it appears
a group of scattered cells of a larger size which, judging from their
fiber connections, are strongly under the influence of the dorso-
medial portion. This latter group constitutes Adolf Meyer’s
dorsal portion of the hippocampus and is the ‘subiculum’ de-
scribed by Johnston (715) in turtles. Except at the very an-
terior end of the hemisphere, the hippocampal formation and
the cortex of the pyriform lobe are separated by the general
pallium. Ventralward, the dorso-medial portion of the hippo-
campus is continuous with a diffuse mass of small cells, the
primordium hippocampi.

In the more anterior part of this dorso-medial region of the
hippocampus, the cells, as seen in Golgi preparations, are goblet-
shaped and are comparable with the cells of the secondary ol-
factory nuclei. They are more nearly related in type to the small
projection cells of the hippocampus. The most anter:or portion
of the hippocampus probably does function to considerable de-
gree at least, as a secondary olfactory nucleus. Other cell types
found in the hippocampus are as follows:

1. Correlation cells (fig. 31). The correlation cells of this
type are found in the dorsal portion of the dorso-medial area at
the anterior part only, so far as is known. They are especially
interesting because of their resemblance to the cells of the hippo-
campal regions in Amphibia (Herrick, 710). These are prob-
ably phylogenetically the oldest of the highly specialized cells
of the hippocampus.

2. Double pyramid cells (figs. 35, 36). These are the cells
which especially give character to the dorso-medial portion of
the hippocampal cortex. Their cell bodies are large and more
or less pyramidal in form. Thick, thorny, bushy dendrites
spread out lateralward and medialward from the cell body but
are especially thick on the medial side, where they can often
be seen breaking up around the terminal arborizations of the in-
coming medial olfactory, parolfacto-cortical and tuberculo-
cortical tracts. Sometimes the impulse reaches the double pyr-
amid cell through an interpolated neurone. The dendrites which
are directed lateralward, receive olfactory impulses from the

THE FOREBRAIN OF THE ALLIGATOR alo

pyriform lobe and the nucleus of the lateral olfactory tract.
These impulses come by way of the alveus which carr es impulses
in both directions, as it does in higher forms. ‘The 'aterally di-
rected dendrites receive short association fibers from the alveus
and perhaps impulses from other ncoming fibers.

The axones of the double pyramids are slender and run lateral-
ward, dividing in many cases into two branches (fig. 36). One
of these branches enters the alveus and can often be traced a long
distance, although it has been impossible as yet to follow any
single fiber all the way into the pyriformlobe. The second branch,
when present, goes to the septum or enters one of the descending
diencephalic tracts (the fornix or tractus cortico-habenularis).

3. Small projection cells (figs. 32, 33). Besides the double
pyramid cells there are other projection cells in the hippocampus.
These are smaller than the ones just described and may be either
pyramidal, oval or nearly round in form. They are usually
either slightly lateral or slightly medial to the double pyramids
and send their dendrites to both lateral and medial surfaces, where
they receive the same sorts of impulses as are brought to the
double pyramids. ‘The axones, like those of the latter, may
divide into two branches (fig. 33), one entering the alveus and
the other running ventralward into the septum and presumably,
in some cases, entering tracts descending to the diencephalon.

4. Small intrinsic cells (fig. 34). The dendrites of the double
pyramid and small projection neurones form a thick feltwork
on each side of the more deeply placed cell bodies. Scattered
through this feltwork are cells of several types, only one type of
which is shown in the figures, which send out relatively short
bushy dendrites and receive collaterals from incoming fibers or
from axones of the hippocampal projection cells and discharge
back into the dendrites of the latter. In this way the whole
of the hippocampus is tied up together and correlated and unified
responses are made possible. Some of these cells are typical
type II neurones, others have longer, less branched processes
and short slender axones. Both of these sorts are apparently
intrinsic to the hippocampus.

354 ELIZABETH CAROLINE CROSBY

Levi (04) has described cells of the double pyramid and small
projection types in the hippocampus of reptiles. The account
given here agrees substantially with the descriptions and figures
given in his article. It is interesting to note that the medial side
of the dorso-medial cortex, as Levi suggests, appears to be con-
cerned mainly with the reception of incoming stimuli from the
lower brain centers, while, on the other hand, the main projec-
tion fibers which connect the hippocampus with the pyriform
lobe (the alveus) and with the diencephalon (fornix and the
tractus cortico-habenularis medialis) leave on the lateral side.
In the turtle the hippocampal cortex lies close to the ventricle.
Presumably, in that case, many of the efferent fibers leave on the
medial side, but so far as is known, there is no literature on that
subject. It is certain, however, that the dorso-medial region
of the hippocampal cortex of the alligator represents a higher
differentiation than the corresponding region in the brain of the
turtle and that this differentiation is marked, not only by a more
definite cell arrangement and possibly by a more specialized cell
form, but also by a new position of the cell mass produced by a
biotactic movement of the cell group away from the ventricular
wall and in the direction of the incoming impulse.

Meyer (92) and Levi (’04) claimed that the dorso-medial
portion of the reptilian hippocampus was gyrus dentatus. This
contention was denied by Ramon y Cajal, who, in an elaborate
series of histological studies, showed that from its cell types and
manner of connection, it could not be considered pure gyrus
dentatus. In the Arris and Gale lectures (710), Elliot Smith
ac'mits the correctness of the Cajal observations but says that the
dorso-medial portion is undergoing a differentiation toward the
production of gyrus dentatus and that it is the forerunner of
that structure. This seems a fair statement of the conditions.

The dorsal portion of the hippocampal cortex does not show
the regular arrangement of cell layers found in the dorso-medial
portion, and in general, its mass of cells shows a lighter staining
reaction to toluidin blue. Figure 31 shows one type of corre-
lation cell found in this dorsal region. This dorsal part appears
to be concerned chiefly with the association of impulses rather

THE FOREBRAIN OF THE ALLIGATOR 355

than as a receptive center, its main incoming impulses, so far as
known, coming in through the alveus. As Elliot Smith and
Levi have suggested, this is probably the forerunner of the hip-
pocampal cortex as distinguished from the gyrus dentatus.
Johnston (15), however, regards this dorsal cortex as the fore-
runner of the mammalian subiculum. From the region of the
infolding of the primordial general cortex (fig. 6) in the alligator
this dorsal part of the hippocampal cortex is continuous with the
general cortex.

The presence in the alligator of a primordium hippocampi,
such as Johnston (’13 and 715) has described in turtles, has al-
ready been mentioned. In the turtle that author has shown the
presence, on the medial wall of a fimbrio-dentate sulcus (Elliot
Smith’s suleus limitans hippocampi) between the dorso-medial
portion of the hippocampus and the primordium hippocampi
and a sulcus limitans hippocampi and a cell free zone between
the primordium hippocampi and the parolfactory area (Her-
rick’s ('10) septal area) in the anterior part of the brain. On
the ventricular side of the medial wall in the turtle are two sulci
which correspond to those on the lateral wall and separate the
same areas. In the alligator in the material which was studied,
no well defined sulci are in evidence on the medial surface of the
hemisphere in these regions but the ventricular sulci are present in
positions corresponding to those in which they are found in the tur-
tle; and the primordium hippocampi, in the more anterior part of
the hemisphere, is separated from the septal or parolfactory area
by a cell free zone. An interesting fact, but one whose significance
is not clearly understood, is the presence on the ventricular surface
of a relatively thin ependymal layer over the dorso-medial por-
tion of the hippocampus and of the primordium hippocampi,
which becomes thickened over the septal or parolfactory region.
Under the head of the parolfactory area, the relative positions
and the relations of the primordium hippocampi and the lateral
parolfactory nucleus have been discussed.

Johnston (13, figs. 23 to 27, pp. 446-447) has shown that the
primordium hippocampi extends forward in the hemisphere con-
siderably anterior to the hippocampus proper. In the alligator

356 ELIZABETH CAROLINE CROSBY

the dorso-medial cortical area extends forward into the region
just posterior to the olfactory crus and the primordium hippo-
eampi, though quite plainly present, is relatively smaller than
in the turtle. This means that in the alligator the hippocampus
in this region is more highly specialized than in the turtle. The
cells making up the primordium hippocampi are small and are
arranged in a diffuse mass. They are not impregnated in the
Golgi preparations which were studied.

Pyriform lobe (figs. 3 to 10, 12, 14 to 19). The pyriform obe
has important functions both as a secondary olfactory center
and as a correlation mechanism of high order. By means of
connections with the olfactory bulb and the basal and cortical
centers of the hemisphere it receives both correlated and uncor-
related olfactory material. By means of its connection with the
tuberculum olfactorium and, also, through short correlation fibers
from the somatic centers of the hemisphere, t receives corre-
lated somatic material. It receives impulses from the other
cortical centers by way of the alveus. Consequently it serves,
in part at least, as an olfacto-somatic correlation center of high
order.

In the anterior end of the hemisphere, in the region of the ol-
factory lobe, the pyriform lobe cortex and the hippocampal cor-
tex lie in close relation with each other dorsally. They are soon
separated, however, by the general pallium which intervenes
between them throughout the remaining extent of the hemis-
phere. Ventrally the pyriform lobe les in close relation with
the tuberculum olfactorium, separated from it only by some
scattered cells of the anterior division of the nucleus of the lateral
olfactory tract. Behind the tuberculum olfactorium, the cortex
of the pyriform lobe is bounded ventrally by the nucleus of the
diagonal band of Broca and by the nucleus of the lateral olfac-
tory tract. Near the posterior end of the hemisphere, this latter
nucleus, which there occupies all the ventral surface excepting
the portion occupied by the ventro-medial nucleus, acquires
a cortex-like arrangement of its superficial cells (fig. 12) which
layer is continuous with the cortex of the pyriform lobe and to
all intents and purposes is a part of that area since the pyriform

THE FOREBRAIN OF THE ALLIGATOR 357

lobe cortex itself arose as a differentiation of the neurones of the
nucleus of the lateral olfactory tract (see Johnston 715 and the
general discussion at the end of this paper).

Medialward the pyriform lobe cortex is bounded by the dorso-
lateral area and by the anterior part of the nucleus of the lateral
olfactory tract. ‘This anterior division bears about the same rela-
tion to the pyriform lobe cortex that the primordial hippocampus
bears to the hippocampal cortex proper, i.e., it consists of cells
which have practically the same type of connections as do the
cells of the pyriform lobe cortex. It represents the general
area from which the specialized pyriform lobe cortex has de-
veloped. In this paper it has been considered as the anterior
part of the complex of the nucleus of the lateral olfactory tract,
but it might equally as well be termed primordial pyriform lobe
cortex or the small celled portion of the pyriform lobe complex,
as Johnston (15) has called it in turtles. (For a further descrip-
tion of this anterior division of the nucleus of the lateral olfac-
tory tract see the description of that nucleus).

The writer has not been able to identify the suleus endo-
rhinalis and the sulcus rhinalis is slight but does show in some
preparations. The cell type illustrated in figure 39 is found in
the anterior end of the pyriform lobe. It resembles the cells
found in the secondary olfactory centers, which is not surpris-
ing since the anterior end of the pyriform lobe cortex itself prob-
ably serves to a considerable degree as such a secondary center.
The Golgi material available for study does not show the cell
types found in the more posterior part of this cortex.

General cortex (figs. 4 to 10, 12,16to19). As has been stated,
the cortex of the pyriform lobe and the hippocampal cortex are
separated from each other by the general cortex except at the
anterior end of the brain in the region of the olfactory lobe.
In many reptiles this cortex forms a definite lamina separated
from the other cortical areas by distinct limiting zones but in
the alligator, at least in the material studied, no such sharp
limiting zones are visible. Medialward as in the turtle, it grades
over into the thicker dorso-medial part of the hippocampal cortex
(Johnston’s subiculum, 715). Lateralward it is continuous with

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

358 ELIZABETH CAROLINE CROSBY

the cortex of the pyriform lobe. The rhinal fissure is demon-
strable in some of the material but is relatively slight.

In the anterior end of the dorso-lateral area of the hemisphere
there is a ridge of cortex-like cells which has the appearance of
being a fold of the general cortex. This has been termed in the
present paper, primordial general cortex. Between the primor-
dial portion and the general cortex proper, at the medial border,
there is a small space (fig. 6) which permits association fibers
of the alveus system to reach the former portion. Johnston
(15) has described in turtles a ridge of cells similar to the pri-
mordial general cortex of this description. He calls it the dorsal
ventricular ridge but says that it belongs to the general pallial
complex (the general cortex of this description). In a later
paper (’16) he shows that both the ridge and the general cortex
are derived during embryonic development from the dorso-
lateral area. Phylogenetically the general cortex complex arose
under the influence of at least two types of fibers. 1. The one
type consists of fibers carrying impulses from the somatic centers
of the diencephalon to the dorso-lateral area of the forebrain by
way of the lateral forebrain bundle. 2. Into this dorso-lateral
area association fibers from the hippocampal and pyriform lobe
complexes also distribute. As these latter areas differentiated
a higher type of integrated impulses was brought in and, within
the dorso-lateral area, neurones exhibiting a cortex-like type
of differentiation appear. In this way within the basal dorso-
lateral area, a primordial general cortex is probably formed.
What the factors were which caused this primordial cortex to
become more superficial in position and to separate from the basal
dorso-lateral area to take on a true cortical form, of course is
not certainly known. Perhaps one cause lies in the neurobio-
tatic influence of the association fibers, the neurones migrating
out along their dendrites toward the source of their stimulation.

The whole of the general cortex complex is a step toward the
differentiation of a neopallial area. To be sure, this complex is
still closely linked with cortical olfactory areas, but it has a rela-
tively large somatic component and its connection with the basal
somatic areas is intimate. As maintained by Elliot Smith and

THE FOREBRAIN OF THE ALLIGATOR 359

others, it cannot be regarded as true neopallium, but rather
represents a process of differentiation in that direction.
Johnston (16a) by a series of experiments on the turtle brain
has reached the conclusion that there is some degree of cortical
localization in the general pallium of that reptile. The writer
at present has not sufficient data to determine whether or not
there is any localization pattern in Alligator mississippiensis.

Centers of the diencephalon

The diencephalon may be divided into the three usual divisions
(1) the epithalamus; (2) the thalmus; (8) the hypothalamus.
It is not the purpose of this report to go into the question of
nuclear localization in these regions nor to attempt to describe
the character of the cell groups. There has not been sufficient
work done to justify such an attempt. Only a few of the more
outstanding facts of especial interest in connection with the
discussion of the forebrain will be mentioned.

At the end of his 1913 paper, DeLange has given a series of
outlines of the diencephalon of the alligator in which the posi-
tions of the various nuclei and their topographic relations to the
various fiber tracts are indicated. These have been of the great-
est help.

Epithalamus. The part of the epithalamus which is_par-
ticularly concerned with the reception of olfactory impulses
is the habenula (figs. 11, 12, 21). This nucleus lies at the dor-
sal surface of the diencephalon and projects into the ventricle.
The stria medullaris brings impulses to this nucleus. It con-
sists of three smaller nuclei; a medial one of closely packed cells,
a dorsal and more anterior one which apparently receives part
of the tractus cortico-habenularis medialis and, lastly, a ventral
one of larger cells that, farther caudad, connects with the cell
mass of nucleus magnocellularis. The habenulae of the two
sides connect with each other by means of the commissura haben-
ularum (fig. 12).

Thalamus. There are really three types of nuclei in the thal-
amus proper: a medial group which connects chiefly with the

360 ELIZABETH CAROLINE CROSBY

visceral centers, a lateral group which is the place of termina-
tion for the somatic impulses brought in by the optic and lem-
niscus systems and, intermediate between these two groups, a
third nucleus which receives fibers of both the visceral and
somatic type. This nucleus is the nucleus medialis or the
nucleus rotundus of some authors.

In the medial group are the nucleus anterior and the nucleus
magnocellularis. The nucleus anterior (figs. 10 and 11, 20), as
its name implies, lies at the very anterior end of the thalamus.
It is dorsal in position and its cells are smaller and more closely
packed together than are the cells of the lateral nucleus. It re-
ceives fibers from the hypothalamus and is connected with the
small celled ventro-medial part of the hemisphere by means of
a fiber tract.

The lateral group includes the nucleus lateralis, a special
derivative of this nucleus—the pulvinar—and another optic
center which most writers have termed the corpus geniculatum
laterale. The nucleus lateralis is conspicuous because of the
large size of its neurones. The cell bodies of these neurones
(figs. 41, 42, 43) are large, goblet or triangular in shape, and have
thick thorny dendrites which extend out in every direction from
the cell bodies. The axones enter the lateral forebrain bundle.
This nucleus is lateral in position, being lateral and somewhat
ventro-lateral to the nucleus anterior and lateral to the nucleus
medialis (or rotundus). It receives lemniscus fibers and some
optic fibers and, with the lateral thalamic optic centers, repre-
sents the beginning of the neothalamus (Edinger) of higher
forms, i.e., that lateral portion of the thalamus which serves
as a place of synapse for nervous impulses passing to the neo-
pallium and which develops parallel with the development of
the neopallial cortex. In the more posterior part of the thala-
mus, a lateral portion has begun to differentiate away from this
nucleus and to form a beginning of the pulvinar. This separate
nucleus is developed under the direct influence of the incoming
optic fibers.

There are other cell masses in the thalamus proper, as for
example the nucleus reuniens figured in the alligator brain by

THE FOREBRAIN OF THE ALLIGATOR 361

DeLange (’13); but the writer knows too little of their relation-
ships or significance at present to discuss them.

Hypothalamus. The hypothalamus of the alligator is highly
developed. An examination of figures 11 and 12 will show
that a number of cell groups are present. In his 1913 paper
DeLange has named these different groups. No attempt has been
made to do so in the present paper because of a lack of knowledge
of the fiber connections of the different groups.

FIBER CONNECTIONS

With the foregoing descriptions of the cell groups as a basis,
attention can now be turned to the courses and terminations
of such of the fiber tracts as have been worked out. Papers
published by C. L. Herrick, Edinger, Adolf Meyer, Kappers,
Unger, DeLange, and Johnston contain descriptions of the fiber
connections of the reptilian brain. These descriptions in al-
most every case, have been based on adult material, the work
being done with Weigert preparations which bring out the myelin
sheaths. On the other hand, the work for this paper has been
done chiefly with Cajal and Golgi material, which bring out the
unmyelinated fibers and, in many cases, the axis cylinders of
the myelinated ones. Repeated attempts to prepare a series
stained by the Weigert method were not successful so far as the
forebrain was concerned. These failures, of course, may have
been due to faulty technique, but only extremely young mate-
rial was available and in such material many of the myelin
sheaths may not have become mature. C. J. Herrick (’10)
has figured on pages 537, 539, and 541 some cross sections of
the forebrain and the thalamus of Alligator mississippiensis
showing the fiber tracts and the positions of some of the centers.
These drawings were made from Cajal material and were of
much help. A series stained with Ehrlich’s haematoxylin and
an imperfect series prepared by the Leuden van Heumen method
were used to check the results obtained by the Cajal methcd.

362 ELIZABETH CAROLINE CROSBY
' Tractus olfactorius

Following the human terminology, the writer has considered
this tract to consist of three divisions, a medial, an intermedi-
ate, and a lateral, although the first two are very closely asso-
ciated and have both been considered by many authors under
the name of the medial olfactory tract. (For a diagram of the
distribution of these tracts see figs. 44 to 46). The data given
here have been obtained, partly by the study of sections
prepared by the use of Ehrlich’s haematoxylin and by the
Leuden van Heumen method and partly by work with a Cajal
series in which the axis cylinders of any myelinated fibers, as
well as the unmyelinated fibers, were brought out. The data
in all probability are not complete.

Tractus olfactorius medialis (figs. 18, 14, 44). This tract has
been described in reptiles by Edinger (’88), Adolf Meyer (’92),
C. L. Herrick (’93), Unger (06), DeLange (11), and Johnston
(15). As its name implies, it hes medial to the ventricle of ‘the
bulb and arises, in general, from the more medially and ventro-
medially placed mitral and granule cells. Throughout the bulb,
this tract is lateral to the mitral cells. In the crus many of its
fibers end in the nucleus olfactorius anterior or send their col-
laterals to that nucleus. The projection cells of the nucleus,
in turn, send axones to join the tract. Thus the medial olfac-
tory tract is made up of fibers from both primary and secondary
olfactory centers.

In the anterior end of the hemisphere the tractus olfactorius
medialis has come to lie along the medial surface and it occupies
this medial position as it passes caudad, discharging at various
levels into the dendrites of the intrinsic cells, the double pyramids
and the small projection cells of the hippocampus.

Tractus olfactorius intermedius (fig. 14). This fiber tract,
arising from mitral and granule cells and not distinguishable
from the medial tract until the hemisphere is reached, ends in
the nucleus olfactorius anterior and the medial part of the tuber-
culum olfactorium. Other fibers pass farther caudad and ap-
pear to enter the nucleus of the diagonal band of Broca. It is

THE FOREBRAIN OF THE ALLIGATOR 363

joined by fibers from the nucleus olfactorius anterior to the
tuberculum olfactorium. Part of its fibers pass through the
anterior commissure to the other side and end there in the nu-
cleus olfactorius anterior and, probably, partly in the tuber-
culum olfactortum. These connections have been described by
nearly all the later workers on the reptilian brain.

Johnston (15) in the turtle has considered the medial and
intermediate tracts both under the name of the medial olfac-
tory tract. He describes a very interesting bundle of this medial -
tract which runs caudad with the fiber bundle of the diagonal
band of Broca to the nucleus of the lateral olfactory tract. Quite
probably this tract is present in the alligator but the material
available does not permit of its identification.

Tractus olfactorius lateralis (figs. 13 to 19, 44 to 46). Edinger
(88), Adolf Meyer (’92), C. L. Herrick (’93), Unger (’06), Kappers
and Theunissen (’08), DeLange (’11), and Johnston (’15) have
described this tract in reptiles. In general it arises from the more
laterally placed mitral cells and projection granule cells of the
bulb but some of the fibers come from the dorso-medial portion
and cross over to join the lateral tract. Like the medial tract,
this lateral one at first lies internal to the mitral cell layer. While
still in the bulb it begins to swing out to the surface and in the
crus and lobe it lies mainly along the lateral border of the hem-
isphere. Near the anterior end of the hemisphere it divides
into an outer and an inner division. The inner division en-
ters the nucleus of the lateral olfactory tract and distributes
to it throughout its whole extent. The outer division ends in
synaptic relations with cells of the pyriform lobe and sends
some fibers to the more lateral portions of the tuberculum olfac-
torium. The tractus olfactorius lateralis not only sends fibers
to the pyriform lobe but, in the crus and the anterior end of the
hemisphere, at least, it also receives fibers from it. This tract,
then, carries both secondary and tertiary impulses.

Tractus tuberculo-corticalis

From the tuberculum olfactorium a band of fibers runs along
the medial border of the hippocampal cortex and discharges

364 ELIZABETH CAROLINE CROSBY

into the dendrites of the hippocampal cells. This is the tractus
tuberculo-corticalis (figs. 14, 15). Some fibers swing to the
lateral side of the hippocampus; these may be cortico-tuber-
cular fibers. Farther cephalad some olfacto-cortical fibers from
the nucleus olfactorius anterior join this tract.

Parolfacto-cortical tracts

Tractus parolfacto-corticalis (figs. 16. 17). Fibers swing up-
ward from the medial parolfactory area of the septal region to
the dorso-medial cortex of the hippocampus, entering this latter
mainly, at least, on the medial side. In view of the fact that
the medial surface of the hippocampus is mainly concerned,
as far as one can judge from the impregnations of its cells (see
discussion of the hippocampal cortex), with the reception of im-
pulses, it seems quite probable that these fibers are concerned
chiefly in carrying impulses from the parolfactory area to
the cortex, i.e., they are parolfacto-cortical fibers. Since the
medial parolfactry nucleus receives fibers from the medial
olfactory tract and short fibers from the tuberculum olfac-
torium and since it is connected by way of the diagonal band of
Broca with the lateral olfactory area and with the hypothala-
mus by way of the medial forebrain bundle, this nucleus prob-
ably serves as an olfacto-visceral correlation center and discharges
the resultant of this correlation into the hippocampus by way
of the parolfacto-cortical tract. just described. -

Tractus cortico-parolfactorius (fig. 17). Accompanying the
lateral border of the fornix longus (see the account of the fornix
beyond) as it swings ventralward from the hippocampus, there
are relatively numerous fibers which enter the more dorsal por-
tion of the lateral parolfactory nucleus and, spreading out, dis-
tribute to approximately all parts of this cell mass. Fibers can
be traced from this nucleus passing out medialward and ventral-
ward to join the medial forebrain bundle. Since the lateral
side of the dorso-medial part of the hippocampus is concerned
mainly with the discharge of nervous impulses (see the dis-
cussion of the dorso-medial part of the hippocampal cortex),

THE FOREBRAIN OF THE ALLIGATOR 365

it is probable that the majority of fibers between the lateral
parolfactory nucleus and the hippocampus conduct in the de-
scending direction and that the nucleus functions as a place
of synapse between the hippocampal cortex and the lower brain
centers.

So far as the present data go they appear to suggest a division
of labor between the medial and the lateral parolfactory regions
(medial and lateral septal nuclei of some authors) and to suggest
a motive for their differentiation, viz., that the medial nucleus
is a way-station for ascending impulses going toward the hippo-
campus and the lateral nucleus is a similar station for descend-
ing impulses coming from the hippocampus. The writer is
aware that the data are insufficient for a definite conclusion and
that experimental researches or even more favorable Golgi ma-
terial may prove these suggestions erroneous.

Commissures of the forebrain

There are two large commissures in the forebrain, the hippo-
campal commissure and the anterior commissure.

Commissura hippocampr (figs. 18, 19). The fibers of this com-
missure arise as axones of the projection cells of the hippocam-
pus, which run ventralward and across the mid-line just above
the anterior commissure. After crossing, some of the fibers
appear to end in the nucleus commissuralis, but most of them
pass dorsalward and end in synaptic relation with the cells of
the opposite hippocampus. Thus the hippocampi of the two
sides are put into connection with each other and enabled to work
in a correlated way.

The commissura hippocampi has been the cause of much dis-
pute among the earlier neurologists. Osborn (’87) identified
it as the corpus callosum and for a time this interpretation was
generally accepted. Adolf Meyer (’85) showed it to be the com-
missure of the medial and dorso-medial wall, which regions he
identified as hippocampus. Elliot Smith (’03) claimed that in
reptiles and monotremes there were no eallosal fibers in the
dorsal commissure. Johnston (13a, pp. 402-404) is quite

366 ELIZABETH CAROLINE CROSBY

certain from a series of experiments performed on the opossum
that in this form'there are callosal fibers in the hippocampal
commissure. He believes that callosal fibers are present in that
commissure in reptiles also, although he does not have the ex-
perimental proof for their presence there.

In the alligator, some of the fibers entering into the hippo-
campal commissure appear to come from the region of the gen-
eral cortex and so to favor Johnston’s conclusions, but of course
nothing definite can be settled in this regard until some further
degeneration experiments have been carried out. Unger, Kap-
pers, DeLange, and others have described and figured the me-
dullated fibers of the hippocampal commissure.

Commissura anterior (fig. 18). The following components of
this commissure have been identified: :

a. Stria terminalis fibers. The course of these fibers through
the commissure is described under the account of the fiber sys-
tems (see account of stria terminalis pars commissuralis).

b. Fibers from the tractus olfactorius intermedius to the
tuberculum olfactorium and the nucleus olfactorius anterior of
the other side. These are myelinated.

c. Also, short fibers from the nucleus olfactorius anterior and
the tuberculum olfactorium of one side to the corresponding
centers of the other side.

d. So-called ‘commissura epistriata’ fibers (DeLange 711).
These are included under the description of the stria terminalis
fibers. This component consists of true commissural fibers of
the stria terminalis, which connect the pyriform lobe and the
nucleus of the lateral olfactory tract of the two sides of the brain,
and of decussating fibers of other types.

Tract of the diagonal band of Broca

These fibers connect the region of the nucleus of the lateral
olfactory tract with the parolfactory region and the nucleus
commissurae hippocampi of the same side. These fibers pass
ventrally of the basal forebrain bundles close to the surface of
the brain. Caudalward many of the fibers end in the ventro-

THE FOREBRAIN OF THE ALLIGATOR 367

medial nucleus and a few of them enter the anterior end of the
nucleus preopticus. This fiber tract has been seen and more
fully described by Johnston (715, p. 407) in the brain of the
turtle. Because of the deposit of silver on the outer surface of
the Cajal material in this region, it has been impossible to study
this fiber tract in the alligator as carefully as would be desirable.
Apparently, however, it is made up, in part, of short fibers which
form synapses among the cells of the diagonal band (see the de-
scription of this nucleus). The significance of this tract les in
the opportunity it gives for a close connection between the lat-
eral and medial olfactory areas of the hemisphere (figs. 16 to 19).

Stria terminalis

This stria consists of two divisions.

The commissural portion (St. term. p. com., fig. 18). Slight-
ly anterior to the level of the anterior commissure fibers may be
seen passing from the region of the pyriform lobe, the nucleus
of the lateral olfactory tract (particularly its dorsal portion),
and the extreme ventro-lateral portion of the dorso-lateral area,
directly medialward over the dorsal surface of the medial fore-
brain bundle (VM. F. B.). Most of these fibers cross to the oppo-
site side through the anterior commissure and distribute to the
corresponding regions of the other half of the brain. Some of
the fibers end in the bed nucleus of the anterior commissure of
the same and the opposite side.

The preoptic portion (St. term. p. preop., figs. 19 to 21). This
part is formed by fibers which distribute to the region of the
pyriform lobe, the ventro-lateral part of the dorso-lateral area
and the nucleus of the lateral olfactory tract from the posterior
end of the region reached by the commissural portion of the
stria terminalis to the caudal end of the basal portion of the
hemisphere. This preoptic division turns medialward and cau-
dalward, lying ventral to the posterior division of the lateral
cortico-habenular tract and dorso-lateral and dorsal and in close
relation with the olfactory projection tract of Cajal and its
accompanying cell band—the interstitial nucleus. This preop-

368 ELIZABETH CAROLINE CROSBY

tic portion of stria terminalis does not cross in the anterior com-
missure but passes caudad of it and distributes to the preoptic
region of the same side.

Alveus

A large number of the alveus fibers arise as axones of the
double pyramid and small projection cells of the hippocampus
and run dorsalward then lateralward and then ventro-lateral-
ward around the outer border of the ventricle to the pyriform
lobe (figs. 15 to 21). They distribute during their course to the -
general cortex, the cortex of the pyriform lobe and at least to
the anterior end of the nucleus of the lateral olfactory tract
(the part which is a derivative of Johnston’s small celled portion
of the pyriform lobe). From the pyriform lobe and quite
possible from these other regions, axones enter the alveus.
Probably they distribute to the general pallium and hippocam-
pal cortex.

A small number of alveus fibers at the anterior end of the fore-
brain swing outward between the hippocampus and the primor-
dial neopallium and distribute along the outer surface of the lat-
ter. These association fibers between the two cortical areas have
been very significant in determining the evolution of the prim-
itive neopallium.

Fimbria

This is a term applied to the fibers which border the hippo-
campal cortex along its ventro-medial boundary (figs. 18 to 21).
Behind the foramen of Monro these fibers also border the place
of attachment of the choroid plexus. In the alligator fibers to
the fornix, to the tractus cortico-habenularis medialis, and asso-
ciation fibers between the cortex of the pyriform lobe and the
hippocampus are found in the fimbria.

Fibrae tangentiales

These are short association fibers which tie up the medial and the
dorso-medial portions of the hippocampus (figs. 15 to 21). They

THE FOREBRAIN OF THE ALLIGATOR 369

are on the superficial or pial side of the layer of cortical cells.
Near the anterior end of the hemisphere short association fibers
pass between the dorso-medial portion of the hippocampus and
the general cortex.

These short superficial association fibers convey the nervous
impulses which probably have operated in the course of the
phylogeny to draw the cells of the primordial neopallium from
the ventricular to the superficial position (neurobiotaxis; cf. the
preceding discussion of the general cortex).

Fornix

In the fornix system of the alligator three parts have been dis-
tinguished. The first of these is the commissura hippocampi
(commissura fornicis), which has already been described. The
other parts are the columna fornicis and the fornix longus.

Columna fornicis (figs. 18 to 21, F). The fibers making up
this division of the fornix are mainly axones of the double pyra-
mid cells and small projection cells of the hippocampus. As
has been said before, the fornix fibers join the hippocampal com-
missure and the tractus cortico-habenularis medialis. They
swing first slightly lateralward and then medio-ventralward.
Below the foramen of Monro the columna fornicis fibers sepa-
rate trom those of the hippocampal commissure and, running
caudad, enter the hypothalamus. They are accompanied by
the fibers of the olfactory projection tract of Cajal (figs. 19 to
2

The description here given agrees with the relations brought
out by C. J. Herrick (10). The myelinated fibers of the fornix
have been described again and again by workers on the reptil-
ian brain, among the number being Rabl-Riickhard (’81), Edin-
ger (88 and ’96), C. L. Herrick (’93), Adolf Meyer (’92), Unger
(06), and DeLange (11).

Fornix longus (figs. 16, 17, F.L.). This term is applied in
mammals to a diffuse collection of fibers of mixed character pass-
ing in the medial wall of the hemisphere between the precom-
missural hippocampus (and adjacent parts of the cortex) and

370 ELIZABETH CAROLINE CROSBY

the basal centers of the septum and hypothalamus. In the alli-
gator there is a broad connection between the medial forebrain
bundle near its anterior end and the overlying hippocampus
which is probably in a general way comparable with the mam-
malian fornix longus. Since these fibers connect chiefly with
the lateral or ventricular side of the layer of cortical cells, they
probably are mainly descending projection fibers for the septum
and hypothalamus.

Stria medullaris

The stria medullaris is made up of a number of fiber tracts
running from secondary and tertiary olfactory centers to the
habenula. (figs. 11, 20, 21). The terminology here used follows
Herrick (10). The following components were identified and
traced out: .

1. Tractus cortico-habenularis medialis (figs. 18 to 20). This
tract arises for the most part from the axones of the double
pyramids and small projection cells of the hippocampus. Its
fibers leave the hippocampus at the same level as those for the
columna fornicis and the commissura hippocampi. All three
bundles run ventralward together, the more lateral belonging
to the tractus cortico-habenularis medialis, the intermediate
ones to the columna fornicis, and the medial ones to the commis-
sura hippocampi. After a time the commissural fibers run more
toward the mid-line and become separated from the general
fiber mass, while the cortico-habenular fibers turn dorso-lateral-
ward, and, passing caudad into the diencephalon, enter the stria
medullaris.

Part of the fibers of this medial cortico-habenular tract arise
among the cells of the nucleus commissurae hippocampi. Scat-
tered cells of this nucleus accompany the tract through the
greater part of its course. Accordingly the tractus cortico-
habenularis medialis receives impulses from the hippocampus
of the same and the opposite side, impulses coming from the lat-
ter by way of the commissura hippocampi and its nucleus.

2. Tractus cortico-habenularis lateralis anterior (figs. 16 to 21).
The fibers of the anterior division of the lateral cortico-haben-

THE FOREBRAIN OF THE ALLIGATOR 371

ular tract arise from the more anterior part of the nucleus of
the lateral olfactory tract, from the cortex of the pyriform lobe
in that region, and possibly from the nucleus of the diagonal
band of Broca. The fibers run medialward in the ventral part
of the forebrain, mingling in part with the fibers of the diagonal
band of Broca, which lie on the ventral surface of the brain just
external to them. Near the medial border of the hemisphere
the anterior cortico-habenular tract turns dorsalward over the
ventro-medial nucleus and occupies a position in the angle be-
tween that nucleus and the lateral forebrain bundle (figs. 20,
21). In this angle it is joined by a fiber band which extends
along the medial surface to the caudal portion of the nucleus
ventromedialis among the cells of which nucleus a part of the
fibers can be traced (fig. 20). These two components of the
tract are joined on their medial surface, at this angle between
the ventro-medial nucleus and the lateral forebrain bundle, by
the lateral olfacto-habenular tract and the tracts run dorsalward
together and enter the stria medullaris and so reach the
habenula.

Accompanying the fibers of the anterior division of the lateral
cortico-habenular tract from the nucleus of the lateral olfac-
tory tract and the pyriform lobe is a small bundle of fibers aris-
ing from the same regions, passing dorsal to the ventro-medial
nucleus. Instead of entering the angle, however, between that
nucleus and the lateral forebrain bundle, this band of fibers
runs farther medialward and joins the medial forebrain bundle
(figs. 20, 21). It runs caudalward in this bundle. Its posterior
distribution is not certainly known as its fibers cannot be dis-
tinguished from others of the medial forebrain tract. Unless
it changes its relative position, however, it probably ends in
the hypothalamus, but nothing definite is known of its ending.
In its connections within the hemisphere and in its relative po-
sition in respect to the forebrain bundles, this fiber tract from
the pyriform lobe region shows several points in common with
the tractus pallii of lower forms (Herrick, 710). It is possible
that it and the olfactory projection tract of Cajal may be the

?

372 ELIZABETH CAROLINE CROSBY

representatives of that tract in reptiles. It has been termed in
this account, the ventral olfactory projection tract.

The portion of this tract which arises from the pyriform lobe
and associated regions is evidently the same tract as that de-
scribed by Kappers and Theunissen (’08, p. 225) for the lizard,
Iguana, under the name tractus olfacto-habenularis (see figures
21 and 22 of their paper). Farther forward these authors de-
scribe it as turning lateralward to connect with the ‘lateralen
Lobusrinde’ (fig. 20), which is apparently the pyriform lobe region
of the present account.

There are probably other components of this fiber complex
which have not been impregnated in the preparations studied.

3. Tractus cortico-habenularis lateralis posterior (figs. 20, 21).
This large system of fibers arises from the nucleus of the lateral
olfactory tract and the ventro-lateral part of the dorso-lateral
area. Some of its fibers may arise from the overlying cortex of
the pyriform lobe. These fibers pass medialward, at the same
time sweeping dorsalward to avoid the area of distribution of
the stria terminalis. At the lateral border of the thalamus they
run parallel with and dorsally of the stria terminalis fibers (figs.
20, 21) and here they turn abruptly dorsalward to enter the
stria medullaris thalami.

4. Tractus olfacto-habenularis medialis (figs. 20, 21). This
tract arises from the more posterior portion of the nucleus pre-
opticus, runs dorsalward medial to the medial forebrain bundle
and turns forward and forms the most anterior part of the stria
medullaris.

5. Tractus olfacto-habenularis lateralis (figs. 20, 21). This
tract has its origin from the more anterior portion of the nucleus
preopticus. It runs first lateralward on the extreme ventral sur-
face of the brain ventrally of the basal forebrain bundles, then
backward and dorsalward, joining the tractus cortico-haben-
ularis lateralis anterior in the angle between the ventro-medial
nucleus and the lateral forebrain bundle and passes dorsalward
with it to enter the stria medullaris. (See description of trac-
tus cortico-habenularis lateralis anterior for a further account
of the relations.)

THE FOREBRAIN OF THE ALLIGATOR 313

6. Tractus olfacto-habenularis posterior. This tract arises
near the posterior end of the hemisphere from the nucleus of
the lateral olfactory tract and the ventro-medial nucleus in the
region illustrated in figure 12. It passes directly dorsalward
into the stria medullaris.

Olfactory projection tracts

The entire secondary olfactory area is broadly connected with
the hypothalamus by way of the medial forebrain bundle. De-
scending impulses are carried from the medial (septal) wall of
the hemisphere through the tractus parolfacto-hypothalamicus
(tr. septo-hypothalamicus of some other authors), as described
beyond in the account of the medial forebrain bundle. The
connections between the olfactory centers in the lateral wall. of
the hemisphere and the hypothalamus may in the aggregate
be termed the olfactory projection tracts, following the usage of
Ramon y Cajal. The application of the term projection tracts
to these fibers finds its justification in the intimate relation be-
tween the secondary or basal olfactory centers and the olfactory
cortex of the pyriform lobe in the lateral wall.

There are two of these tracts which enter respectively the
ventral and the dorsal sides of the medial forebrain bundle (figs. 19
and 20), which together probably correspond approximately with
the so-called tractus pallii of fishes and amphibians.

Ventral olfactory projection tract (figs. 16 to 19). This tract
has already been mentioned in our account of the anterior di-
vision of the lateral cortico-habenular tract. It arises from cells
of the pyriform lobe and the nucleus of the lateral olfactory
tract. It runs with the anterior division of the lateral cortico-
habenular tract until it reaches the ventral part of the medial
forebrain bundle, which latter it accompanies caudad. It
- probably ends in the hypothalamus.

Olfactory projection tract of Cajal (figs. 19 to 21). The fibers
of this olfactory projection tract pass directly dorsalward from
the ventro-medial nucleus, then curve medialward and _ pass
caudad lying dorsally of the forebrain bundles and between them

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

374 ELIZABETH CAROLINE CROSBY

and the preoptic portion of the stria terminalis. Some of the
fibers arise probably from cells of the interstitial nucleus and
fibers from cells of the ventro-medial nucleus probably send
collaterals into the interstitial nucleus. The fibers of this
great olfactory projection tract as they swing medialward come
into relation with the descending fibers of the columna fornicis
and there turn sharply caudad and run with the latter bundle
backward, medialward and ventralward to the mammillary body
(figs. 20, 21). (Dr. C. J. Herrick first called the writer’s at-
tention to the fact that fibers of this olfactory projection tract
join the fornix fibers and accompany them ventralward).

Ramon y Cajal (11, vol. 2, pp. 722-723, fig. 462) has de-
scribed and figured this tract and its associated nucleus in the
mouse. Johnston (’15) described the tract in the turtle. He
considers it to be the characteristic connection of his medial
large celled nucleus of the amygdaloid complex (the ventro-
medial nucleus of this description), but does not mention the
interstitial nucleus which accompanies it.

Basal forebrain bundles

Medial forebrain bundle (figs. 9, 16, to 21, M.F.B.). Thisis the
tractus septo-mesencephalicus of Unger and DeLange. It arises
from the parolfactory (septal) nuclei and runs, accompanied by
fibers of the. fornix longus, medialward and ventralward until
it meets the lateral forebrain bundle, which lies farther laterally.
The two bundles can be distinguished from each other for a long
distance because of a difference in the angles at which the fibers
are running. Finally the two become closely mingled and it re-
quires careful study to distinguish them, although such a dif-
ferentiation is quite practicable. According to DeLange (13)
and Unger (’11) the medial forebrain bundle runs to the midbrain.
In the alligator in material prepared by the Cajal method a
part of the fibers appear to end in the hypothalamus (tractus
olfacto-hypothalamicus of the literature), while others pass
caudad to the midbrain (tractus olfacto-peduncularis).

There is no direct evidence in the material studied regarding
the direction of conduction, but the probability is that impulses

THE FOREBRAIN OF THE ALLIGATOR a1

pass in both directions. It serves, then, partly as a discharge
path from the parolfactory areas (tractus parolfacto-hypotha-
lamicus and tr. olfacto-peduncularis) and perhaps also from the
tuberculum olfactorium, and partly as a pathway by which vis-
ceral impulses from the hypothalamic region may reach the
medial parolfactory area (tractus hypothalamo-parolfactorius)
and, either with or without a synapse there, the hippocampus.
Fibers connecting the parolfactory areas and the hippocampus
run on the medial and lateral borders of the medial forebrain
bundle.

Lateral forebrain bundle (figs. 9, 16, to 21, 37, 45, 46, L. F. B.).
This bundle is made up in part of axones arising from the pro-
jection cells of the striatum. It runs ventro-medialward, joins
the medial forebrain bundle on its lateral side, and then passes
caudad into the diencephalon. ‘This is the tractus strio-thalam-
icus of DeLange and Unger.

Besides these components of the lateral forebrain bundle which
carry impulses from the striatal region, there are fibers from the
lateral and medial nuclei of the thalamus which run ventral- |
ward and join the other fibers of this bundle and then go for-
ward to the striatum. These facts are known because axones
er the cell bodies of the lateral nucleus and nucleus rotundus
have been seen to join this bundle (tractus thalamo-striaticus,
or thalamic projection tracts).

There is a second thalamo-striatal path which runs from the
anterior nucleus of the thalamus to the ventro-lateral small
celled part of the hemisphere (that part which is Johnston’s
nucleus caudatus). This has been described by Johnston,
DeLange, and others.

GENERAL DISCUSSION

The problems of forebrain morphology and especially those
dealing with the evolution of the cortical areas have always had
a peculiar fascination for the comparative neurologist. The
broad lines and many of the details of forebrain development
throughout the vertebrate series have been brought out by such
observers as Edinger, Elliot Smith, Johnston, Herrick, and
Kappers.

376 ELIZABETH CAROLINE CROSBY

It is with considerable hesitation that the writer has under-
taken the analysis of the anatomical data given on the preced-
ing pages. Insufficient time and knowledge and the lack of
experience have been very clearly realized and the following state-
ments are offered merely as suggestions or as possible interpre-
tations of some of the changes occurring and the factors opera-
ting during forebrain evolution.

Following the type of interpretation of Edinger, Herrick, Kap-
pers, and Johnston, centers of the alligator hemisphere may be
classified under two general heads which may be subdivided as
follows:

1. Centers dominated by olfactory impulses
A. Basal centers
1. Medial olfactory area
Nucleus olfactorius anterior (in part)
Nuclei of the septum (in part), or parolfactory nuclei
2. Lateral olfactory area
Pyriform lobe complex (in part)
Amygdaloid complex (in part)
3. Intermediate olfactory area
Tuberculum olfactorium
Nucleus olfactorius anterior (in part)
Nucleus of the diagonal band
4. Correlation centers between telencephalic and diencephalic regions
Tuberculum olfactorium (in part)
Parolfactory nuclei (in part)
Nucleus commissuralis hippocampi
Bed nucleus of the anterior commissure
Nucleus preopticus
Interstitial nucleus of Cajal
Amygdaloid complex (in part)
B. Cortical centers (archipallium of Edinger)
1. Hippocampal formation
Small celled non-laminated part of hippocampus (the primor-
dium hippocampi of Johnston, ’13 and ’15)
Dorso-medial cortex (primordial gyrus dentatus, Elliot Smith,
°96, Meyer, 792, Levi, ’04)
Dorsal cortex (hippocampal cortex, subiculum of Johnston ’13)
2. Lateral cortex (pyriform lobe)
3. General cortex (to some slight degree)
11. Centers dominated by ascending somatic impulses from the thalamus
C. Basal centers
1. Dorso-lateral area
2. Intermedio-lateral area
3. Ventro-lateral areas (comparable to corpus striatum of Johnston ’15)

THE FOREBRAIN OF THE ALLIGATOR Byi7h

D. Cortical centers
1. General cortex (in part)
Primordial general cortex (a special portion of this area in close
relation with the dorso-lateral area)

The basal olfactory centers of the telencephalon will be seen
to be separated into two broad groups. In the first group are
those of the medial, intermediate and lateral areas which serve
primarily as secondary olfactory centers. These are old in type,
having their representatives in the hemisphere from cyclostomes .
(Johnston, ’12, Herrick and Obenchain 713) up through the ver-
tebrate series to man. They were originally simply a place of
synapse and consequent redistribution of incoming olfactory
impulses.

The second group of basal olfactory centers includes those
which have developed within the hemisphere later in the phylo-
genetic history as a place of correlation between olfactory and
non-olfactory impulses. It is significant that some of the centers -
(as for example the tuberculum olfactorium), judging from
their fiber connections, are both secondary olfactory nuclei
and correlations centers for olfactory and non-olfactory impulses.
It is the forward growth, then, of non-olfactory fibers from the
diencephalon into the secondary and tertiary olfactory centers
of the hemisphere which has given the impulse toward differ-
entiation to the telencephalon. These nuclei of the hemisphere,
which serve as correlation centers for the olfactory and non-ol-
factory impulses, represent the beginning of that: higher differ-
entiation. Yet these basal centers do not form true cortex.
In the Amphibia (Herrick, ’10) in the ventro-medial part of the
hemisphere, centers showing such type of correlation are pres-
ent and the medial forebrain bundle, which opens the possibil-
ity of connection between the olfactory centers and the visceral
centers in the hypothalamus, is well developed. In the dorso-
medial part of the hemisphere of Amphibia the material, which
is the primordium of the hippocampus, is present; it is under the
influence of olfactory fibers and, to some extent, of fibers of the
ventro-medial area of mixed function as just indicated. But
here no clearly developed cortex is found and it is not until the

378 ELIZABETH CAROLINE CROSBY

basal olfactory and non-olfactory correlation areas are well de-
veloped, as in reptiles, that true hippocampal cortex begins to
appear.

Johnston has emphasized the fact that the hippocampus is
an olfacto-visceral center, although in a later paper (’15, p. 412)
he has said that there are olfacto-visceral correlations in the subic-
ulum as well. It is well to notice that these types of nervous
impulses are not first assembled in the hippocampus. On the
other hand, this cortex simply brings together material already
correlated, partly in the hypothalamus and more completely
within the basal telencephalic centers. Three types of centers
concerned with olfactory impulses are represented then within
the hemisphere.

1. Those basal centers concerned with the distribution of ol-
factory impulses and their summation and correlation among
themselves.

2. Those basal centers concerned with the correlation of
olfactory and non-olfactory impulses.

3. Those centers which receive impulses from correlation cen-
ters of the second type or from similar non-olfactory correlation
centers and integrate these impulses. This integration of ma-
terial already correlated is characteristic of the reptilian cortex.
Into the hippocampus come impulses from the parolfactory area
and the tuberculum olfactorium on the one hand, and from the
pyriform lobe cortex by way of the alveus on the other hand.

In Amphibia (Herrick, 710) the primordium hippocampi oc-
cupies the dorso-medial portion of the medial wall of the hemi-
sphere. This region has all the characteristic fiber tracts of the
hippocampus (cf. Herrick, ’10, p. 480) but there is no differen-
tiated cortex in this region except possibly to a small degree in
Anura, where there is a row of cells close to the surface of the
ventro-medial wall which send out wide spreading dendritic
processes among the incoming fibers and which resemble in cell
characteristics those cells found in the alligator at the anterior
end of the hippocampal formation.

In lower reptiles the dorso-medial area begins to form true
hippocampal cortex. In the turtle, although, as has already been

THE FOREBRAIN OF THE ALLIGATOR 379

pointed out (see discussion of hippocampus), there is a clearly
defined arrangement of a considerable part of the hippocampal
formation into definite cortex-like layers, these layers have not
moved out from the ventricle as in higher forms, but still form
a ventricular mass.

The hippocampal cortex of the alligator represents another
step in advance in differentiation, for here the cortex has moved
away from the ventricle and accompanying this differentiation
has been the specialization, at least to a considerable extent,
of its medial aspect to serve as the afferent side of the cortex and
its lateral aspect to serve as the efferent side. One of the causes
at least, for the outward migration of cells of the dorso-medial
area to form the hippocampal cortex is probably to be found in
the operation of the law of neurobiotaxis (Kappers, 714). Ac-
cording to this law, cell bodies tend to migrate along their den-
drites toward their. source of stimulation. The medial olfac-
tory tracts and other tracts bearing afferent impulses to the
hippocampus are on the medial surface of the hemisphere and
the cells of the developing cortical layers move out toward the
surface of the hemisphere in order that they may come into
eloser relationship with the incoming impulses.

To recapitulate, the following steps appear to have lead from
the primordial hippocampal type to the relatively simple type
of cortex found in part of the hippocampal area in the alligator.
In Amphibia (Herrick, ’10) the afferent and efferent fibers
spread out all through the dorso-medial area. Following a
higher differentiation of the diencephalic and telencephalic sub-
cortical correlation centers, there is a higher differentiation in
the dorso-medial area so that the arrangement of the cells into
cortex-like layers, such as we find in the turtle, occurs. This
second step is followed in other reptiles by a further specializa-
tion of a part of the hippocampal cortex, so that it has an afferent
medial side and an efferent lateral one.

The non-olfactory diencephalic fibers, which enter the telen-
cephalon for the purpose of forming correlations with the incom-
ing olfactory impulses, are partly visceral and partly somatic
in type. Those ascending from the hypothalamus by way of

380 ELIZABETH CAROLINE CROSBY

the medial forebrain bundle to reach the medial wall of the hemi-
sphere carry mainly visceral impulses and the dominant, al-
though not the only, type of correlation in this wall is prob-
ably olfacto-visceral.

Impulses of a like kind reach the pyriform lobe region from
the hypothalamus by way of the ventral olfactory projection
tract (figs. 16 to 19). Thus, this series of steps which seems to
have lead to the development of the hippocampus, can no doubt
be duplicated in the development of the cortex of the pyriform
lobe through the interrelation existing between the hippocampal
cortex and the pyriform lobe cortex on the one hand and the in-
terrelation between that latter cortex and the amygdaloid com-
plex on the other, although the details are not very well known.
In Amphibia (Herrick, ’10) the dorso-lateral area of the hemi-
sphere receives lateral olfactory tract fibers and presumably is
the primordial material for the formation of pyriform lobe cortex
and perhaps for part of the amygdaloid complex. Moreover,
in Amphibia, the somatic area is ventro-lateral in position and
receives and sends out fibers through the lateral forebrain bundle.
In reptiles, this somatic area has increased in size because of the
greater number of somatic fibers that it receives; for accompanying
the telencephalic changes there has been an increased growth and
differentiation of the thalamic regions, particularly of the lateral
portions which receive the fibers of the incoming optic and leminis-
cus systems. This differentiation of the lateral part of the
thalamus (the neothalamus of Edinger) is correlated with an in-
crease in the number of fibers sent forward into the hemisphere
from this region. This increase in the incoming fibers has lead
to a change in the nuclear pattern among reptiles as compared
with the pattern found among Amphibia. Some of the for-
ward extending somatic fibers have begun to pass dorsalward
of the old limits of the striatum and in the turtle (Johnston, 15)
and in the alligator, perhaps in other reptiles also, the dorsal part
of the lateral wall is chiefly a somatic correlation center. The
lateral and ventro-lateral portion of this dorsal wall, however,
is occupied by the cortex of the pyriform lobe and the anterior
part of the nucleus of the lateral olfactory tract. The main part

THE FOREBRAIN OF THE ALLIGATOR 381

of the latter nucleus is found on the outer surface of the ventral
part of the lateral wall external to the striatum complex.
What the factors were which produced these changes in form
relation between these amphibian and reptilian brains it is quite
impossible at present to say. The following account is offered
as a possible suggestion of some of the ways in which these changes
were brought about. Even in Amphibia one would expect the
lateral part of the dorso-lateral area to be particularly closely
tied up with the olfactory tract, for there the dorsal division of
the lateral olfactory tract ends (Herrick, ’10, p. 523, fig. 40, tr.
olf. d. lat.). In more highiy differentiated forms the same process
probably occurred which is known to have happened in other
parts of the brain, namely, that part of the cells will migrate
outward, away from the general cell mass, in order to form a
special receptive center for the incoming olfactory fibers (a nu-
cleus of the lateral olfactory tract) while the remainder will come
less directly under their influence. In Amphibia (Herrick, ’10)
some thalamic somatic fibers reach the dorso-lateral area, al-
though they are few in number compared with the olfactory fibers
reaching that region. As one passes from amphibians to reptiles,
there is a great increase in differentiation of the somatic thalamic
regions, as has been said before, and this differentiation is ac-
companied by an increase in the number of somatic fibers sent
forward into the hemisphere by way of the lateral forebrain
bundle. Some of the somatic fibers, passing dorsalward of the
old limits of the striatum come into synaptic relation with the
neurones corresponding with the old amphibian dorso-lateral
area. Part of such fibers will form synapses with olfactory fibers
and so a somatic-olfactory center, whose later representatives
are the amygdaloid complex and the pyriform lobe cortex, will
be formed. Others of these somatic fibers come into synaptic
relations with the more medially placed neurones of this dorso-
lateral area (i.e., those neurones less directly under the influence
of the olfactory fibers). The entrance of this new mass of somat-
ic fibers and the resultant somatic correlation will lead to an
increase in both the cell number and cell dfferentiation and in
this way a non-olfactory somatic correlation center can well

382 ELIZABETH CAROLINE CROSBY

grow up within areas primarily olfactory in type. With the en-
trance of a larger and larger number of somatic fibers within the
area and the corresponding increase in the number and size of
the cells, two changes in form relations occur in the lateral hemi-
sphere wall. One change is the pushing outward and down-
ward of the cell masses associated with the lateral olfactory tract
so that they come to occupy secondarily a position superficial
to the striatal region (the area occupied in the figures by the
pyriform lobe and the nucleus of the lateral olfactory tract).
The other change is the bulge medialward into the ventricle of
the dorso-lateral somatic area which is so characteristic of the
forebrains of the turtle and alligator.

The cortex of the pyriform lobe has arisen as a differentiation
from the general cell mass of the forebrain which serves as a
nucleus for the lateral olfactory tract. In the lower reptiles this
cortex has appeared, although it is less differentiated than the
olfactory cortex of the medial wall. Johnston (’16) has given very
briefly some of the main features of the embryonic development
of the lateral olfactory area and the cortex of the pyriform lobe
in turtles. He finds the olfactory areas differentiating in the
ventro-lateral part of the hemisphere and believes that the pyri-
form lobe cortex arises from cells of this region which have pro-
liferated and perhaps migrated dorsalward, so that they came to
lie external to the dorsal ventricular ridge. He isnot certain, how-
ever, that they have not developed in situ. So far as the writer is
aware, the history of the embryonic development of the pyriform
lobe cortex in the alligator is unknown, but in all probability it
is very similar to that of the development in the turtle. Of
course, the question at once arises as to the factors operating to
produce the specialized pyriform cortex from the general nucleus
of the lateral olfactory tract and the reason for its new migration ~
dorsalward (if that occurs as Johnston believes). In attempt-
ing to find a solution for the question one must look for the en-
trance into this region of fibers carrying a different type of im-
pulse, for differentiation within an area is not dependent upon
an increase in the number of fibers bearing the same sort of im-
pulse but upon the introduction into the region of fibers bearing

THE FOREBRAIN OF THE ALLIGATOR 383

a new type. Such a new type is introduced into the primordial
pyriform lobe by the alveus, which carries the association fibers
from the other developing cortical areas, particularly from the
hippocampus. These impulses brought by the alveus are the
resultants of a relatively high type of progressively advancing
integration and give the physiological conditions which the
writer has conceived of as being important in the development.

Attention has already been called to the presence of a large
basal somatic area in the dorso-lateral region of the forebrain in
at least some of the reptiles. This region has been named the
dorso-lateral area (figs. 2, 7 to 10, 12, 16 to 19, 44 to 46, DI.A.,
in this paper.- A similar if not entirely homologous area has
been termed the dorsal ventricular ridge by Johnston (15) who
in a recent paper (’16) has given an account of its embryological
development. He finds that the dorsal portion of the embryonic
brain in the turtle first gives rise by a process of cell proliferation
to the general pallial cortex which then occupies a more super-
ficial position. By a secondary proliferation from cells of the
dorsal area, if the writer has understood Johnston (716) correctly,
the dorsal ventricular ridge is formed. ‘The lateral portions are
in relationship with the general pallial cortex and have the ap-
pearance of being an infolding of that area in adult material.
In the adult turtle, the dorsal ventricular ridge extends to the
caudal end of the hemisphere, showing throughout its extent this
relationship with the general pallium. In the alligator the ridge
of cells (termed here primordial general cortex) is found only in
the anterior end of the hemisphere where it has practically the
same relationships as in the turtle. Farther caudad the dorso-
lateral area, of which the general cortex is a differentiation, is cut
off from the latter region by the outward and downward growth
of the ventricle. The dorsal and dorso-medial portions of this
dorso-lateral area, however, receive somatic fibers throughout
practically their whole extent as far as the caudal end of the area.
Association fibers from the hippocampus and the pyriform lobe
distribute not only to the general cortex but also to the primor-
dial general cortex in the anterior end of the dorso-lateral area.
These superficial tangential association fibers have probably been

384 ELIZABETH CAROLINE CROSBY

responsible for the migration of the cells of the general pallium
from their original ventricular position to form a more super-
ficial cortical lamina, for their neurobiotactic influence (Kap-
pers) would have this tendency.

SUMMARY

To recapitulate, it appears to us that the following factors are
involved in giving the morphological form and typical functional
activity of the alligator forebrain:

1. This forebrain is very largely under the dominance of the
olfactory system.

2. Its differentiation into basal and cortical centers is due,
directly or indirectly, to the entrance of non-olfactory dien-
cephalic impulses.

3. These diencephalic fibers are partly for synaptic relations
with the olfactory fibers; and consequently basal centers for the
correlation of olfactory and non-olfactory impulses are present.
A variety in the type of the incoming diencephalic impulses has
lead to the differentiation of a number of different basal nuclei,
for it has not been the number of synapses through which an im-
pulse has passed, nor the number of fibers coming into a nucleus,
but the variety in the types of stimulation received which has
lead to the differentiation of the telencephalic centers.

4. In the lateral wall of the hemisphere the primordial striatum
is present, which is practically free from olfactory influence and
is under the influence of somatic fibers from the thalamus. The
somatic area is larger than in lower forms and correlated with
this increase in size is an increase in size and differentiation of
the lateral part of the thalamus.

5. There are three primordial cortical areas represented and
they all have certain characteristics in common. All of these
are primarily for the integration of impulses already correlated
in the basal centers or transmitted to them by way of association
fibers from the other cortical centers. A certain proportion of
comparatively pure olfactory impulses enters the hippocampus
and the pyriform lobe; but, as has already been discussed, these

THE FOREBRAIN OF THE ALLIGATOR ISH:

are primitive and, in themselves, insufficient for the formation
of cortex.

Again, all the correlated material brought to each of these corti-
cal areas contains olfactory and non-olfactory elements, the lat-
ter including visceral and somatic types. ‘The differences in
significance of the areas are due to a preponderance of a given
type of correlation in each case. In the hippocampus the olfac-
to-visceral elements are very large and dominate the situation;
in the pyriform lobe there is a considerable amount of corre!a-
tion of the olfacto-visceral type, but there is a sufficient propor-
tion of somatic impulses to give this lateral cortex a different
physiological importance from that of the hippocampus. The
olfacto-visceral types of correlation are small in the general cor-
tex and the somatic types predominate.

The great significance of this general cortex in the alligator is
the appearance of a somatic center having a high cortical type
of integration. Nevertheless, since the general cortex is under
tolerably direct olfactory influence from the adjacent hippo-
campal and pyriform cortex (and possibly from other sources),
it cannot be regarded as fully differentiated neopallium, though
it is undoubtedly the immediate precursor of that type of cortex.

LITERATURE CITED

Casau, 8S. Ram6n y 1909-1911 Histologie du Systéme Nerveux. Paris, 2 vols.

Epineer, Lupwie 1888 Untersuchungen iiber die vergleichende Anatomie des
Gehirns. 1. Das Vorderhirn. Abh. Senckenb. Ges., Bd. 15.

1896 Ill. Neue Studien tiber das Vorderhirn der Reptilien Abh.
Senckenb. Ges., Bd. 19.

1899 1V. Studien itiber das Zwischenhirn der Reptilien. Abh. Senck-
enb. Ges., Bd. 20.

Gace, SusaANNA PHeLps 1895 Comparative morphology of the brain of the
soft-shelled turtle (Amyda mutica) and the English sparrow (Passer
domesticus). Proce. Am. Mier. Soc., vol. 17, pp. 185-238.

Heap, Henry anp Hotmes, Gorpon 1911 Sensory disturbances from cere-
bral lesions. Brain, vol. 34.

Herrick, ©. Jupson 1910 The morphology of the forebrain in Amphibia and
Reptilia. Jour. Comp. Neur., vol. 20, no. 5, pp. 413-547.

1913 Some reflections on the origin and significance of the cerebral
cortex. Jour. of Animal Behavior, vol. 3, no. 3, pp. 222-236.

386 ELIZABETH CAROLINE CROSBY

Herrick, C. JuDSON AND OBENCHAIN, JEANNETTE B 1913 Notes on the anat-
omy of a cyclostome brain: lchthyomyzon concolor. Jour. Comp.
Neur., vol. 23, no. 6, pp. 635-675.

Herrick, C. L. 1890 Notes upon the brain of the Alligator. Jour. Cincin-
nati Soc. Nat. Hist., vol. 12, pp. 129-162.
1891 Contributions to the comparative morphology of the central
nervous system. ll. Topography and histology of the brain of certain
reptiles. Jour. Comp. Neur., vol. 1, pp. 14-387.
1893 Contributions to the comparative morphology of the central
nervous system. 11. Topography and histology of the brain of certain
reptiles (Continued). Jour. Comp. Neur., vol. 3, pp. 77-106, 119-140.

Jounston, J. B. 1898 The olfactory lobes, forebrain, and habenular tracts of
Acipenser. Zool. Bull., vol. 1.
1912 The telencephalon in cyclostomes. Jour. Comp. Neur., vol. 22.
1913 Nervus terminalis in reptilesand mammals. Jour. Comp. Neur.,
vol. 23, pp. 97-120.
1913a The morphology of the septum, hippocampus, and pallial com-
missures in reptiles and mammals. Jour. Comp. Neur., vol. 23, pp.
371-478.
1915 The cell masses in the forebrain of the turtle, Cistudo carolina.
Jour. Comp. Neur., vol. 25, no. 5, p. 393-468
1916 The development of the dorsal ventricular ridge in turtles.
Jour. Comp. Neur., vol. 26, no. 5, pp. 481-505.
1916a Evidence of a motor pallium in the forebrain of reptiles. Jour.
Comp. Neur., vol. 26, no. 5, pp. 475-479.

Kapprrs, C. U. Artens 1906 The structure of the teleostean and selachian
brain. Jour. Comp. Neur., vol. 16, pp. 1-109.
1914 Phenomena of neurobiotaxis in the central nervous system.
XVIIth International Congress of Medicine. London.

Kapprrs, C. U. ARIENS AND THEUNISSEN, W. F. 1908 Die Phylogenese des
Rhinencephalons, des Corpus Striatum und der Vorderhirn-commis-
suren. Folia Neurobiologica, Bd. 1, pp. 173-288.

DeLance, 8. J. 1911 Das Vorderhirn der Reptilien. Folia Neurobiologica,
Bd. 5, pp. 548-597.
1913 Das Zwischenhirn und das Mittelhirn der Reptilien. Folia Neu-
robiologica, Bd. 7, pp. 67-138.
1913a L’Evolution Phylogénétique der Corps Strié. Le Névraxe,
vol. 14, pp. 105-122.

Levi, Guisepre 1904 Sull’ origine filogenetica della formazione ammonica.
Archivio di Anat. e di Embriol., vol. 3, pp. 234-247.

Meyer, Apotr 1892 Uber das Vorderhirn einiger Reptilien. Zeitsch. wiss.
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bei den Reptilien und Siugern. Anat. Anz., Bd. 10, pp. 474-482.

Osporn, H. F. 1887 The origin of the corpus callosum. -Part 11, Morph.
Jahr., Bd. 12, pp. 530-543.

THE FOREBRAIN OF THE ALLIGATOR

Rasu-RijickHarRD 1878 Das

Centralnervensystem des

387

Alligators. Zeitsch.

wiss. Zool., Bd. 30, pp. 336-373.
1881 Uber das Vorkommen eines Fornixrudimentes bei Reptilien.

Zool. Anz., vol. 4.

Reese, ALBERT 1908 The development of the American alligator.

Smith-

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Institution.

1910 Development of the brain of the American alligator.

Smith-

sonian Mise. Coll., vol. 54, no. 1922, published by the Smithsonian

Institution.
SHELDON, R. E.

1912 The olfactory tracts and centers in teleosts.

Jour.

Comp. Neur., vol. 22, no. 3, pp. 177-339.

SmitH, G.
126.

Exuiot 1896 The fascia dentata.

Anat. Anz., Bd. 12, pp. 119-

1903 On the morphology of the cerebral commissures in the Ver-
tebrata with special reference to an aberrant commissure found in

the forebrain of certain reptiles.

Trans. Linnean Soc. of London,

2d series, Zool., vol. 8, pp. 455-500.

1908 The cerebral cortex in Lepidosiren.

20 and 21, pp. 513-540.

1910 The Arris and Gale Lectures.
The Lancet, Jan. 1, 15 and 22:

the evolution of the brain.

Anat. Anz., Bd. 33, nos.

On some problems relating to

Uncer, Lupwie 1906 Untersuchungen iiber die Morphologie und Faserung

des Reptiliengehirns.
Bd. 31, Heft 94.
1911 Idem. II.
Wien, Math-nat.

I. Das Vorderhirn von Gecko.

Das Vorderhirn des Alligators.
Klasse, Bd. 120, Abh. III.

Anat. Hefte,

Sitzb. k. Akad.

ABBREVIATIONS

A., alveus

ax., axone

C.A., commissura anterior

C.H., commissura hippocampi

C.Hab., commissura habenularum

Cor.C., correlation cell

D.B., diagonal band of Broca

DI.A., dorso-lateral area

D.pyr.C., double pyramid cell of hip-
pocampus

F., fornix

F.B., forebrain bundles

Fib.tang., fibrae tangentales

Fim., fimbria

F.L., fornix longus

G.C., general cortex

glom., glomerulus

Glom.L., glomerular layer

Gran.L., granular layer

H., hippocampus

Hab., habenula

Hem., hemisphere

H.p.d., hippocampus, pars dorsalis

H.p.dm., hippocampus, pars dorso-
medialis

Hypth., hypothalamus

I.Gran.L., inner granule layer

Interm.l.A., intermedio-lateral area

Inters.n., interstitial nucleus

Intr.C., intrinsic cell

L.F.B., lateral forebrain bundle

L.Gob.C., large goblet cell

388

L.P., lobus piriformis

M.C., mitral cell

M.C.L., mitral cell layer

M.F.B., medial forebrain bundle

N.acc., nucleus accumbens

N.ant.thal., nucleus anterior thalami

N.c.a.+, nucleus commissurae anterioris

N.c.h., nucleus commissurae hippo-
campi

N.d.b., nucleus of the diagonal band
of Broca

N.lat.thal., nucleus lateralis thalami

N.olf.ant., nucleus olfactorius anterior

N.parolf.lat., nucleus parolfactorius
lateralis

N.parolf.med., nucleus parolfactorius
medialis

N.preop., nucleus preopticus

N.tr.olf.lat., nucleus tractus olfactor-
ius lateralis

N.vent.med., ventro-medial nucleus

O.Gran.C., outer granule cell

O.Gran.L., outer granule layer

OLf.B., olfactory bulb

Olf.C., olfactory crus

Olf.proj.tr.(Cajal), olfactory projec-
tion tract of Cajal

Op.ch., optic chiasma

Op.tr., optic tract

P., pulvinar

Plex.L., plexiform layer '

Prim.G.C., primordial general cortex

Prim.h., primordial hippocampus

Proj.C., projection cell of the ventro-
lateral area

S.Gob.C., small goblet cell

S.proj.C., small projection cell

St.C., stellate cell

St.med., stria medullaris

ELIZABETH CAROLINE CROSBY

St.term.p.com., stria terminalis pars

' commissuralis

St.term.p.preop., stria terminalis pars
preopticus

Taen.c., taenia chorioidea

Taen.f., taenia fornicis

T.olf., tuberculum olfactorium

Tr.cort.hab.lat.ant., tractus cortico-
habenularis lateralis anterior

Tr.cort.hab.lat.post., tractus cortico-
habenularis lateralis posterior

Tr.cort.hab.med., tractus cortico-hab-
enularis medialis

Tr.cort.parolf., tractus cortico-parol-
factorius

Tr.olf., tractus olfactorius

Tr.olf.cort., tractus olfacto-corticalis

Tr.olf.hab.lat., tractus olfacto-habenu-
laris lateralis

Tr.olf.hab.med., tractus olfacto-haben-
ularis medialis

Tr.olf.hab.post., tractus olfacto-haben-
ularis posterior

Tr.olf.interm., tractus
termedius

Tr.olf.lat., tractus olfactorius lateralis

Tr.olf.med., tractus olfactorius medi-
alis

olfactorius in-

Tr.parolf.cort., tractus parolfacto-cor-
ticalis
tuberculo-corti-

Tr.tub.cort., tractus

calis
Vent.olf.proj.tr., ventral

projection tract

olfactory

V1.A.(l.c.), ventro-lateral large celled
area

V1.A.(s.c.), ventro-lateral small celled
area

THE FOREBRAIN OF THE ALLIGATOR 389

Gab tase”

Fig. 1 The brain of Alligator mississippiensis, as seen from the dorsal sur-
face. Drawn from a specimen 55 em. long. X 3.

Fig. 2. A lateral view of the same specimen as in figure 1. A part of the
lateral wall has been removed so as to expose the lateral ventricular surface of
the dorso-lateral area. 3. The line A-A represents the plane of section of
figure 44; the line B-B that of figure 45.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27 No. 3

390 ELIZABETH CAROLINE CROSBY

H.p.dm

N.tr olf lat:
N. olf ant. VILA. (5.¢)

Vi A.(s.cY

Tolf.

Figs. 3-12 A series of transverse sections through the hemisphere of Alli-
gator mississippiensis. Toluidin blue. X 19. The serial numbers of the sec-
tions figured are appended to the descriptions.

Fig. 3 Section through the posterior part of the olfactory crus showing the
anterior part of the pyriform lobe and the hippocampus (14: 286)

Fig. 4 Section slightly caudad to the preceding, showing the primordium
of the general cortex (16 : 318).

Fig. 5 Section illustrating the characteristic appearance of the general cor-
tex (18 : 353).

Fig. 6 Section somewhat caudad to the preceding (19: 370).

Fig. 7 Section through the posterior part of the primordium of the general
cortex, showing the basal nuclei of the lateral and medial walls in that region
(22 : 416).

Fig. 8 Section slightly caudad to figure 7 (23 : 436).

THE FOREBRAIN OF THE ALLIGATOR 391

N.tr olf. lat L
VIA. (lc)
VLA.(.c)

N. ace.

D.B.

N. parolf med.

N. d.b.

392 ELIZABETH CAROLINE CROSBY

Be

ecg EMSS ST

bPrim.h.

N. parolf
lat

MFB

DI. A:

Lies}
VI. A..c)

N.d.b.

Fig. 9 Section through the level of the diagonal band of Broca, showing the
relations of the parolfactory nucleus and primordium hippocampi (27: 1, 4).
(A-A’ and B-B’ show the orientation of figure 30.)

Fig. 10 Section through the anterior end of the thalamus. Note the relative
positions of the ventro-lateral, small-celled area and the nucleus anterior thalami
(29 : 3, 1).

Fig. 11 Section through the nucleus lateralis thalami. Note the large size
of the cells (31:: 2, 3).

THE FOREBRAIN OF THE ALLIGATOR 393

N. ant. thal

N. vent.-med
Tr: olf. hab. post

Inters. n.~

N. lat thal.

N. ant. thal.

Hypth.

394 ELIZABETH CAROLINE CROSBY

ead
Pat
ae . d
Tete 38,
el URS patie:
° shea 5
ee!

N. te olf, lat 7
Op. tr.

N.vent-med:

R
N. lat. thal.

12

wpa eet

Fig. 12 Section through the habenular commissure. (32:3, 3).

Figs. 13-21 Transverse sections prepared by the Cajal method. Sections
from two different series were used in preparing this series of drawings. X13.

Vig. 13 Cross section through the left olfactory bulb anterior to the olfactory
ventricle. The characteristic groupings of the internal and external granule
cells and the ring-like arrangement of the mitral cells are clearly shown. The
incoming fila olfactoria and the glomeruli are shown in the figure (3: 3, 2).

THE FOREBRAIN OF THE ALLIGATOR 395

BEM peers Me A
= AM o

LP

A. A.
Fib. tang
H.p. dm
Tr. parolf.
* cort

Miele off lat

Re pauel ct ‘Tr: cort, hab.
ie ant. Vent
Nisaral A eae is olf proj. tr
ae N.parolf mea. ay WI. Toff lat
# ; Nolf. ant N. ace ESB:
x : Trolf-cort.+ Ir tub.cort.
14 \t, alf interm. 16 Ndb
BEB Had.
DLA.

H.p.d.

Prim.G.C. Fib.tang.

ot >
=D

Stee

Tr parolf.cort,

Tr: cort paroffy(y’

Tr: olf.-lat+ Ir. olf.interm. L ie yy Le laf

15
pone N.trolf lat
Mi eeaae|| / Tr cort. hab.lat:
N. parolfimedy" “J ant +Vent olf proj tr.
\ VLA. Cc)
\s VLA (sc)
I7 Ndb

Fig. 14 A transverse section through the posterior part of the olfactory

crus where it is broadening out into the hemisphere (3: 254).
Fig. 15 A transverse section through the right hemisphere at the anterior

end of the neopallial primordium (8 : 3, 3).
Fig. 16 A section near the anterior end of the medial forebrain bundle, M.

EB 2 cAs Ly
Fig. 17. A section a short distance anterior to the hippocampal commissure

(14 : 2, 3).

396 ELIZABETH CAROLINE CROSBY

: GC. Ir cort—hab.med

N. ch.

DLA.

VLA.
(lc)

IELVILA.

(s.c)

Va
Wy Tre olf. lat.

AN trolf lat.

a MIZE Tr: cort-hah
tr, lat ant
7 RB:

N.vent-med.

Hpd

oe ) VIA. (sc
pn teat lat.
LP

SW pia.
a Vi-A.(le)
)

Libis LY rolf. lat.
GZ, St-ferm.p. preop.

Zi
Za Olf proj, tr. (Cajal)

SR Tr. cort hab. lat. ant.
MIRIB: ee

Vent. olf. proj. tr 19 D.B:

\N. vent. -med

Figs. 18-21 These figures were drawn from a transverse series prepared
after the Cajal method and loaned by Dr. P. S. MeKibben. X 13.

Fig. 18 A section through the anterior part of the hippocampal commissure
(11 : 780).

Vig. 19 A section through the posterior part of the hippocampal commissure
and the beginning of the stria medullaris (11 : 788).

THE FOREBRAIN OF THE ALLIGATOR 397

A.
H.p.dm
Fib Tang.
Fim.
Trolf-hab. med.

N.ant. thal.

Olf proj. Tr. (Caj al)

Ir cort hab.laf. post.

St term.p.preop

IN SS
“D S iB N.ventimed,
Y ‘a GAB
Tr. olf-hab. lat. M.F.B.
Infers.n. “ 2 Op.ch
N. preop. S
20 yi
Vitec Ps] St med.
Fib. tang. Whe ‘ei
y N. lat thal.
A
H. p. dm.
Fim —

Olt proj. tr. (Cajal)
St term.p. preop.
Mesut a eh

CLL
Tr cort-hab lat ant/ BYR N vent med
Tr. olf-hab. lat! gf OILEB
iz Mae

N. Preop. 2| Op. ch.

Fig. 20 Section through the anterior part of the thalamus, showing the rela-
tions of the fiber tracts (12 : 805).
Fig. 21 Section through the anterior part of the habenula (12 : 823).

398 ELIZABETH CAROLINE CROSBY

—

LGob C

et co
Meth cule =
Nolf ant 7 Bo
} “29a

N\: Mai remany =o Gran.
\ *\ Jo ;
i Sen
26 \\ tt .
y) } vp
‘4 Hf
Aqgiom ~~

THE FOREBRAIN OF THE ALLIGATOR 399

Figs. 22-43 Characteristic cells from various parts of the forebrain and
thalamus of Alligator mississippiensis as seen in Golgi preparations. 90.

Fig. 22 A diagramatic sketch of the positions and relations of the various
cell types found in the olfactory bulb.

Fig. 23 Small mitral cells of the olfactory bulb (GI : 60).

Fig. 24 Large mitral cell of the olfactory bulb (GL : 73).

Fig. 25 Large goblet cell of the olfactory bulb (GI : 64).

Fig. 26 Small goblet cell of the olfactory bulb (GI : 55).

Fig. 27 Group of internal granule cells of the olfactory bulb. Note that
one of the stellate cells sends its dendrites down into the plexiform layer and
into the region, at least, of the glomeruli. The other does not send its dendrites
outward beyond the mitral cells (Gl : 55).

Fig. 28 A small stellate cell of the olfactory bulb (G1 : 96).

Fig. 29 A goblet cell of nucleus olfactorius anterior (GI : 103).

Fig. 292 A diagram showing the orientation of figure 29.

Fig. 30 A diagram showing the orientation of the hippocampal cells. ‘The
positions of the hippocampal cells figured (figs. 31-36) are shown here.

Fig. 31 Correlation cell found in the dorsal part of the hippocampus at the
anterior end of the hemisphere (GI : 104).

Figs. 32 and 33. Small projection cells of the dorso-medial part of the hippo-
campus (GI : 139; GI : 140).

Fig. 34 Intrinsic cell of the hippocampus (GI : 140).

Figs. 35 and 36 Double pyramid cells. These are the specialized derivatives
of projection cells of the dorso-medial portion of the hippocampus. The cells
figured are probably imperfectly impregnated (GI : 140; GL : 139).

400 ELIZABETH CAROLINE CROSBY

Fig. 37 This isa diagram of a transverse section through the hemisphere at
the level of the primordial infolding. The cells of this primordial general cortex
are round or goblet shaped (fig. 40) and have their dendrites directed outward
and their axones inward and downward into the striatum. The axones come into
relationship with the projection cells of the striatum, and, after a synapse, the
impulse is carried by the axones of these projection cells through the lateral fore-
brain bundle to the lower centers. Impulses reach the primordial general cor-
tex from the hippocampus, the pyriform lobe and the thalamus (by way of the
lateral forebrain bundle). The interpolated neurone (Intr.C.) pictured in the
diagram was not brought out very clearly in the Golgi sections, for, although
neurones of that type were seen in the sections, they were never clear enough
for high power drawings. Several types of neurones can be distinguished in the
toluidin blue sections and the cell labeled ‘intrinsic cell’ is a guess at one of their
probable functions.

401

THE FOREBRAIN OF THE ALLIGATOR

Nlatthal

41
it
N lat thal a / NC
Fig 43 He i
N. lat thal. — ~
Fig 42 ~ eal |
43a ‘Fig 41

Fig. 38 Projection cell of the ventro-lateral area. For orientation see figure

37 (Gl : 126).
Fig. 39 Cell from the anterior part of the pyriform lobe (G1: 99).
Fig. 39a Diagram for the orientation of figure 39.

Fig. 40 Cell from the primordial general cortex (GI : 105). For orienta-

tion see figure 37.
Figs. 41-48 Cells of the nucleus lateralis thalami (GI: L60; GI : 159;
Cola159))-

Fig. 48a Diagram for the orientation of figures 41—43.

N.latthal:

GC

Tr olf. lat:

Bis Tr olf lat. SS
N.trolf lat T olf SS Olf B
——— = SAS
SS

ch.
‘ x \s

46

Fig. 44 A diagram of the connections of the olfactory tracts and the lateral
forebrain bundle of the alligator, based on a longitudinal section of the hemis-
sphere in the plane indicated by the line A-A’ of figure 2. The olfactory tracts
are printed in red, the lateral forebrain bundle in black.

Fig. 45 <A diagram similar to figure 44, but taken farther ventral and in a
somewhat different plane, passing through the level of the hippocampal com-
missure as indicated by the line B-B’ of figure 2.

Fig. 46 A diagram of a longitudinal section through the forebrain of the alli-
gator taken in a parasagittal plane, to illustrate the relations of the olfactory
and somatic centers. Olfactory fibers red, somatic fibers black.

402

THE NUMBER, SIZE AND AXIS-SHEATH RELATION
OF THE LARGE MYELINATED FIBERS IN THE
PERONEAL NERVE OF THE INBRED ALBINO RAT—
UNDER NORMAL CONDITIONS, IN DISEASE AND
AFTER STIMULATION

M. J. GREENMAN

The Wistar Institute of Anatomy and Biology

In a study of regenerated peripheral myelinated nerve fibers
and their controls (Greenman, 713) the question arose as to
whether peripheral myelinated nerve fibers undergo changes in
sectional area or changes in the axis-sheath relation during ex-
cessive physical exertion or in diseased animals.

Tashiro (13) reported that resting nerve fibers, in vertebrates
and invertebrates, warm blooded and cold blooded animals, give
off CO.; that nerve fibers increase their production of CO, about
21% fold when stimulated by an electrical, chemical, thermal or
mechanical stimulus.

He concludes (1) that the nerve fiber has a metabolism and
that this metabolism is modified by the state of excitation, (2)
and that the increased CO, production accompanying stimula-
tion can be used as a new criterion for protoplasmic excitability.

The experiments here described were conducted for the pur-
pose of measuring, if possible, any morphological changes which
might take place in a nerve fiber by reason of some general patho-
logical conditions or by reason of activity and also of obtaining
some information as to the origin of the myelin, its significance or
the influences which increase or diminish it.

In these experiments the observations have been confined to
the large myelinated nerve fibers of the right and left peroneal
nerves of the inbred albino rat.

403

404 M. J. GREENMAN

PRELIMINARY TESTS

A preliminary test was made upon two female gray rats (Mus
norvegicus) of practically the same body weight, 236.5 and 226.5
grams, respectively. One rat, No. 323, was used as a control
while the other, No. 322, was placed in a revolving cage operated
by a motor and exercised continuously for four hours. At the
end of this period the animal was given water but no food and
left in the cage until the following day when the cage was again
started and the animal exercised for a further period of fifteen
minutes. At the end of this brief period the animal died.

The unexpected death of the animal complicated the test to
some extent, since from previous experience with rats in revolvy-
ing cages the amount of exercise given this animal should not
have resulted in death. It is possible that the animal was un-
healthy, though no external signs of disease were noted. No
autopsy was made. However, as the object of the test was
to detect changes in nerve fibers due to fatigue, I proceeded to
remove the right peroneal nerve for comparison with that of the
control animal.

Following the method of previous work on this nerve, 10 mm.
of the right peroneal nerve just distal to the sciatic bifurcation
were excised, in both the exercised (No. 322) and the control
animal (No. 323), fixed in 1 per cent osmic acid, embedded in
paraffine and cut into sections 7 micra thick. The sections from
the middle zone of each nerve were examined. The number of
myelinated fibers was determined and the sectional areas of the
twenty largest fibers were measured.

In making these determinations Hardesty’s (99) method for
counting fibers was employed; after making a photograph of
each section to be counted, each fiber was automatically recorded
and counted by pricking a hole in each fiber image of a photo-
graphic print, while the original section was observed under a
Zeiss 2 mm. apochromatie objective with a No. 4 compensating
ocular and a tube length of 160 mm.

The largest fibers of each section were selected by carefully
going over each successive zone of the section. The sectional

MYELINATED FIBERS——PERONEAL NERVE OF RAT 405

areas of fibers, axis and sheath were determined by measuring with
a compensating planimeter an outline of each fiber and of its con-
tained axis, magnified 4000 diameters. These outlines were
drawn on finely ground glass in the plate holder end of a specially
constructed rigid camera. A Zeiss 2 mm. apochromatic objec-
tive with No. 4 compensating ocular constituted the optic ap-
paratus of the drawing camera.

In order to keep the personal factor as low as possible all
counts and all planimeter records were made by my assistant,
while all drawings were made by myself.

For the skillful technical work and the accuracy of the counts
and planimeter determinations I am indebted to Miss F. Louise
Duhring.

Table 1 presents the summarized data of this preliminary
test.

TABLE 1
eee AVERAGE SECTIONAL AREA OF
NUMBER AT TIME OF NESE 20 LARGEST FIBERS
Fiber Axis Sheath
BOemO? (EXEreIsed)).- onsen. ee. 226 1996 84.1 35.3 48.8
gee. (COMMOL)J +. 2.2204. 25.) 236 2054 105.6 40.3 | 65.2

The control fibers average 20.4 per cent larger than the fibers
of the exercised animal.

The average axis-sheath relation in the control fibers is 38
per cent axis to 62 per cent sheath, while the average axis-sheath
relation in the exercised animal is 42 per cent axis to 58 per cent
sheath, showing a very slight difference in the percentage of
sheath.

This preliminary test suggested that peripheral nerve fibers
may be affected in area of section by excessive exercise and that
possibly the axis-sheath relation may be thereby modified.
However, as the animals were not known to be of the same litter,
and as the limits of individual variation as to fiber areas were
unknown, the test could only be regarded as an encouragement
to pursue the work further.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 3

406 M. J. GREENMAN

A second test was therefore made using, in this case, an albino
rat suffering from so-called ‘pneumonia.’ This animal had
dropped from 264 to 184 grams in body weight in 66 days. Au-
topsy showed badly infected lungs with hemorrhagic areas and
pus cavities.

A brother from the same litter was used as a control for this
animal. The control animal had increased in body weight from
267 to 315 grams during the same period of 66 days. Autopsy
negative.

TABLE 2

NUMBER OF
FIBERS IN PERO-
WEIGHT| NEAL NERVES

AVERAGE SECTIONAL AREA OF FORTY LARGEST
FIBERS IN SQUARE MICRA

KILLING Right Left

Right:||" Weft). 2 ee ee

Fiber | Axis | Sheath] Fiber | Axis | Sheath

3520) 251 184 | 2240 | 2280 | 100.2) 39.6 | 60.6 | 122.2) 45.9 76.3

(pneumonia)
Combined average of right and left
Fiber 111.2 Axis 42.7 Sheath 68.4
3530 251 315 | 2240 | 2296 | 124.9) 46.8 | 78.1 | 108.8) 43.1 | 65.7
(control)

Combined average of right and left

Fiber 116.8 Axis 44.9 Sheath 71.9

Table 2 gives the summarized records of the examinations
made of both right and left nerves from both the ‘pneumonia’
animal and its control. In this case the technique followed was
the same as in the first test. Forty of the largest fibers of each
nerve were measured in this instance, giving the sectional area
of the entire fiber, its axis and its sheath in square micra.

It will be seen from table 2 that in the diseased animal the
average sectional area of the 40 largest fibers from the right
nerve is less, while the average sectional area of the 40 largest
fibers from the left nerve is greater than in the corresponding
fibers of the control animal. If the averages from the right and

MYELINATED FIBERS—-PERONEAL NERVE OF RAT 407

left nerve fibers of each animal are combined it will be seen that
the fibers of the diseased animal are slightly less in area than
those of the control animal. The difference, (5.6 square micra)
however,.is slight and probably insignificant. The axis-sheath
relation in both animals is the same—38.5 per cent axis to 61.5
per cent sheath.

This test left the matter in doubt, but it seemed desirable to
examine other cases of diseased animals.

‘PNEUMONIA’ RATS

For this further work, three groups of so-called ‘pneumonia’
rats were examined—each group being controlled by a healthy
animal from the same litter.

TABLE 3
Series 14. Pneumonia rats and controls from same litters
Pneumonia rats

S 3 AVERAGE SIZE OF FORTY LARGEST
a ei NUMBER FIBERS IN SQUARE MICRA
tal ES OF
S FIBERS :
STRAIN Saltese AUTOPSY Right peroneal Left peroneal
: e8| 88
a on qj |
2] Q jst S| =] be he oe)
a ae BE | aH Sf Se ae ele le lee
2 PUES? eee A z [ent 4 ca <i n cs < nD
352|/o7| 7/251|/Ext.-inbred* 184 | Pneumonia |2240/2230/100 . 2/39 . 6/60. 6/122 .2/45 .9|76.3

424) 9} 29/335|Ext.-inbred | 213/177 | Pneumonia |2122/2000/137 .3/57.2/80.1/161.2|66.7)/94.4
425| | 29/335/Ext.-inbred | 395/282 | Pneumonia |2145/2006/133 .5/53.7/79 .8)135.9|55.6/80.4

Control rats

445| 7| 35/454| Inbred 342/201 | Pneumonia |2047|1988/128 . 2/53 .0/75.1)119.3|47 .9/71.4
447) | 35/454| Inbred 331/292 | Pneumonia |2040)1916/115. 4/47 .5|67 .9)132.9|57.4/75.5
AV@nagenasin masta sir)s Wade ar5l ohio bes ieee 2119/2028/122 .9|50.2/72.7|134.3|54.7/79 .6
40% |60% 40% |60%

* Extracted inbred albino rat.

353/o"| 7/251) Ext.-inbred 315 Neg. 2240|2296)124 9/46 .8/78.1/108.8/43. 165.7
426|o7| 29/335) Ext.-inbred | 355/347.5 Neg. 2122)/2165/139 . 8/52 .5|87 .3/180.8/52.2/78.5
446| 9 | 35/454| Inbred 248/208 Neg. 1973|1863|109 9/44 .4/65.4)127.5)55.1/72.4
Apenpe Atak... x. ol eee RANT ERTS 2111/2108|124 .9}47 .9/76 .9|122.3/50.1/72.2
38% |62% 41%|59%

AOS M. J. GREENMAN

The results of these examinations are given in table 3. In
this table the animals are arranged in the order of their increasing
age. In all four pneumonia cases where the comparison can be
made it will be observed that there was a marked loss from a
previous maximum body weight, due most likely to the disease.

The normal weight for King’s inbred strain of albino rats is
313.8 grams at 243 days, 332.3 grams at 334 days, and 358.7 grams
at 455 days of age. These weights are from H. D. King’s un-
published records.

It will be seen that the rats were all very much under normal
weight at the time of killing.

If, for greater accuracy, we omit from our consideration the
females No. 424, a ‘pneumonia’ animal, and No. 446, a control
animal, and compare the average number of fibers from the right
‘and left peroneal nerves of No. 352, a ‘pneumonia’ animal with the
average number of fibers from the right and left peroneal nerves
of No. 353, its control, we find the ‘pneumonia’ animal presents
an average of 2235 fibers while its control gives an average of
2268 or 33 more fibers. If, in ike manner, we compare the aver-
age number of fibers from No. 425 a ‘pneumonia’ animal with the
average number from No. 426, its control, we find the average
in the ‘pneumonia’ animal to be 2075 fibers while the average of
its control is 2143 or 68 more fibers.

When we compare the average sectional areas of the fibers of
the right peroneals of the ‘pneumonia’ rats with the correspond-
ing averages of their controls, we note that these averages are
practically the same, while in case of the left side the ‘pneumo-
nia’ rats show somewhat larger fibers. The combined averages
of right and left fibers of the ‘pneumonia’ rats differ very slightly
from the combined averages of their controls.

The axis sheath relation in both ‘pneumonia’ rats and their
controls (members of same litter) is practically 40 per cent axis
to 60 per cent sheath, a relation found to exist in a group of 15:
normal inbred rats 150 days of age, as shown by table 4.

Examination of this small group of animals reveals no signif-
icant changes in the sectional area of fibers or in the axis-sheath
relation as the result of disease (pneumonia).

MYELINATED FIBERS—PERONEAL NERVE OF RAT 409

Examination of the number of fibers on both right and left
sides reveals the fact that the older the animals in both the ‘pneu-
monia’ and control groups the less is the number of myelinated
fibers. Thus members of litter 7, aged 251 days, have an aver-
age of 2251 fibers when right and left nerves of both animals are
considered. Members of litter 29, aged 335 days, show in like
manner, an average of 2093 fibers, while members of litter 35,
aged 454 days show an average of only 1971 fibers.

If, for greater accuracy, the females are omitted, a similar re-
sult is obtained; the 251 day rats average 2251 fibers, the 335
day rats 2109 fibers, and the 454 day rats 1997 fibers.

Dunn (12) observed that old rats (640 days of age) showed a
decrease in size of medullated fibers in the ventral roots of the
second cervical, and states that her observations were made on
albino rats of widely varying weights but not in good health.

Table 3 shows that in the eight animals examined the pero-
neal fibers of the greatest sectional areas occur in animals 335
days of age, or at the end of the first third of the entire span
of the rat’s life. In this series animals both younger and older
than 335 days have fibers of less sectional areas.

EFFECTS OF ELECTRICAL STIMULATION

In further pursuit of the problem of changes in the myelinated
fibers in animals in good health, the experiment was modified and
instead of subjecting animals to involuntary exercise the nerve
under examination was electrically stimulated in the following
manner:

Kach animal was etherized, both right and left sciatic nerves
were exposed and cut in quick succession. The wound in the
left leg was closed, while a small portion of the right peroneal
(a branch of the sciatic) was exposed, kept moist with normal
salt solution and stimulated for 25 to 30 minutes by being touched
at one second intervals by platinum electrodes carrying a weak
interrupted current. At the end of this period little or no mus-
cular contraction, following each contact of the electrode, was
apparent. The stimulated (right peroneal) nerve and the
control (left peroneal) nerve were then removed, fixed in | per

410 M. J. GREENMAN

cent osmic acid, dehydrated, embedded and sectioned in the
usual manner. The stimulated nerve and its control were pre-
pared in the same solutions, thus receiving identically the same
technical treatment.

Twelve animals of the same sex and strain and of about the
same ages were used in these experiments and the results are
here presented in table 4.

TABLE 4

Series 12. All males. Effects (volumetric) of electrical stimulation on the pertph-
eral nerve fibers. (Peroneal nerve.)

AVERAGE SIZE OF FORTY LARGEST
FIBERS IN SQUARE MICRA

NUM-|riprer| age |werent| strain | AUC Hight peroneal (shel eas

~ a ao

‘wo | 2 & a | g a |B
ee ees ree ee Pema es Reeser ce MANES
350 162 | 203 | Inbred 2253/2123] 96.0|38.3|57.7|101 .9/39.2/62.7
363 156 | 261 | Inbred 2080/2143] .68.5|30.2/38.3] 94.5/42.8/51.7
369} 11 154 | 243 | Inbred | Neg. |2013|2090| 84.3/39.2/45.1/105.8)44.7/61.1
370} 11 154 | 245 | Inbred | Neg. |2125|2093} 91.3/41.6/49.7/ 90.7|38.9|51.8

373 - | 150 | 230 | Inbred | Neg. |1912/1903)/107.1|46.7/60.4) 95.9|37.9|58.0
374| 12 150 | 223 | Inbred | Neg. |1920/1870) 94.2/38.8/55.4) 99.5)41.7/57.8

375| 12 150 | 218 | Inbred | Neg. |2163/2117| 99.4)44.9/54.5) 95.6)43.2|52.4
376 149 | 236 | Inbred | Neg. |2058|2038| 88.3/38.6|49.7) 93.643 .9|49.7
389) 16 155 | 251 | Inbred | Neg. |2020/2122| 94.9)/43.6/51.3/105.7/45.4/60.3
390} 16 155 | 230 | Inbred | Neg. |2044/2066|) 82.1/36.3/45.8) 83.6)37.2/46.4
437) 33 150 | 248 | Inbred | Neg. |2166/2150/101 .8|40.4/61.4)110. 6/43 .5/67.1
438} 33 150 | 237 | Inbred | Neg. |2103)/2113)116.2|52.2/64.0/103 3/43 .4/59.9

Average...| 153 | 235 2071/2069] 93.7/40.9/52.8) 98.4/41 .8/56.6

44%|56% 43%|57%

The age of one animal was 162 days, all the others range
between 149 and 155 days, and the group for this purpose may
be considered of one age—an average of 153 days. Autopsies
made on ten of these animals proved to be negative. ‘Two were
not autopsied, but showed no signs of disease. It will be noted
that the average number of fibers on the right and left sides is
practically identical—right 2071, left 2069—adding further proof
of symmetry so far as number of fibers is concerned..

MYELINATED FIBERS—PERONEAL NERVE OF RAT 411

The variability in number is shown by the following deter-
minations.

In making these determinations the formulas given by Daven-
port (’99) have been used for the standard deviation, c, the coeffi-
cient of variability, C, and the probable errors of these, as well
as of the mean, M.

Number of fibers in right peroneal nerve—average of 12 cases. Average age
153 days.

M (mean) 2071 Nie — es
o 96 ee ee
C 4.6 C.= + 0.60

Number of fibers in left peroneal nerve—average of 12 cases. Average age
153 days.

M (mean) 2069 Nie — aly,
o 87 Oo. = + 11.9
G 4.2 . Ce = + 0.54

In examining the sectional area of fibers we note that in
8 of the 12 cases the fibers of the right or stimulated nerve are
less in sectional area than those of the left nerve. In one case
(No. 363) the difference is large.

Taking the average sectional area of the stimulated fibers
we find it to be 93.7 square micra, while the average of the left
fibers is 98.4 square micra. The difference here shown between
the averages is 4.7 square micra or approximately 5 per cent of
the smaller number.

An examination of these measurements by the usual statis-
tical methods gives the following results, table 5.

TABLE 5
Right peroneal Left peroneal
Stimulated fibers Control fibers
M (mean) 93.7 sq. micra M, = += 2.3 M (mean) 98.4 sq. micra M, = + 1.1
. 11.8 G= = 1.66 5.9 oc = +08
G 12.6 Ca LC 6.0 Caso

The difference obtained between the stimulated and the con-
trol fibers is 4.7 square micra. The probable error of this determi-
Mation 1602.5.

412 M. J. GREENMAN

It is seen that the probable error of the determination is more
than one-half the difference found between the stimulated and
the control fibers. Assuming that the areas of the largest fibers
in the nerves of the right and left sides are similar under
normal conditions then the difference should be more than three
times the probable error in order to be significant.

On further analysis of the data presented in table 4 it is found
that when the first six entries are separately considered the aver-
age sectional area of the right peroneal fibers is 7.8 square micra
less than that of the left, while in the last six entries the average
sectional area of the right peroneal fibers is only 1.6 square micra
less than that of the left.

Furthermore, in considering the entire group, if No. 363, which
presents the greatest deviation from the mean, is omitted, the
average sectional area of the right peroneal fibers becomes 96.0
instead of 93.7 and the average sectional area of the left perme
fibers becomes 98.8 instead of 98.4.

The right peroneal fibers thus average 2.8 square micra 1n-
stead of 4.7 square micra less in sectional area than the left.

Assuming that the largest fibers of the right and left peroneal
nerves are normally similar in sectional area, it would therefore
seem that the slight difference in average sectional area noted
between the right and left peroneal fibers was not due to the
electrical stimulation of the right peroneal fibers, but that this
difference is a deviation coming within the limits of normal
variation.

The axis sheath relation in this series is nearly the same for
both right and left nerves—about 48 per cent axis and 57 per
cent sheath.

In a previous paper (Greenman, 713) it was stated that the
intact nerve of an operated animal contains fewer medullated
fibers than the same nerve from a normal animal of the same age,
and that there is a loss in sectional area of fibers of the intact
nerve of an operated animal.

From table 4 it will be seen that normal inbred albino rats,
of an average age of 153 days, have an average of 2070 fibers in

MYELINATED FIBERS—-PERONEAL NERVE OF RAT 413

their peroneal nerves. In a group of seven operated stock albino
rats of an average age of 161 days, referred to in the paper cited,
the average number of peroneal fibers in the control nerves was
2025.

It is thus apparent that the normal animal, even at a slightly
earlier age, has more fibers in its peroneal nerves than-were found
in the intact (peroneal) nerves of operated animals.

The average sectional area of 10 largest peroneal fibers from
normal inbred albino rats of 153 days average age is 108.6
square micra (taken from the original records and not shown in
Table 4). In the previous study (Greenman, 713) the average
sectional area of 10 largest peroneal fibers from the intact side
of operated animals, of 189 days average age, was shown to be
65.7 square micra. Thus it is seen that the normal animal even
at a younger age has peroneal fibers of greater sectional area

than those found in intact nerves of operated animals.

' The present data support therefore the previous conclusion
that in the operated rats the size of the fibers in the intact nerve
was reduced.

NUMBER OF PERONEAL FIBERS; SIZE OF LARGEST PERONEAL
FIBERS IN A NORMAL ANIMAL

The next step was to examine the largest fibers of both right
and left peroneal nerves in a series of normal inbred animals
and to determine whether symmetry of the right and left sides
exists as regards the size of the largest fibers.

Table 6 presents the data bearing upon this point. Here are
given the measurements and counts of the fibers in the right
and left peroneal nerves of 15 animals of same sex and strain and
of about the same age.

Here again it will be noted that the average number of fibers
on the right side is practically identical with the average number
on the left side—2038 on the iight side and 2032 on the left side.

414

M. J.

GREENMAN

The variability is shown by the following determinations:

Number of fibers in right peroneal nerve—average of 15 cases.

150.9 days.

Number of fibers in left peroneal nerve—average of 15 cases.

150.9 days.

o
Cc

o
C

M (mean) 2038
97

4.7

M (mean) 2032
80

3.8

Average age

Pp er ee
Ce = = 0.57

Average age

mM, = + 13
sa = == 9.9
Ce= = 0.47

A comparison of the average sectional areas of the 40 largest
fibers of the right and left peroneal nerves shows that in four

TABLE 6

Series 15. Controls. Normal number and size of myelinated fibers in peroneal nerve

2 AVERAGE SIZE OF FORTY LARGEST
oR NUMBER FIBERS IN SQUARE MICRA
ad an
P c FIBERS
STRAIN z E Right peroneal Left peroneal
: 5 AEE é 2 gy aracaliiee | cs | eee es
P lel E§ Riess Boia ose) | Naess ea ee
325 | o| 1 |150) Inbred |242/34.5 1959|1892) 95.3/32.8/62.5} 99.9/34.3)/65.6
328 o| 2 |151| Inbred |210/45.0 2279)2068/113 . 4/40. 7/72. 7/103 .1/39 .0|64.1
340 | o&| 3 |152| Inbred |239/46.5) Neg. |2175|2131| 98.3/36.9|61.4) 97.8/36.0/61.8
342 o| 4 |151) Inbred |274/69.5| Neg. |2078/2158|103 .5/38.6/64.9} 75.5/29.5/46.0
347 | o| 6 |147| Inbred |245|55.0| Neg. |2040/2018) 77.4|30.0/47.4| 70.0/26.5|43.5
380 | o| 13 }151| Inbred |256|55.5| Neg. |2123/2088] 98.7|39.0/59.7| 92.1/38.2|53.9:
381 o| 13 |151| Inbred |283/53.5} Neg. |1985/2029|101 .3/43.7|57.6) 95.438 .2|57.2
384 o| 14 |153; Inbred |276|33.0 Neg. 1931/1925] 99.4/44.0/55.4| 81.5|31.8/49.7
385 | o| 14 |153| Inbred |256|35.0| Neg. |1887/2085| 89.7|/36.8/52.9| 89.5/39.0/50.5
388 | 15 |151| Inbred |238/60.0| Neg. |1967|1985|101 .0/41 .4/59 6/103 . 5/41 .3/62.2
394 | 18 |150| Inbred |276/53.0| Neg. |2045/2048/100. 2/40 .3/59.9| 97.1|37.2/59.9
395 | o| 18 |150| Inbred |314/78.0| Neg. |2091/2134| 91.4/85.2/56.2| 90.6/35.2)/55.4
403 o'| 21 |151| Inbred |266|85.5) Neg. |1982/2003|) 92.2/85.4/56.8)101 0/41 .9/59.1
439 | 34 |151| Inbred |233/43 .0/ Neg. |1989]1897| 97.1/40.5/56.6) 82.9/32.2|50.7
443 o'| 34 |151| Inbred |200/41.5] Neg. |2039|2025| 92.6/41.8/50.8] 92.9|37.4155.5
Average 151 2038)2032| 96.8|/38.5|58.3] 91.5)/85.8|55.6

40%

39% |61%

60%

MYELINATED FIBERS—-PERONEAL NERVE OF RAT 415

cases the two sides are within 1 per cent of one another; in 8 of
the remaining eleven cases the average of the 40 largest fibers is
greater on the right side.

The axis sheath relation here is practically 40 per cent axis to
60 per cent sheath.

Taking the 15 cases together, the average size of fibers of the
right peroneal is 96.8 square micra, the average of the left 91.5
Square micra, the difference being 5.2 square micra, indicating
that the largest fibers of the right peroneal nerve are more than
5 per cent greater than those of the left.

Examining these results by statistical methods we obtain the
following table 7.

TABLE 7
Right peroneal fibers Left peroneal fibers
M (mean) 96.8 sq. micra Mz = + 1.3 M (mean) 91.5 sq. micra M, = = 1.6
o 7.6 Go = +09 o 9.7 G6 = = 11
C 7.9 Ce= += 0.9 C 10.6 Ce = += 1.8

The observed difference between the average sectional area
of right peroneal fibers and left peroneal fibers as shown by
table 7 is 5.2 square micra.

The probable error of this determination is +2.1; a little more
than one-third of the difference observed.

If, however, the first seven entries of table 6 be considered
separately the average of the right peroneal fibers is found to be
7.7 square micra greater than the average of the left peroneal
fibers, while in the last eight entries this difference is only 3.1
square micra. If No. 342, which presents the greatest difference
between the right and left fibers, be omitted, then the average
of the right peroneal fibers becomes 96.4 instead of 96.8 and
the average of the left peroneal fibers becomes 92.7 instead of
of 91.6, and the difference becomes 3.7 instead of 5.2 square
micra.

From the statistical examination of table 6 and this further
analysis of its contained data we may safely assume that the
difference here shown between the sectional areas of the largest
fibers of the left peroneal nerve and those of the right peroneal

416 M. J. GREENMAN

nerve are within the limits of normal variation and that there
is practical symmetry between the largest fibers of the right and
left peroneal nerves.

Further support of this conclusion is to be furnished by an
examination of tables 2, 3, 4 and 6, where it will be found that
in the six instances the average of the largest fibers appears
greater in three instances on the right side and in three instances
on the left side. i

INCREASE AND DECREASE IN NUMBER OF FIBERS WITH
ADVANCING AGE

In table 8 are brought together certain data from tables 2, 3, 4
and 6 giving the ages of the animals and the number of fibers in
the right and left peroneal nerves.

These entries are arranged according to the age of the animal
from 147 to 454 days. .

They are divided into six age groups, the first group including
animals from 147 to 150 days of age, the second, animals from
151 to 154 days of age, the third, animals from 156 to 162 days
of age, the fourth, animals 251 days of age, the fifth, animals
335 days of age, and the sixth, animals 454 days of age. The
average number of fibers in both right and left peroneal nerves
was determined for each age group. The table shows that be-
tween 147 and 251 days of age there is a steady increase in the
number of peroneal fibers from 2037 in the first age group to
2251 in the fourth age group, roughly about two fibers per day;
from 251 days to 454 days represented by two groups of three
animals each, the number of fibers decreases to 1971 fibers, the
number at 335 days being intermediate. Between 251 days
and 335 days there are no records to show when the maximum
number of fibers is reached.

SUMMARY OF RESULTS

A preliminary test, in which a gray rat was forced to run con-
tinuously for four hours, showed the sectional area of the pero-
neal fibers to be 20.4 per cent less than in the corresponding
fibers of the control animal. The axis-sheath relation showed

MYELINATED FIBERS—PERONEAL NERVE OF RAT 417

3.7 per cent less sheath with a corresponding increase in axis
The animals were of about the same

in the exercised animal.

TABLE 8

Data from tables 2, 3, 4 and 6

NO.

347
376
394
438
395
437
325
375
374
373

342
380
381
388
403
439
443
328
340
384
385
370
369

363
350

352
353

424
425
426

445
446
447

AGE

147
149
150
150
150
150
150
150
150
150

151
151
151
151
151
151
151
151
152
153
153
154
154

156
162

251
251

335
335
330

454
454
454

NUMBER PERONEAL FIBERS

Right

2040
2058
2045
2103
2091
2166
1959
2163
1920
1912

2078
2123
1985
1967
1982
1989
2039
2179
2175
1931
1887
2125
2013

2080
2253

2240
2240

2122

2145
2122

2047
1973
2040

Left

2018
2038
2048
2113
2134
2150
1892
2117
1870
1903

2158
2088
2029
1985
2003
1897
2025
2068
2131
1925
2085
2093
2090

2143
2123

2230
2296

2000
2006
2165

1988
1863
1916

AVERAGE OF
RIGHT AND LEFT
PERONEAL FIBERS

2037

2040
2124

2251

2093

1971

418 M. J. GREENMAN

weight, but were not known to be of the same litter or of the
same age.

Comparing male ‘pneumonia’ animals with male controls the
total number of right and left peroneal fibers of the diseased
animal is in every case less than the total number of right and
left peroneal fibers of the control; this difference is 5.8 per cent.

No significant differences were observed between the sec-
tional areas of fibers from ‘pneumonia’ rats and those from the
controls. The axis-sheath relation in both ‘pneumonia’ rats
and their controls is 40 per cent axis to 60 per cent sheath.

In this group of rats from 251 to 454 days of age, the older the
animal the fewer the myelinated fibers found in their peroneal
nerves. This applies to both ‘pneumonia’ and control rats. The
fibers of the greatest sectional area occur in those animals 335
days of age, the younger and the older animals present fibers of
less sectional area.

Data are presented to show that from age 147 days to age
251 days the peroneal nerve gradually acquires more fibers at
the rate of about two fibers per day. In two groups of three
animals each, aged 335 days and 454 days, respectively, the
number of fibers is shown to decrease with advancing age. Be-
tween 251 and 335 days of age the rat acquires its largest and
its greatest number of peroneal fibers.

In a series of twelve animals in which the right peroneal nerve
of each rat was electrically stimulated for thirty minutes the sec-
tional areas of the stimulated nerve fibers were slightly less when
compared with those of the left or intact nerve, but this dif-
ference may be regarded as insignificant.

Fifteen albino rats were examined to determine the normal
size of peroneal fibers of the right and left sides. This exami-
nation of the largest fibers showed that there is practical sym-
metry between the right and left peroneal fibers.

Twelve normal inbred albino rats of 153 days average age have
an average of 2070 fibers in their peroneal nerves and the aver-
age sectional area of the ten largest fibers of these peroneal nerves
is 108.6 square micra.

MYELINATED FIBERS—PERONEAL NERVE OF RAT 419

CONCLUSIONS

In the five series of albino rats here examined the axis-sheath
relation varies but slightly. Its range is from 38 per cent
axis: 62 per cent sheath to 43 per cent axis: 57 per cent sheath;
an average of 40 per cent axis: 60 per cent sheath.

The symmetry as to number of fibers in the right and left
peroneal nerves is almost exact, the difference shown in one
group of twelve and another of fifteen animals being less than
0.3 per cent.

Pathological ccnditions like the so-called ‘pneumonia’ which
is more or less acute and occurs during the later period of growth
in albino rats, appears to lessen the number of myelinated pero-
neal fibers, but produces no measurable change upon the sec-
tional area of the largest fibers.

In the examination of fifteen rats measurements show that
symmetry as to sectional area of the largest peroneal fibers
exists between the fibers of the right and left peroneal nerves.

Electrical stimulation of a peroneal nerve for thirty minutes
appears to have no measurable effect upon the sectional area of
nerve fibers.

In the data presented there is confirmation of the work of
Dunn (712) that sectional area of myelinated fibers decreases
slightly in old age; of the work of Greenman (’13) that the in-
tact nerve of an operated animal loses in both number and sec-
tional area of fibers.

The number of fibers in the peroneal nerve increases with
age until age 250 days is reached and begins to decrease at or
before 335 days of age. After the first year of life the sectional
area of peroneal fibers decreases with advancing age; at 335 days
of age this process of reduction has already begun.

420 M. J. GREENMAN

LITERATURE

Davenport; C. B. 1899 Statistical methods with special reference to biologi-
cal variation. John Wiley and Sons, New York.

Dunn, E. H. 1912 The influence of age, sex, weight and relationship upon
the number of medullated nerve fibers and on the size of the largest
fibers in the ventral root of the second cervical nerve of the albino
rat. Jour. Comp. Neur., vol. 22, p. 131.

GREENMAN, M. J. 1913 Studies on the regeneration of the peroneal nerve of
the albino rat; number and sectional areas of fibers; area relation of
axis to sheath. Jour. Comp. Neur., vol. 23, no. 5, p. 479.

Tasuiro, S. 1912 Carbon dioxide production from nerve fibers when resting
and when stimulated; a contribution to the chemical basis of irrita-
bility. Amer. Jour. Phys., vol. 32, p. 107.

STUDIES ON REGENERATION IN THE SPINAL CORD!

Il. THE EFFECT OF REVERSAL OF A PORTION OF THE SPINAL CORD
AT THE STAGE OF THE CLOSED NEURAL FOLDS ON THE
HEALING OF THE CORD WOUNDS, ON THE POLARITY
OF THE ELEMENTS OF THE CORD AND ON
THE BEHAVIOR OF FROG EMBRYOS

DAVENPORT HOOKER

Anatomical Laboratory of the School of Medicine, Yale University, New Haven,
Connecticut

NINE FIGURES

In the first paper of this series, the attempt was made to ana-
lyse the processes leading to the reunion of the spinal cord in
frog embryos, following its simple but complete severing just
after the closure of the neural folds. The primary purpose of
the experiments upon which this paper is based was to test the
effect. of reversal of a piece of the spinal cord on the healing of
such wounds in embryos of the same age. It soon became ap-
parent that this purpose would be overshadowed in importance
by the evidence which such experiments would give upon, (1)
the nature of the primary responses of the embryos used, (2)
the nature of the early nerve development, both within and with-
out the spinal cord, and (3) the effect of the reversal on the
polarity of the cord and the neurones. The results detailed
below throw light upon some of these points.

Reversal end-for-end of portions of the central nervous sys-
tem has been carried out by several investigators, notably by
Harrison (’98, ’03), and by Spemann (712). As described in his
earlier paper, Harrison grafted young frog embryos together by

1T am indebted to the Loomis Research Fund of the Yale University School
of Medicine for much of the apparatus used in these experiments. This work was
reported in brief before the American Association of Anatomists, 1915 (’16).

421

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 4
JUNE, 1917

422 DAVENPORT HOOKER

their tail buds and, after from two to six days, cut them apart
again, leaving a portion of the tail of one fixed in reversed posi-
tion to that of the other. From this reversed piece a nearly
normal tail regenerated, but the spinal cord of the graft con-
tained ‘‘few or no ganglion cells or nerve fibers’ and remained
in a very rudimentary condition. As described in his later
paper, Harrison made a composite embryo of a head and tail in
normal orientation, but with a reversed middle section. These
embryos were at first helpless, owing to the fact that each com-
ponent reacted independently of the rest. This, however, was
gradually overcome until almost perfect coordination resulted
and in some cases the embryos lived for weeks.

Spemann (’12) removed a portion of the medullary plate as
soon as it became visible and regrafted it, having turned it end-
for-end. As the edges of the wound were carefully apposed,
healing per primum resulted. The portions of the brain anlagen,
which were thus reversed, retained their original polarity.
Spemann worked with embryos of Rana, Bombinator and Triton.

In the present experiments, a piece of spinal cord about 1 mm.
in length was removed, turned end-for-end and grafted into the
space from which it has been taken. In the greater number of
cases the anterior cut passed through the extreme caudal portion
of the medulla, the posterior cut being 1 mm. caudad to it. For
the most part the embryos operated upon were those of Rana
sylvatica, but R. palustris and R. pipiens were also used. All
these embryos healed well and lived for a considerable period.

EXPERIMENTS

Methods. Before each set of operations was performed, a large
number of embryos were freed from the jelly-mass and egg-
membranes. The animals to be used in the experiments were
carefully chosen from this number. The factors upon which the
selection was based were uniformity of size, of stage of develop-
ment and a healthy appearance. Especial care was taken in
matching the normal control animal to each operated specimen.
The greater number of the embryos were operated upon in the
just closed neural fold stage and were from 2.5 to 3.5 mm. long.

SPINAL CORD REGENERATION. II 423

A few averaging 5.5 mm. in length were operated in the stage
having fairly well developed tail buds; a few others at a some-
what later stage, in which the tail fin was just becoming visible,
when they averaged 6.5 mm. in length. The operations on the
5.5 mm. embryos were as successful as on earlier stages, but it
was found impossible to secure good grafts in the 6.5 mm. stage
because the voluntary movements of the embryos prevented
successful healing. This could have been overcome by the use
of anaesthetics, but as the earlier stages fulfilled the requirements
of the immediate problem these were not used.

The operations were performed in 0.4 per cent saline or in clean
tap-water? under a Zeiss binocular microscope. The dorsum of
each embryo was cut through in two places, marking out the
position and length of the piece to be removed. The cuts sev-
ered the skin, cord and the dorsal portion of the myotomes in-
volved, but the notochord was left intact, with a few exceptions.
The embryos were then laid on one side and the two vertical
incisions joined by cutting horizontally through the skin and
myotomes of the upper side. The cord was then carefully sepa-
rated from the notochord, the myotomes and skin of the opposite
side were cut through and the piece freed from the embryo.

If the piece of cord were to be reversed, the excised mass was
turned end-for-end, replaced in the gap in the back of the em-
bryo and held in situ by piling silver wire about the animal.
They were allowed to remain thus until the cut edges of the skin
had reunited (fig. 1, A). This takes from fifteen to twenty-five
minutes. Of 114 embryos operated, all but 12 were treated in
this manner. In these 12 the removed piece was regrafted into
the space from which it had been taken without being reversed.

In some of the embryos with reversed pieces of spinal cord, the
wound surfaces were very carefully brought into close apposition
so that healing per primum resulted. This was presumably the
condition best adapted to produce a reversal of the polarity of the

* Clean tap-water has been found to be.as satisfactory an operating medium
as 0.4 per cent saline (which is isotonic for embryos of this stage) when no very
great area of epidermis is to be regenerated. Consequently, it was used in all
but the earliest of these experiments.

424 DAVENPORT HOOKER

cord, if such a thing were possible. In the rest no attempt at
apposition was made.

Harrison (703) found difficulty in keeping ‘all sylvatica’
composites with a reversed middle piece alive for any great length
of time. No more trouble was encountered in the course of the

Fig. 1 External form of an embryo in which a portion of the spinal cord
was reversed. A, twenty minutes after operation, the stippled area is the piece
which has been reversed; B, twenty-four hours after operation; C, two days after
operation; D, three days after operation; E, four days after operation; F, seven
days after operation. A is a dorsal view, B to F lateral views. These figures
show the peculiar spur-like process of the dorsal fin which is characteristic of
these embryos. Outlines made with a camera lucida. (Embryo IX, 5.)

present experiments in keeping sylvatica specimens alive than the
other species, a circumstance which may be due to the fact that
the piece reversed was smaller than in Harrison’s experiments.
Where the wounds were apposed, few embryos died, but those
animals in which fusion was not produced appear to have a lim-
ited viability and, though many lived for weeks and became

SPINAL CORD REGENERATION. II 425

nearly or quite normal in behavior, a large proportion of them
died.

Each embryo, with its normal control, was placed in a sepa-
rate Syracuse watch glass, was given an individual number and a
separate protocol was kept for it.

The embryos were mechanically stimulated at frequent in-
tervals by lightly touching or brushing the surface of the body
with a soft human hair, according to the method employed by
Coghill (’09 et seq.). This method is very satisfactory, though
it must be borne in mind that by poking the embryo even a
human hair will penetrate the skin and directly stimulate the
myotomes.

The embryos were fixed at intervals of from twenty-four hours
to eight days after operation in sublimate acetic, with the excep-
tion of those that were to be used for the Bartelmez silver nitrate
method, which were fixed in the absolute alcohol and acetic
acid mixture. As the embryos developed very rapidly, the
eight-day specimens were well advanced.

Serial sections of the embryos were stained with Held’s molyb-
die acid hematoxylin and congo red, Ehrlich’s hematoxylin and
congo red, erythrosin and toluidin blue or the Bartelmez silver
nitrate method.

Wound healing. The method of healing a single, severing cut
through the spinal cord has been described in detail in the first
paper of this series.? A careful study of sections of the embryos
with reversed portions of the spinal cord shows that, in the
main, the method of healing is the same whether a piece of the
cord be reversed or whether it be simply severed, but that
in the former case the reversal brings in certain disturbing fac-
tors which tend to mask and limit the processes at work. An
embryo with the cord severed will, in the majority of cases,
re-establish anatomical and functional continuity of the cord
whether the wound surfaces heal per primum or not, but those
embryos in which a piece of the cord was reversed rarely exhib-
ited complete reunion of the cord, though this sometimes oc-

3D. Hooker, 715, pp. 471-486.

426 DAVENPORT HOOKER

curred, if primary fusion of the wound surfaces had not taken
place. Primary fusion of the severed parts of the embryo always
ensued when the wound surfaces were carefully apposed, but
such embryos afford little or no evidence of the individual
processes leading to the healing of the wound, because no active
regenerative changes are visible. Some evidence on this point
is afforded, however, by those embryos in which the continuity
of the cord was interrupted at either or both ends after the epi-
dermis had regenerated sufficiently to cover the cuts and hold the
reversed piece in position. We shall consequently examine
these latter embryos rather in detail.

A period of primary repair follows immediately on the opera-
tion, whether the cord be severed in one place or in two, even
though in the latter case the reversal end-for-end of the tissue
between the cuts accompanies the operation. The epidermis
plays the major part in this repair, covering over the wound
surface so completely that it pushes down between the cut cord
ends if they are not fused. The mesenchyme proliferates rap-
idly and fills up the spaces made by the operation. The open
ends of the neural tube become closed by a shifting of the cells
already present and new composite myotomes are formed by the
fusion of their dorsal and ventral halves.

Sections show that the cephalic cut in nearly all cases passes
through the caudal extremity of the medulla. The second cut is
found from 1 to 1.5 mm. caudad to it. The caudal (originally
cephalic) end of the reversed portion of the cord has usually
somewhat greater diameters than the cephalic (originally caudal)
end, due to the small part of the medulla attached to it. In
many embryos the cut ends of the cord do not lie directly oppo-
site one another, but deviate in various directions from true align-
ment. The piece reversed is so small that it is difficult to place
it exactly in position, but such deviation, unless excessive, does
not necessarily hinder the ultimate reunion of the cord.

During this period of primary repair the embryo as a whole
continues to grow and the characteristic feature of all frog
embryos in which a portion of the dorsum has been reversed
begins to appear. This is the peculiar hump on the back which,

SPINAL CORD REGENERATION. II 427

thick at first, gradually thins out into a typical dorsal fin, but in
reversed orientation (fig. 1, B—-F).

In the first paper of this series, it was noted that the epider-
mis probably plays no réle in the reunion of the spinal cord, but
that further evidence was needed on this point. The close jux-
taposition of the epidermis to the cut ends of the cord renders it
very difficult to settle this question definitely. In both series of
experiments, where the wound edges were not in apposition, a
V-shaped invagination of the epidermis occurred between the
cord ends in the early stages which, on further development,
became a solid fold and was finally withdrawn. In the early
stages of this process, the epithelial cells le in nearly direct
contact with the spinal cord ends and are morphologically in-
distinguishable from the primitive neural cells. The problem is,
therefore, the same in both series of experiments. A careful
study of many embryos brings out certain facts which, if they
do not afford definite proof, at least give strong evidence in
favor of the conclusion that the epidermis contributes no ele-
ments to the regenerating cord.

An examination of the wound region in an embryo twenty-
four hours after operation, of which figure 2 may be considered
typical, shows that, even though the epidermal ingrowth les
almost in contact with the cells of the spinal cord, it has a very
definite boundary and, except for an occasional cell, its connec-
tion with the epidermis covering the body may be clearly seen.
In the lower part of the invagination the condition is somewhat
more confused owing to the presence of cells from the notochordal
sheath, but even here one may differentiate with a fair amount of
accuracy between the two types. The presence of mesenchyme
cells here and there between the epidermal and neural cells also
complicates the problem, but it is usually possible to identify
them. The slight space between the cord ends and the inter-
posed tissue seen on the left in figure 2 is too characteristic and
constant to be a shrinkage space.

This space between the cord ends and the interposed tissue
increases with the growth in length of the embryo, due to the
separation of the cord ends from one another and the withdrawal

428 DAVENPORT HOOKER

of the invaginated epithelium. In this process of withdrawal,
the epidermis maintains no epithelial connection with the cord
by which it could play any réle in contributing elements to its
regeneration. The mesenchyme cells slip in between the epi-

Fig. 2 Condition at site of the wound twenty-four hours after operation.
Epi, epidermis, which has grown down between the cut ends of the spinal cord
and notochord; Mes, mesenchyme cells; Sp, spinal cord. Note the shght separa-
tion on the left hand side of the figure from the epidermal ingrowth and the
definite boundary of the neural cells. N, notochord. Note the large rounded
cells at the cut edges which will proliferate to re-establish its continuity. Y,
mesenchyme cells containing a large amount of yolk.

This drawing is considerably schematized in order that the identity of the
different cells may be more clearly brought out. As a matter of fact, the epi-
dermal cells, shown lightly stippled in the drawing, are very similar to the
neural cells, shown somewhat darker in the figure. Both contain a large amount
of yolk, as do the cells of the notochord and the mesenchyme. The definite con-
tinuity of the epidermal ingrowth and the rounded end of the spinal cord render
identification of the cells possible. This figure represents the condition follow-
ing the stage of primary repair of the wound. With further development the
epidermal ingrowth will be withdrawn and the continuity of the two ends of the
cord re-established. (Mmbryo VIII, 83.)

SPINAL CORD REGENERATION. II 429

dermis and the cord ends as the former is withdrawn, so that
the only connections remaining between the two are mesen-
chymal in nature. The only time at which the epidermis could
contribute elements to the spinal cord is in the earliest stage.
While it is not possible to definitely state that no such ele-
ments are contributed, the continuity of the epithelial tissue and
its separation from the cord ends cast strong doubt on such a
condition. The mesenchymal elements which become mixed
with the developing nerve fibers are cast out at a later stage
by the development of ependymal cell fibers and the wandering
out of neuroblasts in the same manner as has been described for
embryos with severed spinal cord.

In embryos having the cord merely cut in two, the first active
regenerative process which is visible is the growth of nerve fibers
from both stumps of the cord. From the cephalic stump (fig. 3,
I, A) in such cases, there appears a number of rather large nerve
fibers which grow toward the caudal stump. These fibers are
the descending processes (A, dm) of motor neurones situated
within the cord cephalad to the cut. They are the first fibers to
appear at the wound surface. From the caudal stump (fig. 3,
I, B) there appear shortly after the outgrowth of motor fibers
from the cephalic stump, bundles of fibers which are the ascend-
ing processes of motor neurones situated caudad to the cut
(B,am). At a slightly later time, a number of small nerve
fibers appear from the caudal stump growing out from its dorsal
portion toward the cephalic stump. These fibers are the as-
cending processes of the sensory neurones (B, ds). No sen-
sory fibers could be identified as such, growing from the cephalic
stump. ‘They appear to be very essentially centripetal in their
manner of growth.

In embryos having the cord cut in two places, without reversal
of the piece between the cuts, a condition represented in figure 3,
If obtains. There are four cut surfaces, C, D, H and F, of which
Cand F are true cephalic stumps of the cord, corresponding to A
in number I of the same figure, and D and F true caudal stumps
corresponding to Bb in I. The regenerative conditions found in
the wound CD are identical with those in the wound AB in I,

430 DAVENPORT HOOKER

as are those found in the wound EF’, with the exception of the
fact that the nerve fibers from the latter surface appear at a
slightly later time than those from CD. This would be ex-
pected from Coghill’s demenstration of the caudally progress-

fas L

a we Cae salle fe Dee *
U

ac eae Oe Ee D Piero
JE

Fig 3 Diagrams showing the nature of the nerve processes growing out
from the cut ends of the spinal cord. The arrows point toward the head, except
in the segment HD in III, where it points toward the cephalic end of the re-
versed piece; am, ascending motor; as, ascending sensory; dm, descending motor;
ds, descending sensory processes.

I represents the condition found after a single severing cut through the spinal
cord. From the caudal extremity of the cephalic stump, A, only descending
motor processes arise. From the cephalic end of the caudal stump, B, both
ascending motor and ascending sensory fibers are developing.

II represents the condition in a spinal cord which has been completely sev-
ered in two places. The wounds CD and EF each show the same conditions as
the wound AB above.

III represents the condition in the spinal cord of an embryo, the middle sec-
tion of which, ED, has been reversed end-for-end. The stump / shows only
descending motor processes arising from it, while the stump D has both sen-
sory and motor fibers. A comparison of figures II and III shows that by the
reversal of the position of the middle segment no change has been brought about
in the nature of the processes which arise from the surfaces D and E.

ing differentiation of the neurones of the cord. In both of these
wounds the motor fibers from the cephalic surfaces are the first
to appear, those arising from H making their appearance some-
what later than those from C, but before the sensory fibers have
begun to grow out from D. The motor fibers appear in the

SPINAL CORD REGENERATION. II 431

order C, D, HE, F. They are followed by the appearance of
sensory fibers, first from D and later from F.

If the segment DE is reversed in position, as shown in figure 3,
III, the two originally cephalic stumps, C and E, are brought into
apposition, as are the two originally caudal stumps, D and F.
Two possibilities for the outgrowth of nerve fibers from the stumps
in this position present themselves. Either (1) the original polar-
ity of the neurones and the fibers, being an inherent function of
the cells themselves, will be maintained in their reversed position
and the surfaces C, D, FE and F will exhibit the same kind of
fibers as they would have done in their original position, or (2)
the polarity of the neurones and their fibers, if a function of their
position, would be reversed with the reversal of their position,
so that D would show only motor fibers growing from it and EH
both motor and sensory.

Serial sections of a considerable number of embryos in this
stage have been available and they demonstrate that the first of
these possibilities is the one which actually occurs. The posi-
tion of the cord segment is reversed but the polarity of the
neurones within that segment is unaltered. The surface of the
stump C exhibits only nerve fibers which are the descending
processes of motor neurones. The surface D presents fibers
which from their morphological character and from their posi-
tion must be ascending processes of both motor and sensory
neurones. Such a condition is also exhibited by the wound
surface F, while E exhibits only motor fibers.

One most noticeable characteristic of the further development
of these nerve fibers is that they show a marked tendency to
avoid entering the wound surface opposite them. While in the
simple cord severing experiments, it was quite rare to find the
ascending branches of the sensory neurones from the caudal
stump 6b wandering away from and not entering the cephalic
stump A, in the embryos with a reversed piece of the cord, it is
quite rare to find any sensory fibers from F entering D or vice
versa. The motor fibers also exhibit this antagonism, but to a
less degree, so that at least partial motor union between the two
adjacent surfaces is fairly common. It is this avoidance of the

432 DAVENPORT HOOKER

opposite wound surface chiefly on the part of the sensory fibers
which causes the failure of so many of the embryos with primarily
unfused wounds to re-establish continuity of the cord and, in-
deed, to live. This apparent antagonism between ‘like’ wound
surfaces has been noted by many investigators who have worked
on the reversal of the position (and polarity) of parts of organ-
isms. That it exists in those embryos in which the wounds are
caused to heal per primum by close approximation of the cuts
is without doubt, but the very fact of fusion prevents its ex-
pression in so marked a manner as is permitted by the separation
of the cord ends in the embryos under discussion. The fact that
in a very few cases the late stages of complete reunion have been
obtained in these embryos, does not militate against the effective-
ness of the antagonism as a factor which oppose such re-establish-
ment of the continuity of the cord, as, in all these embryos, the
healing is not as clean cut as in those with unreversed cords.

In those embryos in which complete healing is being effected
the same stages as those found in the re-establishment of con-
tinuity after simple severing (outgrowth of ependymal fibers,
wandering out of cells into the fiber mass and elongation of the
canalis centralis) are to be found, though in somewhat more
fragmentary form in the majority of cases. A very few embryos
show complete healing and these differ in no essential respect
from the final stages observed after a single severing out of the
cord beyond the presence of a number of irregularly situated
nerve fibers which run out from the cord into the surrounding
mesenchyme to end there, apparently blindly.

The reversed middle piece. Whether the reversed piece has
fused with the normal cord and medulla at operation, has healed
at one or both ends by the active regenerative processes indicated
above or has failed to establish union at. either end, it stull
possesses certain characteristics which make it readily identifi-
able. The cephalic cut, it will be remembered, passed through
the caudal end of the medulla, leaving a part of the cavity with
the piece to be reversed. This cephalic end, now directed cau-
dad, shows the fragment of medullary ventricle as a small, usu-
ally almost spherical cavity (figs. 4, 5, 6) in all but two cases.

SPINAL CORD REGENERATION. II 433

Where the continuity of the cord was restored by either active
or per primum healing, this cavity opens into the canalis cen-
tralis of the caudal end of the cord. The original, rather wide
funnel-shaped cut edge of the ventricle has been rounded over,
chiefly by the curling downward of the very thin dorsal wall, a
portion of the inferior medullary velum. Where the part of the
medullary ventricle reversed was considerable, the rounding-

Fig 4 Frontal section through a portion of an embryo, the spinal cord of
which was reversed. At the left hand side of the figure the remnant of the
medullary ventricle is visible and in the middle of the figure the slight uneven-
ness of the central canal marks the point of fusion of the cephalic cut. It will
be noted that the walls of the medullary ventricle have become narrowed.

Fig 5 Graphic reconstruction of the central nervous system of the same
embryo. (Embryo IX, 9.)

Fig. 6 Sagittal section through an embryo, a portion of the spinal cord of
which has been reversed. A transposed portion of the medullary ventricle is
clearly to be seen. (Embryo IX, 13.)

434 DAVENPORT HOOKER

over process has been less abrupt and a larger, more fusiform
cavity results (figs. 7, 8).

In one case, a piece of the notochord which was cut at opera-
tion became interposed between the caudal wound surfaces
(fig. 9). Healing was per primum and the end of the noto-
chordal fragment became covered over by ependymal cells, thus
projecting into the canalis centralis. As the fragment was in
continuity with the rest of the notochord, the ventral fiber tracts
were shifted laterally and upward to avoid the obstacle. Func-
tion, in spite of the abnormal distribution of the fibers, became
perfectly normal.

Where the fusion of the cephalic wound has been complete,
the canalis centralis of the originally caudal end of the reversed
piece of the cord, now fused with the caudal end of the medulla,
has undergone enlargement in a few cases. As a consequence,
the medullary ventricle passes over into the canalis centralis of
the spinal cord by a gradually tapering funnel. This is the
normal condition of course, but in these operated embryos the
wall of the funnel is formed in part from the ependymal cells of a
portion of the canalis centralis normally situated a millimeter or
more away. The point of fusion is noticeable in many of the
specimens as a more or less pronounced notch in the ventricular
wall, though in others the fusion is so perfect as to make it
impossible to detect the line of healing. In other cases, however,
it seems that the caudal extremity of the medullary ventricle
has undergone a narrowing to produce this funnel (fig. 4), and
that the canalis centralis of the reversed piece of the spinal
cord has been unaltered in the process. In those cases where
fusion or healing did not occur at the cephalic cut, the caudal
end of the medulla is rounded over and closed. In some of these
cases the ventricle is much enlarged.

Effect of reversal of position upon the polarity of the cord. In
the operative procedure described above, a piece of the spinal
cord has been removed from its normal position, turned end-for-
end and been grafted into the space from which it was removed.
Its position was therefore reversed. The evidence given demon-
strates that the neurones contained within that reversed piece

SPINAL CORD REGENERATION. II 435

continued to develop in their normal orientation to that piece
and that the transposed portion of the medullary ventricle
remained,,with but minor changes, normal. During the early
stages of development and up to a time after the healing has
been completed, there is not a single sign that the polarity of the
cord has been reversed.

Fig. 7 Frontal section through an embryo in which a relatively large part
of the medullary ventricle (seen at the left) was reversed.

Fig. 8 Graphic reconstruction of the same embryo. (Embryo IX, 12.)

Fig. 9 Sagittal section through the central nervous system and notochord
of an embryo in which a small portion of the spinal cord was reversed. The
right hand piece of the notochord marks out the length of the segment reversed.
It will be noted that the central canal of the spinal cord has apparently enlarged
at the point of fusion with the medulla anteriorly. Posteriorly a piece of the
notochord became inserted between the wound surfaces, necessitating the diverg-
ence of the developing nerve fibers from the normal course. In spite of the
cavity in the spinal cord in which this piece of notochord hes, a return to nor-
mal function was obtained. This section is from the embryo shown in figure 1.
(Embryo IX, 5.)

436 _ DAVENPORT HOOKER

In those embryos in which healing has not occurred no sign of
a reversal of polarity ever presents itself. For any evidence
that later adaptation produces anything similar to reversal of
polarity in the older stages of the healed embryos, we must turn
to a consideration of their behavior.

Responses of embryos with primarily fused cords to tactile
stimulation. As previously noted, the embryos were stimulated
with a human hair according to the method of Coghill. Em-
bryos in which a portion of the spinal cord had been reversed
and in which primary fusion had taken place began to exhibit
responses to tactile stimulation from twenty-four to thirty-six
hours after operation. In the case of those embryos operated
when from 2.5 to 3.5 mm. in length reactions to stimulation
appeared in the latter part of this period in most cases, while
those operated at a later stage, averaging 5.5 mm. in length,
usually reacted twenty-four hours after operation. In every case
the first reaction to appear was a decided bending of the head
toward the side stimulated. But a single response followed each
stimulation. Inasmuch as Coghill (09) has described this type
of reaction as occurring only occasionally and very irregularly in
Amblystoma, a large series of normal frog embryos were carefully
stimulated with a human hair in order to obtain information in
regard to the frequency of its occurrence in that animal. The
results of these experiments demonstrated quite conclusively
that in the frog embryo a contraction of the myotomes of the
same side as that which receives the stimulation is the first
normal type of reaction to tactile stimuli. It is of course evi-
dent that direct stimulation of the myotomes would result in a
similar type of response. Great care was therefore taken to be
sure that the pressure of the hair upon the embryo was not
sufficient to directly stimulate the myotomes. ‘To render con-
trol of this point more satisfactory all stimulation was per-
formed while the embryo was being watched with a Zeiss binoc-
ular microscope. It was found that by gently touching the
surface of the embryo with a slow stroke-like movement of the
hair at a point just ventral to the center of the myotomes, with-
out the slightest indentation of the skin of the tadpole having

SPINAL CORD REGENERATION. II 437

been caused by the pressure of the hair upon it, this type of
response could be almost unfailingly obtained. The constant
reappearance of this type of response lends further evidence in
favor of the view that a single quick bending of the head toward
the side stimulated is the most primitive reaction to tactile
stimulation in the freg embryo.

This type of response is quickly followed by a typical ‘avoiding’
reaction. This reaction makes its appearance within two to
three hours after the beginning of sensitivity to tactile stimu-
lation of the embryo and lasts for a varying period.

Embryos from thirty-six to forty-eight hours after operation
almost uniformly exhibit a double C reaction. The term
‘double C’ reaction has been applied to that form of response
in which the embryo on stimulation contracts into an are,
straightens out and contracts into a similar are on the opposite
side. This constitutes the complete reaction to a single stimula-
tion. This is true even of those embryos which do not begin
to exhibit any response until after the first thirty hours. These
embryos which are late in beginning their reaction appear to
hasten through the earlier stages so that they tend to become
uniform in their response at this later period. It was noted
that at the very beginning of this type of response there seemed
to be a tendency on the part of many of the embryos to con-
tract first on the side stimulated, but there is not sufficient
evidence to prove that the first contraction toward the side
stimulated is any more decidedly frequent in appearance than a
first contraction away from the side stimulated. During the
beginning of the appearance of this type of reaction the two
contractions toward opposite sides of the body constituted the
entire response, but within a very short time, frequently not
more than half an hour afterwards, the embryos exhibited a
series of these double C reactions to each stimulation.

This type of reaction lasts for a relatively long time, from six
to twelve hours, and it is followed by a typical S or sinuous re-
action. During the latter part of the double C reaction period,
at a time which apparently bears no relation to the beginning
of the appearance of the § reaction, the embryos exhibit spon-

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, wo. 4

438 DAVENPORT HOOKER

taneous movement for the first time. This is from two and a
half to three days after operation. The 8 reaction, which is of
course the most primitive manifestation of the swimming move-
ment, rapidly passes over into a period in which active locomo-
tion is produced. In these embryos with the reversed spinal
cord the first movements producing locomotion are ill coor-
dinated and the consequent movement of the embryos is far
from normal. As a rule spontaneous movement on the part
of these operated embryos takes the form of the double C reac-
tion for a considerable period after locomotion may be pro-
duced as a result of stimulation. From a careful study of the
movements of these embryos with reversed middle piece it is
evident that there is little or no coordination between the por-
tions and that locomotion is produced by the activity of the
central (reversed) portion of the body which drags the rest of the
organism into activity. It is true that, during these movements
which produce locomotion, both head and tail portions of the
embryos do move by the contraction of the myotomes situated
in them, but there is every indication that the stimulation of
these myotomes in the head and tail is brought about directly
through the pull of the skin over them which is caused by the
movements of the middle piece. In a previous paper (711) I
demonstrated that very slight tension on the skin of an embryo
produced by pressure at a distant point is capable of mechani-
cally stimulating the myotomes to activity. The cause of the
movements in these embryos is apparently identical with that
described in Paper I of this series.

From this time on the embryos show a steadily increasing
sensitivity to tactile stimulation and a steadily increasing
amount of coordination in the responses. As soon as even the
most primitive type of coordination was exhibited in the reac-
tions to tactile stimulation, the embryos began to show spon-
taneous swimming movements, which with the passage of time
gradually improved until five to six days after operation many
of the embryos were almost, if not entirely, normal in their
movements. Indeed in several cases it was only possible to
differentiate the operated embryos from their normal controls

SPINAL CORD REGENERATION. II 439

by the peculiar spur remaining from the dorsal fin upon the
back (fig. 1, F).

Responses of embryos with unfused spinal cords to tactile stumu-
lation. Embryos in which the middle section of the spinal cord
has been reversed and in which the wounds have not been per-
mitted to fuse begin to respond to tactile stimulation approxi-
mately at the same time as those in which primary fusion of the
wound surfaces has taken place. Those operated in the open
neural fold stage seldom show any reaction to tactile stimulation
until about thirty-six hours after operation, while those which
were operated in the beginning tail bud stage begin to respond
about twelve hours earlier. This is undoubtedly due to the
greater differentiation of the nervous system at the time of
operation in the latter. The majority of the stimulations of
these embryos was carefully watched under the binocular, as
the regions are small and consequently their movements diffi-
cult to perceive accurately with the naked eye. In these em-
bryos, of course, there is complete anatomical separation at the
sites of the two wounds in the earlier stages. Care was taken
that the myotomes were not directly stimulated.

The first response to tactile stimulation is exhibited by the
middle piece, irrespective of the age at which the embryos were
operated upon. ‘The earliest visible response is a slight twitch-
ing of the myotomes on the same side as that which receives the
stimulation. Within two hours after this, these embryos ex-
hibit a marked contraction of the middle piece into a well
defined are, the concavity of which is toward the side stimulated.
Attempts were made to carefully stimulate only minute areas
of the middle piece, but contractions of the entire segment resulted
in every case. It was impossible to demonstrate any greater
sensitivity to tactile stimulation of one end of the reversed
piece over the other, or of any tendency on the part of the
myotomes of either end to contract more vigorously than those
of the other. The reversed middle piece reacted throughout as
a unit. If the cut passed through the caudal extremity of the
medulla, the head remained inert for some time after the middle
piece had begun to respond. If the cut, on the other hand,

440 DAVENPORT HOOKER

passed through the cord just behind the medulla, the head ex-
hibited a single reaction toward the side stimulated. The tail
never showed responses to tactile stimulation until a later time
than the middle piece. This type of reaction to stimulation on
the part of the middle piece lasted for several hours.

The second phase of reaction to stimulation on the part of
the embryos was marked by the appearance of contractions of
the middle piece into an are, the concavity of which was away
from the side stimulated. This is of course a typical avoiding
reaction. The entire middle piece again responded to stimula-
tion as a unit. At atime which practically coincided with the
beginning avoiding reaction in the middle piece, sometimes
slightly preceding it, but more usually following it, the head
began to exhibit a single movement toward the side stimulated.
The myotomes involved were those situated at the extreme
caudal end of the head region. The tail in the majority of
cases remained unresponsive to stimulation during this second
phase of reaction, though in a few cases, just before the begin-
ning of the third phase it also exhibited a single contraction
toward the side stimulated.

The third phase of reaction is marked by the passage of the
middle piece from the simple avoiding reaction to the double C
type. Usually at a time which precedes this by not more than
one to two hours, the head has begun to exhibit an avoiding re-
action and, almost coincident with its appearance, the tail be-
gins to respond by a single movement toward the side stimulated.
The tail portion passes through this period of primary response
toward the side stimulated very rapidly and soon enters upon a
period in which it gives a typical avoiding reaction. The char-
acteristics of the third stage may therefore be said to be as
follows: (a), head gives an avoiding reaction; (b), middle piece
exhibits double C reaction; (c), tail passes from a brief stage of
reaction toward the side stimulated to one in which a typical
avoiding reaction is shown.

The fourth stage of reaction to stimulation is characterized
by the appearance of the S reaction in the middle piece and of the
double C reaction in both the heac and tail segments. At this

SPINAL CORD REGENERATION. II 44]

stage locomotion of the embryo as a whole will result from
continued stimulation of the middle piece. The locomotion thus
produced is in every way similar to that exhibited by those
embryos in which the wounds have been primarily fused, but
appears at a slightly later time. An explanation for the apparent
delay in the appearance of the S reaction in the middle piece of
those embryos in which the cord wounds were not permitted to
fuse is not entirely clear, unless it be that the isolation of the
middle segment from the head and tail and the consequent ab-
sence of the support which a continuous skin over the embryo
gives to it does not enable the myotomes to express their con-
traction to as marked a degree.

From this time on, the development of locomotion in response
to stimulation proceeds slowly and is accompanied by the active
regenerative processes which go forward in the embryo. Com-
plete return to a normal condition of coordinated locomotion
was seen only in two or three embryos in which practically com-
plete reunion of the spinal cord had taken place.

Spontaneous movement appears in the middle piece as soon as
the double C reaction makes its appearance and is followed by
voluntary movements of the head when it also passes into the
double C phase. The tail is the last portion of the embryo to
exhibit spontaneous movement.

Correlation between the phase of reaction and stage of regenera-
tion. From the work on embryos in which the spinal cord has
been simply severed, it is apparent that the embryo is capable of
performing swimming movements in response to stimulation
before there is any nervous connection between the two ends of
the cord and that a very simple type of voluntary swimming
movement may arise before these connections have been estab-
lished. It is further to be noted that apparently the only réle
played by the nervous connections in the embryo is that of co-
ordination in the later phases of the swimming movement.
Exactly the same conditions are met with in all embryos in which
a portion of the spinal cord has been reversed, whether the
operation has been followed by the complete fusion of the
wound surfaces or not. It has been noted in these experiments

442 DAVENPORT HOOKER

that, not only may a very primitive type of swimming move-
ment be developed, but that a type which is apparently fairly
well coordinated may be exhibited by embryos in which no ner-
vous connection is present between the cut ends of the spinal
cord. This was noticed particularly in the case of several, em-
bryos in which the reversed middle piece healed in a somewhat
oblique position to the long axis of the embryo. These embryos,
though they lived for a considerable time, long after some sort
of nervous connection had been established in the majority of
other specimens, did not exhibit any connections of a nervous
nature between the ends of the reversed piece of the central nery-
ous system and that contained in the head and tail region.
Nevertheless, the S reaction in these embryos gave place to the
usual uncoordinated swimming movement in due course, which
continued to exhibit all the signs of progressive coordination
which had been previously supposed to accompany the estab-
lishment of nervous connections between the severed cord ends.
These embryos of course never swam in a perfectly normal
manner, but they developed the ability to move over relatively
long distances. Their movements showed a considerable degree
of synchrony between the different nervously isolated portions
of the body and the head and tail regions took part in the move-
ments. It is of course certain that there 1s no nervous con-
nection through the skin in any such sense as Wintrebert (’04)
supposed. Careful examination demonstrated that there are no
nervous connections between the two regions of the body, be-
yond the possibility of the innervation of a pair of myotomes on
either side of the cut surfaces. That this possibility is not a
probability is demonstrated by the fact that such nervous
connections have never been observed in these embryos and that
the heavy mass of notochordal connective tissue which grew
out from the injured notochord in these specimens completely
isolated the cut ends of the cord in the middle piece from the
other portions of the body. It is much more probable that the
tension on the skin of the embryo caused by the movement
of the middle piece has brought about a direct mechanical
stimulation of the myotomes of the head and tail regions which

SPINAL CORD REGENERATION. II 443

has excited them to contraction. This contraction may have
acted as a proprioceptive stimulus to the nervous system of the
head and tail region which caused in turn a contraction of the
opposite side of the body.

It is therefore to be noted that apparently the activity of the
middle piece is responsible for the early movements of embryos
in which this middle piece has been reversed. In a few indi-
viduals the middle piece sloughed out immediately after opera-
tion and these embryos continued their development with this
wide gap in the back. Such embryos never exhibited even an
approach to a swimming reaction. They were never able to
produce locomotion over the most limited distances. Indeed it
can be said that the middle piece is the only one which exhibits
a true § reaction.

On the other hand, it must be remarked that for a normal
swimming movement some sort of nervous connection between
the cut wound ends is essential. From a careful study of the
stages of regeneration in the spinal cord in correlation with the
type of swimming movement exhibited, it seems that motor
connections are alone essential for a normal swimming move-
ment. It is further apparent that the ascending motor processes,
which are the ones which bridge the caudal cut, are capable of
acting as typical descending processes and it is evident that
motor stimuli from the middle piece are transmitted along these
fibers to the tail portion of the embryo. Conversely, it is also
apparent that the descending processes growing out from the
cephalic portion of the piece may also change their functions
and transmit stimuli to the head region of the embryo. In this
sense there is a reversal of the polarity of the neural elements
contained within the reversed portion of the cord. On the other
hand, there is considerable question as to the specificity of these
two types of processes.

The establishment of sensory connections between the isolated
portions of the cord is long delayed in the embryos under dis-
cussion and seems to play little or no réle in the development
of the swimming movement.

444 DAVENPORT HOOKER

DISCUSSION

From the foregoing résumé of the details of the experiments,
it is evident that when a portion of the spinal cord taken from
the cervical or upper thoracic region of the frog embryo is re-
moved, turned end-for-end and grafted into position, the piece
as a whole retains its original polarity. This is what one would
expect from the results obtained by Spemann (712). The fact
that the reversal of position does not affect the original polarity
of the piece reversed is amply demonstrated by the persistence
of the medullary cavity at the originally cephalic end of the
middle piece. Furthermore, the manner in which the develop-
ing nerves grow out from the cut ends of the reversed middle
piece demonstrates that in the beginning at least the reversal
of position does not affect the polarity of the elements contained
within the piece reversed. The nerve processes arising from these
ends are exactly the same in kind as those which would have arisen
if the piece had remained in its original position. This fact
complicates the ensuing attempts to re-establish the continuity
of the cord. After simple severing of the cord, the descending
processes of the motor neurones situated at the various levels of
the cord began their development in normal orientation to the
cord as a whole. There is in reality no true regeneration of
the individual elements, inasmuch as the operations are carried
out before the time when the nerves normally begin to develop.
In consequence of this fact, these processes are subjected to no
other abnormal condition than the necessity of traversing an
area filled wtih connective tissue. The same is true as regards
the ascending processes of the sensory neurones. In the embryos
under discussion, on the other hand, the normal relationship
between the direction of growth of all the processes and the
antero-posterior axis of the embryos has been completely upset
so that the nerve fibers which were originally descending pro-
cesses grow in an ascending direction and vwice versa. AS we
have seen, this brings together at the cephalic wound surface a
series of nerve fibers growing in opposite directions which are all
descending processes and at the caudal wound a number growing

SPINAL CORD REGENERATION. II 445

in both directions which are ascending processes. As we have
noted there is apparently an antagonism between these ‘like’
surfaces, demonstrated by the marked tendency on the part
of the nerve fibers to avoid entering the opposite wound surface
in those embryos with open wounds. In spite of this fact some
of these fibers do enter the opposite cut surface and we may be
very certain, in the case of those embryos in which per primum
healing took place, that all of the fibers from the reversed middle
piece grew in an abnormal direction.

It is of course doubtful whether there is any real specificity
of ascending and descending processes and the physiological
result obtained certainly demonstrates that a considerable de-
gree of adaptation has taken place here, in that the descending
processes must certainly function as ascending processes and
vice versa. In this sense therefore, we must conclude that
there is a reversal in the polarity of the elements contained
within the reversed piece of the spinal cord, though whether this
reversal in polarity includes anatomical reversal of the cells
themselves is very doubtful. It is much more probable that
only the direction in which the stimuli travel along the processes
is the reverse of its usual course.

Not only do the nerve cells situated within the reversed piece
of the spinal cord show a considerable degree of adaptability, but
the piece as a whole adapts itself in a remarkable degree to its
new environment. This is shown at the anterior wound surface
in several embryos. Here we note that the canalis centralis of
the reversed piece of the spinal cord (originally situated a milli-
meter or more away from its present position) has become
enlarged to form a rather typical funnel shaped outlet for the
medulla, or the medullary ventricle has become contracted for
the same purpose. In the first case, this has been accomplished
by the thinning out of the dorsal portion of the spinal cord to
such an extent that, in one or more cases, the point of union
between the cord and the inferior medullary velum is indistin-
guishable. In the latter case, the more usual one, the walls of
the medullary ventricle have become thickened.

446 DAVENPORT HOOKER

A more extreme example of the capability for adaptation on
the part of the spinal cord is demonstrated in another embryo
(fig. 9) in which a portion of the notochord became inserted be-
tween the posterior wound edges at operation. In this case,
we have a resulting fistula into the canalis centralis occupied
by the end of the piece of notochord, but the embryo appar-
ently suffered no ill effects from the consequent diversion of the
nerve tracts from their normal course. In fact, this embryo
was one of the best in point of executing the various movements
and was only distinguishable from normals by the spur of the
dorsal fin. In spite of the large cavity in the ventral surface of
the spinal cord, the connections between the original caudal end
of the reversed piece and the medulla were apparently completely
re-established. This necessitates, of course, an increase in the
number of fibers which pass laterally along the side of the cord
and which must have bridged an open gap to re-establish the
continuity.

The results of the observations on the nature of the primary
responses in these embryos with a reversed middle piece demon-
strates certain very important facts in regard to their origin. It
is to be noted that each portion of the embryo goes through a
graded series of responses to tactile stimulation which is as
follows: (1), contraction of the myotomes on the same side as
that stimulated, giving a bending of the body toward the side
stimulated; (2), a contraction of the myotomes on the opposite
side from that which receives the stimulation, giving an avoiding
reaction; (3), an alternate contraction of the myotomes on oppo-
site sides of the body, giving a double C reaction; (4), a primi-
tive swimming movement. Furthermore it is to be noted that
the middle segment of the body, which has been reversed in
these experiments is the region which actually determines the
locomotion of the animal as a whole and it is in this region that
this succession of responses to tactile stimulation is best to be
observed. In the head region we note that the reaction toward
the side stimulated and the avoiding reaction are of the same
nature as in the other parts of the body. The double C reac-
tion consists of a side-to-side swaying of the head, for owing to

SPINAL CORD REGENERATION. II 447

its shortness there is not sufficient length to give a marked arc.
For the same reason there is no true § reaction in the head re-
gion. The tail region exhibits all the above phases except that
the S reaction is very much later in making its appearance than
is the case with the middle section of the body.

The reactions in the middle piece of the body can not be lo-
calized in any portion of it, either as regards the sensitivity of
the receptors or the functioning powers of the effectors. We
must therefore consider that this region of the frog embryo,
about one mm. in length and extending from the caudal end of the
medulla backward, is a unit not only in its reaction to stimuli,
but also as regards the development of the primary nervous
connections within the cord. It would seem then that in the
frog embryo there is a larger area in which the primary nervous
connections are developed simultaneously than is found to be
the case in Amblystoma (Coghill).

SUMMARY

1. Reversed portions of the spinal cord heal per primum when
the edges of the cut have been carefully apposed. When they
have not been apposed, the wounds may heal according to the
same principles and by passing through the same stages as do
wounds caused by simple severing of the cord.

2. No healing has been obtained in embryos of a later stage
than that having a fairly well developed tail bud.

3. The reversed portion of the spinal cord retains its original
polarity.

4. The neurones begin their development in normal orienta-
tion to the reversed piece, but the direction of the transmission
of stimuli is apparently reversed at a later time. Their mor-
phological polarity is therefore unaffected by their reversal in
position but adaptation causes a subsequent reversal of the
functional polarity.

5. Embryos in which a portion of the spinal cord has been
reversed are not as a rule as viable as those in which the cord
was simply severed.

448 DAVENPORT HOOKER

6. A marked tendency on the part of the nerve fibers to avoid
entering the opposite wound surface of the spinal cord is notice-
able in these embryos.

7. These embryos render an analysis of the primary responses
to tactile stimulation possible. The middle piece is the first
portion of the body to respond to tactile stimulation and it
reacts as a unit.

8. The first type of response to tactile stimulation in these
embryos is a single contraction of the myotomes on the same
side as that which receives the stimulation.

9. In spite of the reversal of a portion of the cord the responses
to tactile stimulation of these embryos are normal in charac-
ter, but are somewhat tardy in their appearance. This is par-
ticularly true of those movements which are coordinated.

10. Embryos with a nervously isolated reversed middle
piece are capable of developing a fairly coordinated swimming
movement by means of the tension of the skin over the body.

SPINAL CORD REGENERATION. II 449

LITERATURE CITED

Coeuitt, G. E. 1900 The reaction to tactile stimuli and the development of
the swimming movement in embryos of Diemyctylus torosus, Esch-
sholtz. Jour. Comp. Neur., vol. 19, pp. 83-195.
1913 The primary ventral roots and somatic motor column of Ambly-
stoma. Jour. Comp. Neur., vol. 23, pp. 121-143.
1914 Correlated anatomical and physiological studies of the growth
of the nervous system of Amphibia. I: The afferent system of the
trunk of Amblystoma. Jour. Comp. Neur., vol. 24, pp. 161-233.
1916 II. The afferent system of the head of Amblystoma. Jour.
Comp. Neur., vol. 26, pp. 247-340.

Harrison, Ross G. 1893 The growth and regeneration of the tail of the frog
larva. Arch. f. Entw.-mech., Bd. 7, 8. 430-485.
1903 Experimentelle Untersuchungen iiber die Entwicklung der
Sinnesorgane der Seitenlinie bei den Amphibien. Arch. f. mikr. Anat.,
Bd. 68, 8. 35-149.

Herrick, C. J. anp Cocuttit, G. E. 1915 Development of reflex mechanisms
in Amblystoma. Jour. Comp. Neur., vol. 25, pp. 65-85.

Hooxer, Davenport 1911 The development and function of voluntary and
cardiac muscle in embryos without nerves. Jour. Exp. Zodl., vol. 11,
pp. 159-186.
1915 Studies on regeneration in the spinal cord. I. An analysis of
the processes leading to its reunion after it has been completely sev-
ered in frog embryos at the stage of closed neural folds. Jour. Comp.
Neur., vol. 25, pp. 469-495.
1916 Some results from reversing a portion of the spinal cord end-
for-end in frog embryos. Anat. Rec., vol. 10, pp. 198-199.

Spemann, H. 1912 Uber die Entwicklung umgedrehter Hirnteile bei Am-
phibienembryonen. Zool. Jahrb., Suppl. Bd. 15%, 8. 1-48.

WINTREBERT, P. 1904 Sur |’éxistence d’une irritabilité excitomotrice primi-
tive, indépendante des voies nerveuses chez les embryons ciliés de
Batraciens. Comp. rend. Soe. Biol., t. 57, pp. 645-647.

‘

. Fs s hy Abit a, \
= ie a f , j TAR AP a , ;
® = ; a naan Mevivae gig! ave _ on

7 ‘ ibd art a ] ‘9

GLYCOGEN IN THE NERVOUS SYSTEM OF
VERTEBRATES

SIMON H. GAGE
Cornell University, Ithaca, N. Y.

TEN FIGURES (ONE PLATE)

HISTORICAL SUMMARY

The brilliant series of investigations by Claude Bernard on
sugar in the blood which culminated in his discovery and iso-
lation of glycogen in 1857, may justly be characterized as epoch-
making for the understanding of animal physiology, and a
proper correlation of the physiology of animals and _ plants,
and their fundamental similarity.

In this series of investigations, Bernard was disturbed and
puzzled by not finding at any period of the life of animals gly-
cogen in the nervous system. He missed it also in other organs
and tissues, but all the gaps were filled up by one investigator
or another until in 1904 the glycogenic function had been dem-
onstrated in some stage of development for all organs and
tissues except the nervous system.

This is what Bernard himself says (’59) and _ practically
repeats in all of his published papers and books upon Glycogen:

A aucune époque de l’évolution organique, je n’al pu constanter
la matiére glycogéne dans les tissue nerveux. J’ai traité, soit par la
coction, soit par divers autres moyens précedemment indiqués, le
cerveau, la moelle épiniére . . . . chez des foetus d’homme,
de veau, de mouton, de lapin, et 4 aucune age je n’ai pu y constater la
moindre trace de matiére glycogéne.

Barfurth (’85, p. 299) referring to what Bernard says concern-
ing glycogen in the nervous system of vertebrate embryos, says:
“Tech kan diese Angeben lediglich bestitigen.”’

451

452 SIMON H. GAGE

In Pfliger’s book on glycogen (2nd edition, ’05, pp. 159-160)
is the statement with reference to the nervous system of the
adult; that Pavy (1881 had reported the presence of glycogen
in a normal adult brain analyzed by him, and that Cramer (’80)
had found traces in the brain of a person dead of diabetes. On
the other hand Barfurth (’85, p. 297) and others, reporting the
analyses for the normal adult brains of rabbits and dogs, asserted
that no glycogen was found by them.

Pfliiger says further on with reference to glycogen in the
nervous system in embryos:

Auch im Embryonalzustand ist bisher kein Glykogen in der sich
bildenden Nervensubstanz aufgefunden worden, was bereits Claude
Bernard untersucht. In neuester Zeit haben G. Fichera (’04) sowohl

als E. Gierke (’05) das Nervensystem auf Glycogen untersucht und
nur negative Ergebnisse gemeldet.

In 1904 I was fortunate enough to discover the presence of
glycogen in the nerve cells of the central nervous system of
Amphioxus, and began then a systematic investigation of the
nervous system of vertebrates, believing that in some period of
development glycogen would be found in the nervous system of
each form in the ascending series up to and including man. The
following paper is a summarized statement of the results of the
work up to the present. For the more extended study, forms were
selected in which abundant material, in all stages of embryonic
as well as in adult life, could be easily obtained. These forms
are: Petromyzon to represent the available form nearest to
Amphioxus; Amblystoma punctatum, among the amphibians;
the chick (Gallus domesticus) among the birds; and the pig
(Sus scrofa) among the mammals. Other forms, including human
material, were studied whenever opportunity offered.

In a word, it may be stated that the hopes held out by the
discovery of glycogen in Amphioxus were abundantly fulfilled,
for glycogen was found in large amounts in some stage of
development in the nervous system of every form studied.

In carrying on the investigation microchemical methods were
used, and not the usual analyses of entire animals or organs.
On showing microscopical specimens containing glycogen to

GLYCOGEN IN THE NERVOUS SYSTEM 453

chemists it was pointed out by them that where large amounts
of glycogen might be present in some elements of the organ or
animal, the amount of glycogen relative to the entire bulk might
be so small that it would be wholly missed by the usual chemical
analyses. It is also now recognized that chemical analyses by
the aid of the microscope have as great validity as those made in
the usual way, where relatively large amounts of substance and
reagents must be used (Chamot, 715). Furthermore, the micro-
scopical method is the only one by which the exact anatomical
location of the glycogen can be determined. For the precise
steps employed in fixing, imbedding, sectioning, and staining
and mounting tissues for glycogen, see the note at the end of this
paper. It may be stated here that to make sure that the ma-
hogany red substance shown in the nerve cells is glycogen, the
test was made in every case with saliva, which transforms gly-
cogen to sugar and therefore renders it no longer stainable with
iodin; it is believed therefore that the results here given, and
which were obtained over and over on many different specimens,
can be relied upon.

GLYCOGEN IN THE NERVOUS SYSTEM OF AMPHIOXUS AND ASYM-
METRON FROM BERMUDA AND AMPHIOXUS FROM NAPLES

In 1904 while a member of the group of workers at the Ber-
muda Biological Station, under the direction of Dr. E. L. Mark,
advantage was taken of the abundant Amphioxus material there
available to investigate the tissues for glycogen. It was found
rather generally distributed but not in striking amounts except
in the most unexpected situation, viz., in the large nerve cells of
the central nervous system. This was so opposed to the findings
of all previous investigators of glycogen in the nervous system
that it was only after repeated verifications on specimens of
various sizes that it was accepted. While any nerve cell appar-
ently might contain glycogen, this substance was most strikingly
shown in the large nerve cells associated with pigment. Whether
or not there is any connection, it is a rather striking fact that
glycogen in large amount is found in the retinal nerve cells of

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, No. 4

454 SIMON H. GAGE

adult Petromyzon, in some teleosts Ameiurus, and may be found
in the retinae of higher forms when sufficiently -investigated.
Fortunately Bermuda has also in its waters the little Amphi-
oxus discovered by Andrews in the Bahamas (Asymmetron
lucayanum). Asymmetron showed also glycogen in the nerve
cells, agreeing in every particular with Amphioxus. Through
the courtesy of The Wistar Institute, I was enabled to examine
Amphioxus from Naples also, and found glycogen in even greater
abundance if possible in its nerve cells. Probably glycogen is
present in the nerve cells of Amphioxus wherever found.

GLYCOGEN IN THE CENTRAL NERVOUS SYSTEM AND RETINA OF
THE LAKE LAMPHREY (PETROMYZON MARINUS UNICOLOR),
AND THE BROOK LAMPREY (LAMPETRA WILDERI) OF
THE CAYUGA LAKE BASIN

After finding glycogen in the nerve cells of Amphioxus it
seemed probable—at least not improbable—that it might be
found in the nervous system of Ammocoetes (larval Petromyzon
and Lampetra), whose life habits are so similar to Amphioxus,
although living only in fresh water. On returning to Ithaca
from Bermuda, larval lampreys of all sizes were obtained in
nature and put directly into 95 per cent alcohol and some also
in absolute alcohol, exactly as had been done for Amphioxus.
On sectioning and staining this material, glycogen was found as
hoped, and it was in brain cells as well as in those of the myel
(medulla spinalis). Not all nerve cells contain glycogen, but
many of them. In the larval Petromyzon (Ammocoetes) there
was another striking fact brought out in the sections: The
tissue surrounding the central nervous system has the general
appearance of fat; with the glycogen stain almost every cell
showed abundant glycogen in a part of the cell. On using Sudan
III, and osmic acid, as well as the glycogen stain, it appeared
that the cells in the loose tissue enclosing the central nervous
system were most of them filled in part with fat and in part with
glycogen. The cells in the dorsal region of the abdomen around
the mesonephros and gonads were also in many cases partly
surrounded by similar cells filled with fat and with glycogen.

GLYCOGEN IN THE NERVOUS SYSTEM 455

In sections through the entire head of ammocoets the unde-
veloped eyes were sectioned and much glycogen found in the
cones, no rods being present in the petromyzon eye at any stage
of development.

The presence of glycogen in the retinal cones of the frog was
called attention to long ago by Ehrlich (’83), and recently there
has appeared a paper by Brammertz (15) in which glycogen is
asserted to be present in the retinal rods and cones of the frog,
the pigeon, and the rabbit.

In the adult Petromyzon and Lampetra, the eye always con-
tains glycogen, but not in the cones. The glycogen in the func-
tioning eye is in the retinal nerve cells (fig. 4); and very import-
antly as it appears to me, even in the stages of advanced starva-
tion after the spawning. Vision seems to be of the highest im-
portance for the lamprey in the shallow streams during its
spawning period; and that the vision is good every one will be
willing to concede who attempts to catch them. In addition
to the glycogen in the retina proper, the arachnoid layer of the
eyeball near the optic nerve is filled with cells containing a
large amount of glycogen. That is much more marked in the
lampreys during the vegetative, or growing and maturing period
than late in the spawning season.

In passing, attention might be called to a very striking pecu-
harity of the petromyzon retina. The optic nerve, instead of
passing through all the layers of the retina and finally spreading
out on the inside next the vitreous, only extends about half way
through the thickness and then spreads out. As the optic nerve
leaves the retina on its way to the brain, the fibers decussate.
The lamprey eye certainly deserves more attention than has
been accorded to it.

In the course of development of Petromyzon the ova show no
glycogen until the eggs are ripened and ready to be shed and, of
course, immediately afterward; then the glycogen is abundant
and scattered between the yolk granules (fig. 5). It is in very
fine granules and especially abundant near the periphery of the
egg. As the ovum segments, the glycogen is most marked in
the mitotic areas of the cells, and as segmentation proceeds it

456 SIMON H. GAGE

becomes most condensed at the animal pole and finally in
the medullary region which gives rise to the central nervous sys-
tem. In embryos 5 to 10 mm. long it is marked in the central
nervous system. It is at this time present between the gran-
ules of food yolk, also in the myotomes, notochord, connective
tissue, nephric system, cardiac muscle, liver diverticulum, and
epidermis.

In embryos of 10 to 17 mm., in which the food yolk has
mostly disappeared, glycogen is present as in the younger em-
bryos, and has appeared in the brain plexus, retina, and auditory
epithelium. It is also present in the enteric epithelium.

With larger, well fed specimens, besides the nervous system,
the tissues containing glycogen are those of the heart, both
auricle and ventricle, branchial epithelium, thyroid duct, bran-
chial cartilages, and their striated muscles, and the muscle of
the velum, which has only a striated periphery. The glycogen
is in the granular non-striated central part of the velar muscle.
In the digestive tract it is found in the gall duct and in the
liver; also in the intestinal epithelium, especially the terminal
third. In the urinary system it is found in the nephrostomes,
and in both pro- and mesonephros, also in the Wolffian duct.
It is present in skeletal muscles, the notochord, the primitive
skull cartilages, the ear- capsule, the fat cells, around the central
nervous system and that on the ventral side of the notochord,
some of the epidermis, expecially that of the branchial region
and the oral hood, the epithelium of the nose and the ear.

AMBLYSTOMA PUNCTATUM

*

In this salamander, as with most of the Amphibia, the inde-
pendent life of the young commences very early, and on the alert-
ness in escaping enemies and in obtaining food depends its exist-
ence. Going with this early activity is the presence of glycogen
in large amount in the unsegmented egg, and in all stages of
segmentation. During segmentation glycogen is more abun-
dant in the animal than in the vegetative pole of the ovum.
While glycogen is more abundant in the animal pole, as segmen-

GLYCOGEN IN THE NERVOUS SYSTEM 457

tation proceeds and the germ layers are formed it is present in
all germ layers but is especially marked in the neural plate.

Glycogen appears in the first proton or anlage of the eye, ear,
and nose; in the brain and the myel (medulla spinalis) and in all
the organs and tissues of the embryo; that is, in Amblystoma
glycogen is universal in its distribution throughout the body in
the early embryonic condition, but the liver early takes on the
most prominent glycogenic function. It persists for a long time,
perhaps throughout life in the cardiac muscle and in the retina
(rods and cones).

GALLUS DOMESTICUS

In the chick glycogen appears first in the cardiac muscle,
thirty-sixth to the forty-eighth hour of incubation. In strong
contrast with Petromyzon and Amblystoma, the appearance of
the glycogen in the nervous system is late. In the sixth to the
tenth day, it is very abundant in the medulla oblongata and in
the sacral and lumbar myel (medulla spinalis).

In the tenth day it appears in cartilage and in the muscles of
the trunk and limbs, but it is not so abundant in the somatic
muscles of the chick as.in those of Petromyzon and mammalian
embryos., It is also present, to a limited extent, in the epidermis
and the enteric epithelium.

It has already been pointed out by many previous workers
that glycogen is not so abundant in the organs and tissues of the
chick as in the embryos of many other forms, including mammals.
It seems to me that this is true if one deals with the glycogenesis
in all of the organs at any one period. With the large amount of
stored food in the hen’s egg, the chick has the advantage of devel-
oping at leisure, so to speak, and whenever the time arrives to
bring to definitive form or activity any tissue, the glycogenic
builder and energy producer is on hand, but as these perfecting
processes do not occur in all the organs and tissues of the chick
practically at the same time as with Amblystoma, one finds abun-
dant glycogen at any one period only in a limited region of the
body.

458 SIMON H. GAGE

GLYCOGEN IN THE NERVOUS SYSTEM OF MAMMALS

Sus scrofa. The pig was selected for determining the pres-
ence of glycogen in the nervous system of mammals because of
the abundance of material obtainable at all periods of develop-
ment. Other mammals were examined as opportunity offered,
and it was found that whatever occurred in the pig appeared
also in other mammals if taken at the favorable developmental
state.

Up to the present, glycogen has been found by me in the cells
of the dorsal root ganglia of pigs up to a length 15 mm., those of
10 to 12 mm. in length had perhaps the greatest number of nerve
cells with the glycogen. At this time the outgrowing nerves
seem to be wholly free from glycogen, but commencing with
embryos of 30 mm. and as large as 70 mm. and perhaps older
ones, the nerves within and beyond the ganglion are so filled
with glycogen that they appear a deep brown.

In addition to the nervous element proper, the endymal cells
of the relatively free choroid plexus are filled with it in the
older embryos, 1.e., those of 40 to 70 mm. and perhaps older ones.
In addition to the cells on the free plexus, those extending for a
considerable distance upon the ventricular wall are well supplied
with glycogen.

In the fourth ventricle the endymal cells contain glycogen
at a somewhat later stage, viz., in embryos of 50 to 75 mm. in
length.

Abundant glycogen was also found in the olfactory as well
as the respiratory epithelium of the nose; and its presence is
very marked in the epithelium of the cochlear canal opposite the
organ of Corti. The eye has not yet been sufficiently studied,
but the appearance of glycogen at some period is predicted.
Glycogen was found in every organ and tissue in the body of pig
embryos at some period of development. Naturally the heart
contained much of it. For example, in the smaller pigs studied,
i.e., those of 8 to 16 mm., the cardiac glycogen was so abundant
that it made the heart sections almost opaque. In embryos of
70 mm. the amount was relatively less. The liver contained no

GLYCOGEN IN THE NERVOUS SYSTEM 459

glycogen in any of the embryo pigs studied, thus agreeing with
the statements of Bernard that glycogen is relatively late in
appearing in the liver.

In the alimentary canal the glycogen passes as a kind of wave
along the tube, commencing at the mouth and passing in order
to the esophagus, the stomach, and, as the villi commence to
appear, extending along down the small to the large intestine.

The investigation of human embryos for glycogen is carried
on with more uncertainty than is that for other forms owing to
the difficulty of obtaining material in the different stages which
can be fixed in the alcohol before the glycogen becomes dissolved.
However, owing to the courtesy of Dr. Mall, and several of my
old students, some fairly normal human embryos fixed in alco-
hol before all of the glycogen was dissolved, have been studied,
and I have found the glycogen distributed among the organs and
tissues as described for mammals generally.

In the nervous system, the only unmistakable situation in
which it has been found up to the present is in the choroid plexus
of a 19 cm. human fetus and in the choroid plexus and the cells
of the raphé of the medulla oblongata of a 35 mm. human em-
bryo preserved in strong alcohol. The endymal cells showed the
same abundance of glycogen that has been found in the embryo
of the cat and pig. A figure of this human plexus with the
glycogenated endymal cells is given in the accompanying plate
(fig. 10). It is confidently expected that when the proper ma-
terial can be obtained glycogen will be found in the human
nervous system and organs of sense, as with other mammals.

CONCLUSIONS

From the data given above it is believed that the following
conclusions may be fairly drawn:

1. The production and use of glycogen is one of the properties
of nervous as of all other forms of protoplasm.

2. Glycogen is an essential accompanier of nervous as of all
other tissues in their histogenesis, especially in the transition to
their definitive and functional stage.

460 SIMON H. GAGE

3. Glycogen is, then, a builder as well as an energy producer
for nervous as for all other forms of tissue.

4. Its appearance in developing tissue in all forms of verte-
brates depends in part, at least, upon the relative time in which
the tissues must function. For example in Amblystoma that
must have full functional activity very early in its life, the
perfecting glycogen appears correspondingly early, while with
the chick it is late in appearing.

5. After reaching their definitive form the elements of the
nervous system in the lowest vertebrates, Amphioxus and
larval lampreys (Ammocoetes), continue their glycogenic func-
tion in the central nervous system. In the adult lampreys (Pet-
romyzon and Lampetra), this function persists throughout life
in the nerve cells of the retina. With the higher vertebrates,
glycogen in demonstrable amount is not found in the nervous
system after the embryonic period, the liver and muscles then
assuming the main glycogenic function.

That is, specialization of this function keeps pace with dif-
ferentiation of structure consequent upon advance in ‘the
zoological scale.

METHOD OF DEMONSTRATING GLYCOGEN

The fundamental thing is that no liquid is to be used in any of the
steps that will dissolve the glycogen. The most certain medium for
fixing is alcohol. Absolute alcohol is mostly recommended; but, as
glycogen is wholly insoluble in alcohol of 67 per cent and, of course,
in all stronger grades, one has considerable range.

As alcohol diffuses through the tissues slowly, only small animals
and small embryos should be fixed entire. For the organs and tissues
of larger forms small pieces or widely opened and dissected organs in
which the aleohol comes quickly in contact with all the parts containing
glycogen should be used. Plenty of aleohol should be used—fifty
times the bulk of the tissue—and it is well to change it two or three
times. In general it is safer to use alcohol of 95 per cent, then it is
not liable to be diluted by the lymph sufficiently to make the glycogen
soluble.

As alcohol distorts the tissues, it is well to carry along parallel
specimens prepared by the usual fixers. Mercuric chlorid, or mercuric
chlorid and dichromate mixtures are good. Picric alcohol is also good
and it has the advantage of fixing the glycogen as well as the other
tissue elements (it is compbdsed of 67 per cent alcohol, 500 ec.; picric acid, 1

GLYCOGEN IN THE NERVOUS SYSTEM 461

gram). The tissue is fixed in this twelve to twenty-four hours and then
transferred to 67 per cent alcohol the same time; and then for a day or
more in 82 per cent alcohol before the final imbedding. Plenty of
fixer and alcohol should be used.

Other fixers have been recommended for glycogen, and many dif-
ferent ones preserve a part of the glycogen, but as the purpose of any
investigation on glycogen is to find all the elements in which it is
present either in large or in small amounts, a fixer should be used
which experience has shown to be the most precise and certain, and that
fixer is alcohol.

For large embryos, small animals, limbs, etc. containing bone it
was found entirely practicable to decalcify the bone without in any
way disturbing the glycogen. The embryo, animal or part is fixed
with alcohol as usual for glycogen, then it is placed in the nitric acid
decalcifier composed of 67 per cent alcohol to which has been added 3
per cent nitric acid. After the decalcification is complete, the embryo
remains a day cr twe in 67 per cent alcohol, changed two or three times.
It is then transferred to 82 per cent alcohol for a day or more before
dehydrating and imbedding.

' Imbedding and sectioning. Either the collodion or the paraffin
method can be used. The paraffin method or the combined collodion
and paraffin method’has proved most satisfactory in my work. Scme
of the sections should be moderately thick, 10 to 15 u. Sections less
than 5 uv are not serviceable for glycogen investigations.

Staining glycogen. The only fully reliable and satisfactory stain
for glycogen is iodin. As some glycogen is very soluble, a glycogen
stain containing alcohol was found most generally useful (95 per cent
alcohol, 150 ece.; water, 150 cc.; iedin crystals, 1.5 grams or 15 ce. of a
10 per cent alcoholic solution of iodin; iodid of potassium, 3 grams;
sodium chlorid, 1.5 grams). For the aqueous stain, water is used
instead of the alcohol mixture.

For staining, spread the paraffin sections with the iodin stain in-
stead of water. The glycogen in the sections will stain a mahogany
red and the stain will remain in the spread sections for years (10 to
15). If care is taken not to melt the paraffin when spreading the sec-
tions, they can be restained at any time by immersing the slide in the
stain or placing some of the stain on the sections.

Permanent preparations. The permanence of the iodin stain in the
spread paraffin sections gave the clue. For low powers the sections in
paraffin show very well without further preparation, but for high
powers the crystals of paraffin interfere. Various paraffin media
were tried, and finally ordinary yellow vaseline was settled upon
as best. For mounting, the sections are restained by immersing the
slide in the iodin stain for two to three minutes or longer; they are
then dried half an hour or more in the air or in a drying oven, then
immersed in xylene to dissolve off the paraffin. Some melted yellow
vaseline is then put on the sections and a cover-glass added exactly

462 SIMON H. GAGE

as in balsam mounting. As the vaseline does not hold the cover very
firmly, it is best to seal the cover with shellac. .

Specimens so stained may be restained at any time by reversing the
process and then remounting. The stain remains for two to ten years.

A second method was to mount in Canada balsam without a cover-
glass, as with the Golgi preparations. For this dried balsam is pow-
dered and to 25 grams of dry balsam, 50 ec. of xylene is added. The
sections are deparaffined, and the balsam put over them and allowed
to dry in the air. More than one coat of balsam may be needed.

The glycogen stain is not so persistent in the balsam, but for high
power work the finest details are more satisfactory. If a homogeneous
immersion objective is to be used the original immersion liquid, viz.,
Canada balsam of moderate thickness is better than cedar oil. It
need not be removed. The other stains recommended for glycogen
are not so precise as iodin, and are lable, if not checked by iodin, to
lead the investigator astray.

The most exact test for glycogen is saliva. If a section is deparaf-
fined, washed off with alcohol and water, and then saliva put upon
it for half an hour, if the substance is glycogen it will be changed to
sugar by the enzyme of the saliva. If now the section is restained
with iodin no mahogany red glycogen will appear. This test was |
applied to all the work given in the accompanying paper to make
sure that the reddish brown substance in the cells was glycogen and
not something else.

BIBLIOGRAPHY

The literature of glycogen is very extensive, and may be found in the Index
Catalogue of the Library of the Surgeon General’s Office, Ser. I, II, and in
special papers such as Fichera’s with its 311 references and in Pfliiger’s book on
Glycogen. Only the works bearing directly upon the subject of this discussion
are here given.

BarrurtH, D. 1885 Vergleichend-histochemische Untersuchungen iiber das
Glycogen. Arch. f. mikr. Anat., Bd. 25, pp. 261-404. Nervous sys-
tem of vertebrates, pp. 297, 299. Invertebrates, p. 298. While deny-
ing glycogen to vertebrate nervous tissue at any period, p. 299, he
reported its presence in small amounts in the nervous system of
snails.

BerNarp, CLaupr 1859 De la matiére glycogéne considerée comme condition
de développement de certain tissus chez le foetus avant l’apparition
de la fonetion glycogénique du foie. Jour. de la Physiol. t. 2, Pp.
326-337.

1878-1879 Legons sur les phénoménes de la vie communs aux animaux
et aux végétaux. Two volumes.

3ernard wrote many papers upon sugar in the blood and upon gly-
cogen, which he discovered and isolated in 1857. The paper cited
above and the volumes on the phenomena of life give his views very

GLYCOGEN IN THE NERVOUS SYSTEM 463

completely. It may be said in passing that physiologists have not
gone far beyond Bernard. It is however, more widely distributed than
he thought, and its locations have been more completely mapped out
since his day.

BrRAMMERTZ, Wm. 1915 Ueber das normale Vorkommen von Glykogen in der
Retina. Arch. f. mikr. Anat., Bd. 86, pp. 1-7. He reports the pres-
ence of glycogen in the rods and cones of frog, pigeon, rabbit and
between them in the pike, and in the eye of the house fly.

CREIGHTON, CHARLES 1896-1899 Microscopie researches on the formative
property of glycogen. Part I with five colored plates, 152 pages.
Part II (99), Glycogen of snails and slugs. Nine plates, 127 pages.
In Part I he figures and discusses glycogen in the choroid plexus of
the cat embryo, pl. i, figure 3; and a combination of fat and glycogen
in the developing mammary gland, pl. iv, figure 20. All students of
glycogen would do well to consult this work.

Cuamot, E. M. 1915 Elementary chemical microscopy.

Duaaar, B. M. 1911 Plant physiology. This work gives a good account of
starch and its transformations in plants.

Ficnpra, G. 1904 Ueber die Verbreitung des Glycogens in Verschiedenen
Arten. Ziegler’s Beitrige zur path. Anat., Bd. 36, pp. 273-339.
Fichera gives references to 311 papers.

Gags, 8. H. 1905-1906 Glycogen in the nervous system. Reports and dem-
onstrations at the Association of American Anatomists. Am. Jour.
Anat., 1905, pp. xii—xiii; 1906, pp. xiii—xv.

Lusk, GRAHAM 1906 The elements of the science of nutrition. Discusses
carbohydrates, including glycogen, in nutrition.

MENDEL, L. B. anp LEAVENWoRTH, C. S. 1907, The American Journal of
Physiology, vol. 20, pp. 117-126. Chemical studies on growth; III,
the occurrence of glycogen in the pig. They used the brain substance
in rather large amounts and got only negative results. Their final
conclusion is that embryos do not have a large amount of glycogen
in their tissues. The author showed Dr. Mendel the microscopic sec-
tions of embryo pigs and he could not help thinking that a great deal
of glycogen was present. In case of the brain it is entirely possible
that the amount of glycogen in the choroid plexus might not be
sufficient to give results in ordinary analyses, although they are most
striking and convincing in microscopic preparations.

Pritcer, E. F. W. 1905 Das Glykogen und seine Beziehungen zur Zucker-
krankheit. Zweite Auflage. Very many references.

SmirH, Lucy Wricur 1912 Glycogen in Insects, especially in the nervous
system and eyes. Science, vol. 35, March i. Much glycogen is
found in both compound and simple eyes and in the nerve cells of
the ganglia from all parts of the body, but not in the nerve fibers.

Wwe

“I

PLATE 1

EXPLANATION OF FIGURES

Glycogenated nerve cells of Amphioxus.

Nerve cells of Ammocoetes from the brain opposite the eye and ear.
Nerve cells in the myel (medulla spinalis) of Ammocoetes with glycogen;
also glycogen and fat in the same cells.

Retina of an adult Petromyzon showing glycogen in the retinal nerve
cells, and the decussation of the optic fibers.

(a) Edge view of glycogenated retinal nerve cells.

(b) Face view of retinal nerve cells.

Ova of Petromyzon and Amblystoma (be) showing the great amount
of glycogen in the cells of the animal pole. In the developing nervous
system there is much glycogen.

Lumbar enlargement of a ten day chick’s medulla spinalis showing a
prismatic mass of glycogenated cells in the raphé. A similar glycogen-
ated area is present in the medulla oblongata.

Spinal ganglion of a 12 mm. pig embryo with glycogenated cells. (a)
Some of the cells greatly enlarged.

Glycogenated nerve trunks of the brachial plexus of a 30 mm. pig
embryo.

Glycogenated endymal cells of the brain plexuses in a cat embryo. (a)
Endymal cells greatly enlarged.

Choroid plexus of a 19 em. human embryo. (ab) Section through
the cerebrum and medulla of a 35 mm. human embryo to show the
glycogenated choroid plexus and the cells of the raphé in the medulla.

464

GLYCOGEN IN THE NERVOUS SYSTEM PLATE 1
SIMON H. GAGE

465

THE MOTOR NUCLEI OF THE CEREBRAL NERVES IN
PHYLOGENY: A STUDY OF THE PHENOMENA
OF NEUROBIOTAXIS

PART I. CYCLOSTOMI AND PISCES

DAVIDSON BLACK

From the Central Institute for Brain Research, Amsterdam, Holland, and the
Anatomical Department, Medical School, Western Reserve University,
Cleveland, Ohio

FORTY-TWO FIGURES

CONTENTS
introductlonkassen ee nee BS lai: ee a oA eae aed 467
LOirae) keshrras cas 4 ak ao as Sciers he ah aR RES 2c ary DOU pe PL ee OE 470
Motonmuclerines dellostomaydombeyies- pe secrete cae eis ei ere 470
DISCUSS O Tear Rete ee a ets tees. iss, zt <M mse/cODRG sc a ae 476
Sel Sie Wee eee sexe Se ieee cin ne Re ees ce Ns 5 5 oN RE TYR o, con Re eS EE 485
Motorsnucleiminuselachenmaxaimancn. 22 eae sare Mee nnriat a caeriee er: 485
DIS CLISSTO Mtr 3. yoepcr a eae ies Sl «Sid dk eas cinco fot cee ee 494
CLTNOTE Me eee Se oS Soe tS coeiie) Sia ee RE RREORS See cokcc A oere nn e nOiae 502
Motonnuclemnerolyodonispathul aes. seeo seer ee eee eee areca 502
ID ESYOLOVSTSHIG) be Braet hes GRRE rato i, «i Renata yen ERR ORB 1! coh pr 2 SP EPR NEES cE alata 509
UNIX OPS) Weg bees ok eat ee a 8s Se eet Le Dil Rae eA 8 ho eer Ae 522
Motor nuclei in Ameiurus nebulosus and Solea vulgaris................. 522
DTS USSU O MD yee See ce IA rc ots OW et OAS che Ik ote ae aaeR SR Rees oan 535
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INTRODUCTION

In 1907 Kappers first directed attention to the significance
of the phylogenetic displacements of the motor nuclei of the
medulla. In 1908 (60, p. 521) he enunciated more definitely
his doctrine of Neurobiotaxis and set forth the following con-
clusions deduced from the study of nuclear migrations:

1. Wenn in dem Nervensysteme am verschiedenen Stellen Reiz-
ladungen auftreten, so erfolgt das Auswachsen der Hauptdendriten,

467

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL, 27, NO. 4

468 DAVIDSON BLACK

namentlich auch die Verlagerung des ganzen Leibes der Ganglienzellen,
in der Richtung der maximalen Reizladung.

2. Nur zwischen gleichzeitig oder direkt sukzessiv gereizten Stellen
findet diese Dendriten- oder Zellenannaherung statt.

3. Das Auswachsen der Achsenzylinder der sogenannten Zentral-
motorischen Systeme wird nicht primir bedingt durch die motile
Funktion gewisser Zellen, sondern ebenfalls durch synchron cder
sukzessiv gereizte Gebiete (Schaltzellen v. Monakows).

Since that time much work has been done by this author and
his associates in extending and elaborating the field of these
investigations, the importance of which, in view of their wide
application to the problems of morphology, ontology and
physiology of the nervous system, becomes increasingly evident.!

It was my privilege to begin the present work under the
direction of Dr. Kappers in the summer of 1914 with the object
of adding to the data bearing upon the phenomena of neuro-
biotaxis, by the study of new material recently acquired by the
Central Dutch Institute for Brain Research. In accordance
with this object the plan of treatment here adopted corresponds
somewhat to that obtaining in Kappers’ communication of 1912
(66) and the reconstruction charts of the motor nuclei in both
cases are of the same dimensions. With regard to plotting these
charts, it seems hardly necessary to point out that all charts
can be made to correspond approximately in size for the sake of
direct comparison because a difference in magnification does not
in any way alter relative proportion. A description of this
method of reconstruction and a discussion of its advantages and
limitations will be found in Kappers’ earlier papers. A brief
statement concerning the method is also set forth in the present
article on page 474.

Except in the case of Solea vulgaris, all the observations,
drawings and reconstructions were carried out in the laboratories
of the Central Dutch Institute in Amsterdam. My work
being unavoidably interrupted in August, 1914, Dr. Kappers
very kindly gave me the slides of the Solea series so that I might
subsequently complete my observations on this form.

‘ Reference may be had to the chief communications on the subject of Neuro-
biotaxis in the appended bibliography.

MOTOR NUCLEI IN PHYLOGENY 469

It is a pleasure for me to acknowledge my indebtedness to
Dr. Kappers, as well for his help and the stimulating interest
he took in my work as for the generous way in which he placed
the resources of his laboratory at my disposal.

With regard to my material, the present work is based upon
the special study of the motor nuclei in the following represen-
tative forms: Bdellostoma dombeyi, Selache maxima (Ceto-
rhinus maximus), Polyodon spathula, Ameliurus nebulosus,
Solea vulgaris, Rana catesbeana (mugiens), Damonia_ sub-
trijuga, Cacatua roseicapilla, Hypsiprimnus murinus and Pan
satyrus (Troglodytes niger). In addition, Hexanchus, Hep-
tanchus, Ciconia alba and Vesperugo noctula were re-studied,
though the motor nuclei in these forms had already been charted
and recorded elsewhere.’

The specimens of Polyodon spathula were obtained through
the courtesy of Prof. R. J. Terry of Washington University, St.
Louis, and I am also much indebted to Prof. Howard Ayers
who very generously furnished me with specimens of Bdellos-
toma dombeyi from his personal collection. Several of these
brains had been fixed by him by the Cajal method, others fixed
by different methods were subsequently stained in various ways.

The series for the most part were cut transversely at 25
microns. With the exception of Bdellostoma, alternate sec-
tions in each series were arranged on celloidin films and stained
by Pal-Weigert-carmine and van Giesson methods (Kappers,
64). ;

All the technical work in connection with cutting, staining
and mounting this material, was done in the laboratories of the
Central Dutch Institute for Brain Research and I wish to ex-

2 The name ‘Pan satyrus’ is used here for this species of chimpanzee in con-
formity with the recent work of the late D. G. Elliott (21). The claims of
priority also demand the change in nomenclature from ‘R. mugiens’ to ‘R.
catesbeana’ for the bull frog (vide Gadow, 26). In the case of the sharks Noti-
danus cinereus and N. griseus, the old terms Heptanchus and Hexanchus re-
spectively have been retained for convenience in description, while in the case
of the basking shark the name ‘Selache maxima’ rather than ‘Cetorhinus maxi-
mus’ has been used, though both seem equally in vogue (vide Jordan, 54, and
Bridge, 12).

470 DAVIDSON BLACK

press here my sincere appreciation and thanks to Miss de
Lange for the skilled and careful manner in which she carried out
this important part of the work.

CYCLOSTOMI
Motor nuclei in Bdellostoma dombeyt

This form is the common hagfish of the American Pacific
coast which, according to Worthington, is identical with the
Californian variety not infrequently described as B. stouti (100).
Several brains of this form prepared by different methods and
cut in both transverse and sagittal series were studied. The
reconstruction chart (fig. 7) was prepared from one series cut
transversely and stained by the method of Cajal.

Spino-occipital nuclei and roots (Nu. et rad. mot. Nn. spin. occ.)
At the junction of the cord and medulla in Bdellostoma certain
very definitely specialized nerves may be recognized, each
possessing one sensory and two motor roots. The essential
difference between these specialized nerves and the cervical
motor roots lies in the total absence in the former of peripheral
branches supplying dorsal trunk musculature (Worthington,
99). Furbringer’s term ‘spino-occipital’ (24) has been adopted
by Worthington to describe these nerves and this name has
been retained in the present description.’

Both the motor roots of the first spino-occipital nerve have
their superficial origin slightly caudad of the level of entrance
of the sensory root, while the reverse is the case in the second
spino-occipital nerve. In the latter nerve, four motor rootlets
uniting to form two main stems were described by Worthington
(99) but I was unable to confirm this observation.

’ However, it is evident that the nerves in question are derived from segments
more rostrally placed than those from which the first two spinooccipital roots
arise in selachians (Neal, 84, Furbringer, 25, et. al). Thus, both here and in the
subsequent description of this region in selachians and ganoids, the term ‘spino-
occipital’ is used in its broadest collective sense to designate certain precervical
motor elements whose number and segmental relationships are subject to con-
siderable variation in these different groups.

MOTOR NUCLEI IN. PHYLOGENY A471

The motor roots of the spino-ocecipital nerves course obliquely
cephalad and mesiad to reach their nuclei of origin in the ter-
minal rostral portion of anterior horn of the cord (fig. 1). There
is no indication whatever of any separation between these nuclei
and those of the succeeding ventral spinal nerves.

Nu. F.c.

R.sp.occ.s .,.

R sp.oce.m.(1)

Fib.arc.int.

Fib.arc.int.

Figs. 1 to3 Bdellostoma dombeyi. Outlines of transverse sections to illus-
trate the topography of the medulla. Sensory areas and fiber tracts indicated
diagrammatically. Figures 1 to 6 drawn to same scale. Abbreviations: Ar.g.c.,
general cutaneous area; F’.c., fasciculus communis; Fib.arc.int., fibrae arcuatae
internae; Laq., laqueus; s. tractus tecto-bulbaris et spinalis; Nuw.f.c., nucleus
and region of fasciculus communis; Nuw.sp.occ.m., rostral portion of anterior
horn (spino-occipital motor nucleus); Nw.VII.m., caudal end of nucleus motorius
N. facialis; Nu.X.m., nucleus motorius N. vagi; R.sp.occ.m.(1)., first motor
rootlet of first spino-occipital nerve; R.sp.occ.s., part of sensory root of first spino-
occipital nerve; R.X.m.(2)., second motor rootlet of vagus; R.X.m.(5)., fifth
motor root of vagus: 7'r.cb.sp., tractus cerebello-spinalis.

472 DAVIDSON BLACK

Worthington (I.c.) described a continuation upward of the
anterior horn nucleus almost to the rostral end of the medulla
and noted the presence of a number of large cells of ‘Mauthner’
in the upper part of the nucleus. This is probably homologous
with the nucleus that Holm observed in Myxine and to which
he gave the name ‘“‘ganglion centrale nucleus posterior’ (45).
Worthington states, moreover, that the nucleus ‘‘gives fibers to
the vagus,’’ while Holm makes it evident that he did not con-
sider this cell group to be a nucleus of origin for peripheral
motor fibers.

I have examined this cell column rostral to the origin of the
motor spino-occipital roots in Bdellostoma and have indicated
some of its largest Miller cells in figures 1 to 6. I could not
discover any evidence of the direct connection of these cells
with the roots of the vagus series. They do, however, send
processes into the neighborhood of the viscero-motor column
and ventro-lateral area of the bulb.

Thus, in the present connection I cannot consider this cell
group to be a part of the somatic motor column proper; it is,
rather, of the nature of a scattered reticular nucleus of special-
ized coordinative function,’ such as is commonly found near the
raphé in the caudal part of the medulla in all vertebrates (van
Hoevell, 44).

However, this reticular nucleus in Bdellostoma in all proba-
bility is the specialized representative of the somatic column
which has lost, or is in process of losing, its peripheral motor
components but has retained its primitive dorso-mesial position
and its intercalary coordination elements. This view would be
supported by the important observations of Bartelmez on the
nucleus motorius tegmenti in Ameiurus (9) which is apparently
represented in Bdellostoma by the reticular nucleus under dis-
cussion. According to this author the nucleus motorius teg-
menti, together with the nucleus abducentis, constitutes the
somatic motor column of the bulb.

4 The terms ‘coordinative’ and ‘correlative’ are used in the present paper in
the sense defined by Herrick (42, p. 35).

MOTOR NUCLEI IN PHYLOGENY 473

It is quite possible that further study of a large number of
specimens may confirm Worthington’s observations and show
that the spino-occipital nucleus overlaps the vagus area in some
specimens to a greater extent than in the one charted in figure 7.
Individual variation, especially in this part of the brain in
Bdellostoma, is rather to be expected, so that Johnson’s obser-
vation that “the segment of the glossopharyngeus also has a
somatic motor nerve in Bdellostoma”’ (52, p. 194) should be
modified to a certain extent.

Motor vagal nuclei and roots (Nu. et rad. mot. N. X). The re-
maining motor nuclei in this form are visceral in character and
may conveniently be described as forming two columns of cells,
one on each side of the bulb in the ventro-lateral area. Each
column is further definitely subdivided into a rostral and a caudal
portion (vide fig. 7).

The caudal viscero-motor column corresponds to the caudal
atero-ventral column described by Holm in Myxine (lLec.). It
is the motor nucleus of the vagus and consists of a continuous
column of medium sized cells from which five (?) vagus rootlets
arise. It is probable that the first two very small rootlets
should be considered together as the first vagus root, though the
first small rootlet of this series in all probability represents the
motor glossopharyngeus of other authors (q. v.). The pres-
ence of a true glossopharyngeal nerve in Bdellostoma, however,
is highly improbable (vide infra.).

Rostrally the vagus nucleus begins a few sections above the
level of its first motor rootlet, while caudally it extends for
some distance below the level of the upper prolongation of the
anterior horn.

Motor trigemino-facial nuclei and roots (Nu. et rad. mot. Nn.
V-VII). The rostral viscero-motor column (frontal latero-
ventral column of Holm) contains the motor nuclei of the
trigeminus and facialis (fig. 7 C). |

The motor root of the trigeminus consists of two parts, of
which the rostral is the larger. These two portions are only
evident on the periphery of the bulb and at once unite to form a
single trunk. The motor nucleus of this nerve is also divisible

A74 DAVIDSON BLACK

into two parts: a large celled nucleus (nucleus magnocellularis)
extending approximately over the length of the superficial origin
of the motor trigeminal roots, and a smaller celled nucleus
(nucleus parvicellularis) which is incompletely separated from
the former and becomes directly continuous caudally with the
motor facial nucleus (figs. 4, 5 and 6).

I have been unable to demonstrate in Bdellostoma such a
complete correspondence between the two motor roots of the
trigeminal nerve and the two cell groups of its-nucleus of ori-
gin, as was done in the case of Myxine by Rothig and Kappers
(88). It is presumable, however, that the first root takes its
chief origin from the nucleus magnocellularis, though some of
the fibers of the second root appear to originate there also.

The motor facial nucleus may be traced down to within a short
distance of the rostral end of the vagus column. It is difficult
to set a definite limit to this nucleus caudally, as the cells in
this part gradually become scattered. No root fibers from any
source, however, can be traced into this diffuse caudal portion,
which occupies a position somewhat more mediad than the
caudal visceral motor nucleus.

The abducens, trochlear and oculomotor nerves are absent in
Bdellostoma, in which there is no functional eye and conse-
quently no intrinsic nor extrinsic musculature to require
innervation.

Method of reconstruction. The relations described above are
graphically represented in figure 7C. The nuclei and motor
roots in question are plotted as if projected upon the mid-
sagittal plane. In view of earlier discussions of this method of
reconstruction, it is only necessary to mention here briefly the
following points:

1. Only the nuclei of one side of the brain stem (right or left)
are plotted in these charts.

2. The longitudinal extent and relations of the nuclei and
emergent roots are accurately plotted.

3. The dorso-ventral extent and relations of the nuclei are
indicated only approximately. For this reason the charts have
been supplemented by projectoscope drawings of transverse

MOTOR NUCLEI IN PHYLOGENY 475

sections of the brain stem taken at various representative levels
in the series, where these relations can be accurately recorded.

4. For the sake of convenience the general outlines and
shapes of the nuclei are depicted quite diagrammatically. In

Nu.V.mag.m.

Figs. 4 to 6 Bdellostoma dombeyi. Outlines of transverse sections to illus-
trate the topography of the medulla. Sensory areas and fiber tracts indicated
diagrammatically. Abbreviations: Nuw.ac., nucleus acusticus; Nu.V.mag.,
nucleus motorius N. trigemini (magnocellularis); Nu.V.paf., nucleus motorius
N. trigemini (parvicellularis); R.V.m., radix motorius N. trigemini; R.VII.m.,
radix motorious N. facialis; VJIJ., acoustic fibers. Other abbreviations as in
figures 1 to 3.

476 DAVIDSON BLACK

this connection reference should be made to the exact nuclear
reconstructions made by Berkelbach Van der Sprenkel and to
the discussion by this author of the method he employed (92).

In not a few instances, nuclear masses other than those of the
motor nerves have been plotted on the charts. This has been
done, for example, in the case of the superior olive and inferior
olive (nucleus paramedianus) in such forms as they occur in a
sufficiently well differentiated condition to allow a sharp de-
marcation. Similarly the site of the tip of the calamus scrip-
torius is indicated in all forms with the exception of myxinoids,
where a true calamus is absent. The addition of these land-
marks has been made to facilitate the comparison of different
forms one with another by increasing the number of points for
comparison.

Fig. 7 Reconstruction charts of motor roots and nuclei: (A), Petromyzon
fluviatilis (after Kappers, 66). (B), Myxine glutinosa (after! Rothig and Kap-
pets, 88). (C), Bdellostoma dombeyi. Explanation of signs and abbreviations
used in all charts as follows: V/., abducens root; /X., motor glossopharyngeal
root; X., motor vagus rootlets; Sp.occ., motor spino-occipital rootlets. The
arrow indicates the site of the calamus.

= Oculomotor root and nucleus
HUTT Trochlear root and nucleus

Trigeminus motor root and nucleus
SEO Abducens nucleus

Facial motor root and nucleus

Glossopharyngeus motor nucleus

YL Vagus motor nucleus
BEY Spino-occipital motor roots and nucleus

a Nu.olivaris superior
Nu.paramedianus

477

MOTOR NUCLEI IN PHYLOGENY

478 DAVIDSON BLACK

Discussion

On comparing the reconstruction of the motor nuclei in
Bdellostoma dombeyi with that of the nuclei in Petromyzon
fluviatilis (fig. 7), two striking differences are at once apparent:
firstly, the ventral position of all motor nuclei in Bdellostoma,
and secondly, the complete absence in the latter form of the
oculomotor, trochlear, and abducens nerves. In these two
respects Bdellostoma closely agrees with Myxine glutinosa (vide
Eds ES).

In all these forms there is a significant division of the visceral
motor nuclei into two parts: (a), a caudal portion, consisting of
the vagus and glossopharyngeus nuclei in Petromyzon, and of
the vagus nucleus alone in Bdellostoma and Myxine; and (b) a
frontal portion, consisting of the V-VIT nucleus.

It is important to note that the position of the last named
nucleus with reference to the level of exit of the V and VII
motor roots, is almost exactly similar in each of the three forms.

It is strongly probable that in phylogeny all motor nuclei
tend to be situated originally on the segmental level of their
emergent roots and close to, or within the central gray. Kap-
pers has already drawn special attention to this point. Thus, in
Petromyzon the position of the chief bulk of the motor VII
nucleus in the central gray, approximately on the exit level
of its motor root, is indicative of a primitive condition. The
fusion of the V-VII motor nuclei (or it may be, their non-sepa-
ration) Kappers also considers somewhat primitive for various
reasons, though the possibility of such a condition is to a certain
extent no doubt dependent upon the absence of a large gusta-
tory VII-IX—X center in these forms (see especially 61 and 64).

The ventro-lateral displacement of the motor V—VII nucleus
in Myxine has occurred under the dominating influence of the
chief reflex paths (especially the general cutaneous system)
situated in the lateral and ventral area of the medulla (R6thig,
87, Réthig and Kappers, 88).

It will be of interest then to inquire into the arrangement of
the chief reflex connections of the motor V and VII nuclei in

MOTOR NUCLEI IN PHYLOGENY 479

Bdellostoma and consider in how far the position of these nuclei
presents evidence of the operation of neurobiotactic forces.

Worthington has clearly shown that in Bdellostoma the two
chief sources of information concerning its environment are
respectively olfactory and tactile (100).

The whole brain rostral to, and including, the so-called mid-
brain is dominated, almost exclusively, by the olfactory appa-
,ratus. On the other hand, the largest and most important
tract of the hindbrain, one which dominates all the anatomical
arrangement of the medulla and extends from the funicular
nuclei to the level of the large trigeminal sensory root, is the
general cutaneous system (99). Worthington remarks further
that ‘‘this tract,’’ (ascending sensory trigeminal) ‘‘judging from
Johnston (51), is relatively much larger in Bdellostoma”’ than
it is in Petromyzon (I. ¢., p. 160).

The fasciculus communis system in Bdellostoma, though defi-
nitely developed, is small (Ayers and Worthington, 5), while
the same may be said of the acustico-lateral system (Ayers and
Worthington, 4). Both these, like the general cutaneous sys-
tem, give rise to efferent fibers passing either by way of the
fibrae arcuatae internae to the region of the heterolateral vis-
cero-motor nuclei, or directly ventral to the homolateral motor
column.

Moreover these authors have shown that a free and extensive
system of intercommunications between the general cutaneous,
communis, and acustico-lateral areas by means of correlation
neurones, is characteristically developed. In addition to these
connections it would appear from the description and figures of
these authors (4, p. 7 and 8, figs. 38, 40 and 43) that projection
neurones also are related by their dendrites to each of the three
areas mentioned. It follows from this that the secondary corre-
lation tracts in the brain of Bdellostoma can not possess a very
high degree of specificity of function.

Thus, a condition exists in this form which is apparently not
far removed from that obtaining in the medulla of the half-
grown amphibian larva, where each correlation tract ‘‘may be
actuated physiologically at the same time by two or more diverse
physiological systems of the periphery” (Herrick and Coghill, 43).

480 DAVIDSON BLACK

In this connection it is of interest to recall Worthington’s
observations (q. v.) that Bdellostoma possesses but two charac-
teristic reactions toward injury or discomfort, viz., wriggling
(swimming) and casting off slime (c. f. common type of total re-
sponse of Amblystoma larva, Herrick and Coghill, 1. c.).

The fasciculus longitudinalis posterior is but slightly devel-
oped, as may be expected, and practically all the chief secondary
correlation tracts course in the formatio reticularis of the ventro- .
lateral area of the bulb.

The tractus tecto-bulbaris et spinalis (laqueus of Holm) is
without doubt primarily olfactory efferent in character since the
so-called midbrain is comparable in but few, if any, respects
with the structure bearing this name in higher forms. The
chief afferent connections of the midbrain are from the habenular
nuclei and the base of the forebrain, both of which are olfactory
nuclei. The laqueus descends through the brain stem in the
ventro-lateral area and passes into the cord. According to
Holm’s descriptions and figures, this tract in Myxine lies about
midway between the raphé and the viscero-motor column.

The so-called cerebellum has its chief afferent connections by
way of the tractus olfacto-cerebellaris from the floor of the
forebrain and by crossed and direct fibers from the acoustic
nuclei of the medulla. The greatest efferent path from this
region is the tractus cerebello-spinalis which passes through the
upper part of the general cutaneous nucleus and then courses
in the lateral part of the formatio reticularis beneath this
nucleus.

A tract from the interpeduncular region, also olfactory in
character, passes down an indefinite distance in the ventral
formatio reticularis of the medulla.

The arrangement of these tracts, ete., is illustrated diagram-
matically in figures 1 to 6.

The ventro-lateral formatio reticularis thus contains the chief
olfactory projection paths. Dorsal to this region, the great
general cutaneous area and the subsidiary communis and the
acoustico-lateral centers are located. Between the overlying gen-
eral cutaneous area and the ventro-laterally placed olfactory paths,
the whole viscero-motor column in Bdellostoma is situated.

MOTOR NUCLEI IN PHYLOGENY 481

The viscero-motor column is situated in the midst of the
undifferentiated formatio reticularis, through which must pass
the majority of intrasegmental impulses, acting reflexly upon it
from the overlying general cutaneous and associated areas.
The presence of the long olfactory paths in the ventro-lateral
area is more probably the result rather than the cause of the
position of the viscero-motor column.

The ventro-lateral displacement of the V—VII, as well as the
X nucleus, from the primitive dorsal position has thus been
in the direction of the greatest number of incoming impulses in
accordance with the first concept of neurobiotaxis (vide supra).

With regard to the close association of the motor nuclei of the
trigeminus and facialis to form the rostral visceral motor column
it is to be noted that these nuclei innervate musculature derived
from the maxillary, mandibular and hyoid arches which is pri-
marily concerned in the ingestion of food and not in respiration.
During the process of feeding the musculature in question has no
respiratory function whatever and respiration takes place at
such times by ebb and flow of water through the gill clefts.

The chief feeding reflexes in Bdellostoma are primarily initi-
ated by tactile stimuli from the tentacles which are innervated
exclusively by the trigeminal nerve (3, 100).

The fusion (or perhaps the non-separation) of these two
effector nuclei (V-VII) whose coordinate action is initiated by
sunilar and almost simultaneous afferent impulses, may thus
illustrate the second concept of neurobiotaxis (vide supra).

A comparison of the total length of the caudal viscero-motor
column in Petromyzon with that in Bdellostoma brings out the
fact that the column in question is shorter in Bdellostoma than
in Petromyzon by just the length of the IX nucleus. In other
words, the vagus nucleus proper in Petromyzon is almost exactly
the same length as the entire caudal viscero-motor column in
Bdellostoma (fig. 8).

The gap between the rostral and caudal viscero-motor column
is smaller in Bdellostoma than in either Petromyzon or Myxine;
while the caudal viscero-motor column overlaps the anterior
horn (spino-occipital nucleus) to a considerable extent in

482 DAVIDSON BLACK

Bdellostoma, though this is not the case in either Petromyzon
or Myxine.

The number of gill sacs in petromyzonts is seven, while in
Myxinidae the gill sacs seldom exceed six pairs, though in rare
cases there may be seven pairs present (Bridge, 12, p. 422, p.

X. Sp.oce.

Fig. 8 Reconstruction charts to illustrate the reduction of the rostral end
of the caudal viscero-motor column in Bdellostoma and Myxine. (A), Petro-
myzon fluviatilis (Kappers, 66), (B), Bdellostoma dombeyi, (C), Myxine gluti-
nosa (Réthig and Kappers, 88). Signs and abbreviations as before.

281). In Bdellostoma dombeyi, however, the usual number of
gill sacs is eleven to twelve, though variations both above and
below this are not infrequent (Worthington, 99).

With almost double the number of gills to innervate, it is
not surprising to find the caudal viscero-motor column in Bdellos-
toma almost double the length of the homologous structure in
Myxine. It is, however, difficult to understand why the caudal

MOTOR NUCLEI IN PHYLOGENY 483

viscero-motor column in Bdellostoma should not, for the same
reason, be more extensive than the vagus nucleus in Petromyzon
also.

Attention has already been drawn by R6thig and Kappers to
the apparent ‘telescoping’ of the brain in Myxine and to the
seeming crowding of the vagus rootlets by the otic capsule as a
result of such a process (88). Evidences of a similar extensive
‘telescoping’ in the medulla are not lacking in Bdellostoma. It
would appear that the motor and sensory nuclei of the spino-
occipital nerves have migrated rostrad, while the superficial
attachments of their respective roots have suffered but little
disturbance. In other words, the spinal cord appears to be
impacted, as it were, into the medulla.

The peripheral area innervated by the spino-occipital nerves
is directly contiguous to that supplied by the trigeminus. Their
rostral position places the sensory spino-occipital nuclei more
directly in connection with the great general cutaneous area of
the medulla.

This rostral situation has thus given rise to a close associa-
tion of the general cutaneous centers of medulla and cord in
Bdellostoma, similar in all essentials to that brought about in
higher forms by a greater development and specialization of the
tractus spinalis nervi trigemini. <A different method has been
used in the two cases to attain the same result, viz., the close
association of centers whose peripheral areas are subject to the
influence of simultaneous stimulation.

The rostral projection of the spmo-occipital nuclei in front
of the exit level of the first spino-occipital nerve appears to be
less extensive in Myxine than in Bdellostoma, though it is a
curious fact that the distance between the rostral extremity of
the nucleus in question and the level of the facial nerve, is ap-
proximately equal in the two forms (fig. 7).

In Bdellostoma, on account of the larger size of the vagus
column, this nucleus overlaps the spino-occipital nucleus, as
already described. However, but little evidence can be adduced
to indicate any caudal displacement of the vagus column, the
distance between the caudal end of this nucleus and the first

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 4

484 DAVIDSON BLACK

motor spino-occipital root being approximately the same in
Bdellostoma and Petromyzon, and but very slightly reduced in
Myxine (fig. 8).

With regard to the absence of the glossopharyngeal nerve in
Bdellostoma. but little positive evidence can be advanced here.
The first (rostral) two vagus rootlets indicated in figures 7 and
8 are so closely associated at both their origin and exrt that
they should, I believe, be considered as a single root. The
probability of this is increased when it is noted that the first
vagus root in Myxine is much larger than the succeeding ones.
The best evidence on this point, though it be but negative, may
be had by reference to figure 8. The reduction of the frontal
portion of the caudal viscero-motor column in Bdellostoma when
thus compared with Petromyzon is clearly evident, though this
reduction is apparently not so extensive as in Myxine.

Such evidence as the above, together with that furnished by
the similar observations of Rothig and Kappers (88), tends to
confirm Johnston’s conclusion that the so-called glossopharyn-
geal nerve in myxinoids is in reality pup a pharyngeal branch
of the vagus (53).

With regard to the smallness of the gap between the V—VII
nucleus and the vagus column in Bdellostoma, this seems to be
the result of a condensation or ‘telescoping’ of the medulla as a
whole, probably in consequence of the complete loss in this
form of the peripheral glossopharyngeal area (vide Johnston,
53; Stockard, 90).

Finally, I would point out that the arrangement of the motor
roots and nuclei in the medulla of Bdellostoma dombeyi is not a
primitive one. On this account I cannot entirely agree with the
opinion expressed by Worthington (99, p. 188), and by Ayers
and Worthington (4, p. 1) to the effect that myxinoids are the
most primitive craniates known and several degrees lower than
the Petromyzontes. This conclusion is in harmony with that
of Réthig and Kappers based on their study of the motor nuclei
in Myxine (1. ¢.).

MOTOR NUCLEI IN PHYLOGENY 485

SELACHII
Motor nucler in Selache maxima (Cetorhinus maximus)

The basking shark is the largest of living fishes. It is a pelagic
form of wide distribution and often gregarious habit, attaining a
length of 35 to 40 feet (Jordan, Bridge, 1. c.).

The general morphology of the brain is illustrated in figure 9
and requires no further description in the present connection.
Attention may be directed, however, to the marked transverse
folding of the large cerebellum. The complexity of the folding

Fig. 9 Brain of Selache maxima. A, lateral view; B, dorsal view; C, ventral
view. X 3%. Abbreviations: N.l.l., nervus lineae lateralis; sp.occ., spino-occipi-
tal roots; Sp.J., first spinal nerve; VIIJ.+1.1., acusticus and lateralis root;
LI1,UL,IV,V,VI,Vil,IX,X, cranial nerves.

A4S6 DAVIDSON BLACK

of the surface of the cerebellum in sharks varies almost directly
with the size of the organ in conformity with the general laws of
cortical expansion enunciated by Baillarger and modified by
Darests (cf. Kappers 68, 69, 71).

Spino-occipital nuclei and roots (Nu. et rad. mot. Nn. spin. occ.).
Only the first two rootlets of the spino-occipital series are charted
in Selache and these probably represent separate fascicles of the
first spino-occipital nerve (fig. 17C). They pass almost di-
rectly ventrad from their dorsally placed nucleus of origin to the
ventral periphery of the medulla (fig. 10).

As in Bdellostoma, there is no line of demarcation between
the anterior horn of the cord and the motor nuclei of the spino-
occipital nerves. However, the rostral end of the anterior horn
in Selache becomes thinned by the absence of ventrally placed
motor cells, so that in the spino-occipital region the somatic
motor column in this animal is characterized by a more dorsal
position than in the succeeding cervical region. A similar ar-
rangement of the spino-occipital nuclei and roots obtains in all
selachians examined. The possible significance of these rela-
tions in contrast to those obtaining in ganoids and teleosts will
be discussed subsequently.

The rostral limit of the motor spino-occipital nucleus in Selache
is sharply marked and the reticular cells of the medulla are not
arranged in such evident morphological continuity with this
column as in Bdellostoma. The reticular cells form a nucleus
whose elements are irregularly distributed in the formatio
reticularis from a level somewhat rostrad of the entrance of the
trigeminus, downwards to the calamus region. The largest
cells tend to be situated centrally and the nucleus corresponds
in general arrangement to the reticular nucleus of Raja de-
scribed by van Hoevell (1. c.). The latter author recognizes
three more or less distinct parts to this nucleus, viz., (a) nucleus
reticularis inferior which is distributed on the level of entrance
of vagus roots, (b), nucleus reticularis medius situated chiefly on
the level of entrance of N.VIII, and (ce), nucleus reticularis su-
perior distributed over the region rostral to the entrance level of
N.VIII.

MOTOR NUCLEI IN PHYLOGENY 487

The arrangement of the reticularis nucleus in Selache and
Raja thus corresponds both in its general extent and in the extent
of its subdivisions to the nucleus motorius tegmenti described by
Bartelmez in Amelurus (I. c.).

Tr.oc.mot.cruc.
\

R.VII.m.ase.
XN

ee
~\ Fib.arc.ext.

10 ENE

Tr.oc.mot.cruc.
\

R.VII.m.asc.. \

Figs. 10, 11 and 12 Selache maxima. Outlines of transverse sections to illus-
trate the topography of the medulla. Figures 10 to 16 drawn to same scale.
Abbreviations: C.c., crista cerebellaris; F.l.m., fasciculus longitudinalis medialis;
Fib.arc.ext. ventral arcuate fibers of the acusticum; Lob.lin.lat., lobus lineae
lateralis; Lob.visc., visceral lobe; N.VI.. abducens root; Nu.par., nucleus para-
medianus (inferior olive); Nu.sp.occ.m., cells of motor spino-occipital nucleus;
Nu.VII.m., nucleus motorius N. facialis; Nu.X.m., nucleus motorius N. vagi;
R.desc. V., descending sensory trigeminus root; R.l.VIIT., root of the posterior
lateralis nerve; R.sp.oce.m.(1), first spinal-occipital motor root; R.VII.m.asc.,
ascending facial motor root; R.IX.m.asc., ascending glossopharyngeal motor
root; R.X., vagus root; T’r.oc.mot.cruc., tractus octavo-motorius cruciatus;
Tub.ac., tuberculum acusticum.

488 DAVIDSON BLACK

Nucleus paramedianus (Nu. paramed.). The nucleus para-
medianus, or paraseptalis, is well developed in Selache maxima.
The sagittal relations of the right nucleus are indicated in the
reconstruction chart, figure 17 C. This nucleus is bilaterally
symmetrical, consisting of circumscribed collections of grey mat-
ter, on each side of the raphé near the ventral periphery of the
medulla in the neighborhood of the emergent roots of the
spino-occipital nerves (fig. 10). The connections which this
nucleus establishes with other centers are not fully understood,
but Johnston has shown that the neurites of this nucleus in
Acipenser cross the raphé and end among the tract cells of the
lateral column (52) and Goronowitsch considered this nucleus
to the homologue of the inferior olive of higher forms (30). In
general among vertebrates the size and compactness of the
nucleus in question varies directly with the relative develop-
ment of the cerebellum. This has been pointed out by Kappers,
who has shown that a well circumscribed nucleus paramedianus
is present only in those forms which possess a relatively large
cerebellum (64).

Motor facial, glossopharyngeal and vagal nuclei (Nu. et. rad.
mot. Nn. VII-IX—-X). The arrangement of the caudal viscero-
motor column in Selache presents a marked contrast to that
obtaining in cyclostomes (figs. 7 and 17). In Selache the motor
nucleus of the facial nerve is situated a considerable distance
caudad of the level of its root exit and forms the rostral portion
of a continuous dorsally placed cell column composed of the
motor VII-IX--X nuclei. This arrangement of the caudal
viscero-motor column is characteristic of all selachians and the
situation of the VII motor nucleus far behind the level of its
own root exit has necessitated the formation of a long ascend- .
ing. VII motor root in these forms, as already fully set forth by
Kappers (61, 64, 66 and 72).

The relations of this ascending root of the facial nerve in
Selache to its nucleus of origin and to the fasciculus longitu-
dinalis medialis are illustrated in figures 11 and 12.

The motor root of the glossopharyngeus in Selache passes
dorso-mesially from its superficial attachment and _ courses

MOTOR NUCLEI IN PHYLOGENY 489

through the ventral portion of the VII motor nucleus to reach
the lateral side of the posterior longitudinal bundle. Here it
turns and courses caudad to its cells of origin in the posterior
visceral column lateral to the fasciculis longitudinalis medialis.
This mode of origin is essentially similar to that described by
Kappers in Scyllium (72, p. 384, fig. 1.).

In Selache and in Hexanchus the ascending motor VII root
may be traced caudad as far as the motor glossopharyngeal
fibers extend. It results from this that the motor VII nucleus
overlaps that of the glossopharyngeus and the two nuclei are
quite as intimately associated as are those of the sensory VII
and IX roots. This condition is indicated in the reconstruction
charts in figure 17 where the caudal limit of the descending VII
and IX motor roots is marked by a heavy line.

The motor vagus nucleus in Selache extends for a long dis-
tance into the upper part of the cord and in consequence it over-
laps the somatic motor column to a much greater degree than
was the case in Bdellostoma. Its exact caudal extent was only
determined by cutting an additional series of sections of the
cervical cord. For comparison the posterior visceral column in
Hexanchus was re-examined and it was found the vagus nucleus
in this form could indeed be traced further caudad than had
been indicated in previous reconstructions, though its exact
caudal limit was not determined.

Abducens nucleus and roots (Nu. et. rad. N. VI). The abducens
nucleus in Selache occupies a position about midway between the
exit levels of the motor VII and IX roots. Its cells of origin
are scattered among the fibers of the posterior longitudinal
bundle and its fibers converge to form three rootlets which pass
directly ventrad and emerge in series from the ventral periphery
of the bulb (fig. 12). The.nucleus in Selache is not quite so
dorsally situated as in Hexanchus or Heptanchus, but its rela-
tion to the vestibulo-motor fibers is essentially similar in all
three forms.

The position of the emergent roots of the abducens at a
more caudal level than those of the motor VII nerve is character-
istic of sharks and also of ganoids and amphibia. In the case

490 DAVIDSON BLACK

of Hexanchus both glossopharyngeal and abducens fibers may
be cut in the same transverse section (fig. 13).

Motor trigeminal nucleus and root (Nu. et. rad. mot. N. V.).
The motor trigeminal nucleus in Selache is placed in a dorsal
position on the level of its root exit, though it extends some dis-
tance both rostrad and caudad of this plane. This situation is a
characteristic one for this nucleus in sharks (fig. 17).

In the reconstruction chart figure 17 C, the motor root of the
trigeminus in Selache appears to be very small when compared
to that of Hexanchus or of Heptanchus. This results from the

CO Dexia.

Fig. 13 Hexanchus. Outline of transverse section through the medulla to
show the emergence of abducens and motor glossopharyngeal rootlets at the
same level. Abbreviations: N.V/., abducens root; R.[X.m., motor rootlet of
glossopharyngeus. Other abbreviations as in figures 10 to 12.

fact that the root emerges from the medulla in Selache in a very
compact bundle, so that its superficial attachment extends over
fewer sections than on the other forms.

The position of the motor V nucleus in transverse section is
indicated in figure 14. Here it will be seen that the cells show a
distinct tendency to develop processes chiefly in a ventro-lateral
direction and the nucleus as a whole is somewhat more ventro-
laterally placed than that of the motor VII (fig. 11). In this
position the motor V nucleus is placed in close relation to the
reticular grey surrounding the upper end of the descending
sensory V, and the inconspicuous secondary gustatory tract.

MOTOR NUCLEI IN PHYLOGENY 491

Oculomotor and trochlearis nuclei and roots (Nu. et. rad. Nn. IIT
& IV). The trochlear and oculomotor nuclei in Selache maxima
have already been described and charted by Huet (47) and dis-
cussed by Kappers (66). However, a short description of these
nuclei and their roots will be given here for the sake of com-
pleteness.

Tectum

aX
MYYANu.V.m.
‘ 4

Figs. 14, 15 and 16 Selache maxima. Outlines of transverse sections to
illustrate the topography of the medulla. Abbreviations: N.III., oculomotor
roots; N.JV., trochlear nerve; Nu.mag.tect., nucleus magnocellularis tecti;
Nu.III., oculomotor nucleus; Nu.IV., trochlear nucleus; Nu.V.m., motor tri-
geminus nucleus. Other abbreviations as in figures 10 to 12.

492 DAVIDSON BLACK

The trochlear nucleus lies in a crescentic depression upon the
dorsal surface of the fasciculis longitudinalis medialis (fig. 15).
Its large motor cells are more compactly arranged than those of
the oculomotor nucleus with which it is in contact rostrally.
The emergent root passes dorsally in the Sylvian gray and,
after decussation with its fellow of the opposite side above the
ventricle, emerges dorso-laterally between the cerebellum and
the midbrain.

The elements composing the oculomotor nucleus are arranged
irregularly and are not divisible into definite subsidiary groups
(fig. 16). The emergent radicles pierce the posterior longitu-
dinal bundle on their course to the ventral periphery of the
midbrain. No definite arrangement of crossed oculomotor
fibers could be determined. No small reticular elements such
as are characteristically present in higher vertebrates were
found in association with either oculomotor or trochlear nuclei.

The sagittal relations of these nuclei and their roots are charted
in figure 17 C. The oculomotor nucleus occupies a dorsal posi-
tion on the level of its root exit and extends rostrad of this level
some distance on the same horizontal plane. The trochlear
nucleus lies directly behind it but on a slightly more dorsal
plane. In most selachians the trochlear root emerges from the
brain some distance behind the level of the caudal end of its
nucleus (fig. 17 A). In Selache maxima, however, the root
emerges at the level of the caudal third of its nucleus (fig. 17 C)
and in Hexanchus at the level of the caudal end of its nucleus
(fig. 117-3).

Numerous large cells constituting the nucleus magnocellu-
laris tecti may be observed in the ventricular gray of the mid-
brain roof (figs. 15 and 16). Among the cells of this nucleus, the
radix mesencephalica trigemini takes its origin. As this root
in all probability is a highly specialized sensory component of
the trigeminal nerve, it has not been charted (cf. Johnston, 54;
Van Valkenburg, 93 and 94).

Fig. 17 Reconstruction charts of motor roots and nuclei. (A), Heptanchus

(after Kappers). (B), Hexanchus (mod. after Kappers, 66). (C), Selache
maxima. Signs and abbreviations as before (page 476).

493

MOTOR NUCLEI IN PHYLOGENY

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494 DAVIDSON BLACK
Discussion

Spino-occipital complex. Among sharks the spino-occipital
nerves are distributed to both epibranchial and hypobranchial
spinal musculature and the pre-hyal elements of the hypo-
branchial musculature are well developed (25 and 97). Unlike
cyclostomes, there is evidence of a definite specialization of the
rostral end of the spino-occipital motor column in sharks, whereby
this nucleus becomes dorsally placed, loses its ventral elements,
and terminates abruptly a very short distance rostral of the
exit level of the first spino-occipital root. In consequence of
this, the spino-occipital rootlets in sharks pass from their nucleus
of origin almost directly ventrad. These differences between
the spino-occipital complex of selachians and that of cyclo-
stomes, are significant in view of Furbringer’s findings: that the
lower sharks mark a stage in vertebrate evolution where all tend-
ency toward caudal extension of the branchial apparatus comes
to an end, so that from sharks onward, there is a definite tend-
ency toward reduction of gill area together with assimilation
into the head (and consequent reduction) of cervical segments.

The very evident variation in the number of gills among
individuals of Bdellostoma (100) mustexercise no small influence
upon the development and stability of the motor center inner-
vating the somatic structures affected by this variation. This
would partly explain the lack of any appearance of fixation or
specialization of the rostral end of the motor spino-occipital
column in these species.

On the other hand, the relative constancy of the development
of the pre-hyal coraco-mandibularis elements as well as the
epibranchial spinal musculature in sharks (subspinalis and inter-
basales, innervated by Furbringer’s occipital nerves (v), w, x,
y, and z), would tend to produce a relatively constant arrange-
ment of the rostral end of the motor spino-occipital column
from which these nerves arise; for progressive reduction of the
subspinalis and interbasales among sharks becomes evident only
when the higher and lower selachians are compared or when this
region is studied ontogenetically and it would thus appear that

MOTOR NUCLEI IN PHYLOGENY 495

in a given species but slight, if any, individual variation in the
development of this musculature obtains.

In the absence of disturbing individual peripheral variations
it is probable that, as Kappers has pointed out, reflex impulses
passing by way of the well developed fibrae arcuatae dorsales
and the fasciculus longitudinalis medialis from the acustico-
lateral areas, exercise no small influence upon the rostral end
of the somatic motor column and contribute largely to the
dorsal situation of the cells of this nucleus. Further, the absence
of ventrally situated motor elements in the spino-occipital
nucleus in selachians would be in accordance with the compara-
tively slight development in these forms of the ventral reflex
pathways, le., tractus octavo-motorius cruciatus ventralis,
tractus tecto-bulbaris ventralis, etc. (40 and 43).

Motor vagal nucleus (Nu. mot. N. X.). The trapezius muscle
in sharks is a well developed structure which receives its inner-
vation by way of the so-called caudal ascending vagus root.
This root corresponds to the ‘posterior fasciculus’ of the vagus
which Owen described as ‘‘representing the nervus accessorius”’
(85). This statement has been verified in detail by Furbringer
who demonstrated once more the connection between this por-
tion of the vagus and the well developed trapezius muscle in
Hexanchus, ete. (25). It follows from this that the caudal end
of the motor vagus nucleus must represent the nucleus acces-
sorius which here occupies its most primitive position on the
same plane and in undisturbed continuity with the caudal
viscero-motor column (fig. 17).

Among sharks, the extent of the viscero-motor column caudad
of the exit level of the first spino-occipital rootlets, appears to
be more or less directly correlated with the degree of develop-
ment of the trapezius musculature. Among rays, however,
such direct correlation between these structures may not ob-
tain. Thus, according to Kapper’s reconstruction (65, fig. 8, 66,
fig. 12), the viscero-motor column in Raja clavata extends but a
short distance caudad of the exit level of the first spino-occipital
nerve. However, this appearance may be due in part to the
loss of certain occipital segments which, as Furbringer has shown,
are absent among the higher forms of elasmobranchs (rays).

496 DAVIDSON BLACK

Vetter (95) considers that the trapezius is derived from a
primitive superficial visceral constrictor system of muscles
which were innervated by the vagus. In sharks, however, the
trapezius has lost its visceral constrictor character and, though
retaining its vagus innervation, has become a muscle of impor-
tance in the somatic movements of the head and pectoral girdle.

The relations of the nucleus accessorius accord well with this
hypothesis for, though it retains the essential visceral character-
istic of continuity with the caudal viscero-motor column, yet it
has come to lie in the upper cervical region where it is closely
related to the general somatic sensory centers receiving afferent
fibers from the skin area overlying the trapezius. Thus, the
peripheral change in muscular function has been accompanied
part passu by a central change in the reflex connections of its
motor nucleus. In other words, the motor nucleus tends to
migrate in the direction of the most important centers acting
upon it reflexly.

Motor facial and glossopharyngeal nuclei and roots (Nu. et rad.
mot N. VII-IX). The increased importance of the visceral
sensibility in the life habits of the selachians, as compared with
cyclostomes,® is evidenced by the formation in the former ani-
mals of a communis nucleus in which the visceral sensory com-
ponents of the facial nerve terminate in common with those of
the glossopharyngeal and vagus nerves and on the level of the

latter. The association of the visceral sensory nuclei of these

nerves to form a common center situated a considerable distance
caudad of the level of entrance of the sensory VII root, affords
an example of nuclear association under the influence of similar,
simultaneous stimulation (vide supra).

It follows from this, that in selachians, the chief center act-
ing reflexly upon the motor facial nucleus (viz., the nucleus of
its own sensory root) is situated at and caudal to the exit level
of the glossopharyngeus. As a result the motor VII nucleus,
like that of its sensory root, has also become displaced from its

* Taste buds among sharks are more numerous than in cyclostomes, though it
is probable that they are more or less restricted to the pharynx, gills and mouth
in the former animals (vide Kappers, 68).

MOTOR NUCLEI IN PHYLOGENY 497

primitive position on the level of its root exit, and its migra-
tion has been in the direction of the greatest number of im-
pulses acting upon it reflexly in accordance with the first concept
of neurobiotaxis. The long intramedullary, ascending course
over which the emergent motor facial root passes, marks the
path along which the nucleus of this nerve travelled during the
phylogenetic development of this motor nuclear pattern (v.
Kappers, l. c.).

The factors operating to produce the slight displacement of
the motor LX nucleus caudal of the level of its root exit, with
the resulting formation of its ascending emergent motor root,
are essentially similar to those discussed in the preceding para-
graph. For further details of the phylogenetic displacements
of the motor VIJ-IX nuclei, reference should be had to Kappers’
original papers on this subject (1. ¢.).

The respiratory mechanism in sharks in general may be
described as consisting of two opposing muscular complexes
(constrictor group and dilator group) which operate in alternate
rhythmic opposition and which are segmentally innervated
from before backwards by the motor components of ‘branchial
nerves VII, IX, X. The respiratory reflex is initiated chiefly
by visceral afferent impulses arising in the branchial mucosa,
which reach the communis center through the visceral sensory
components of branchial nerves VII-IX—-X (6). Thus, the
formation of the caudal viscero-motor column by the intimate
association of the motor VII-IX—X nuclei and the situation of
this column in close relationship with the communis nucleus,
furnishes a striking example of the important réle played by the
peripheral respiratory mechanism in the production of specific
nuclear (reflex) pattern.®

6 That the primitive segmental nature of the respiratory centers has not
become lost by this adjustment is evident from the observations of Hyde (48).
This author carried out her experiments on the skate, though unfortunately
she has omitted to identify the species of the animals on which she worked.
She has demonstrated, however, that the respiratory centers are segmentally
arranged and bilateral in nature and has indicated physiologically the charac-
teristic anatomical arrangement of the ascending motor and descending sensory
facial roots (1. c., p. 247, fig. 3).

498 DAVIDSON BLACK

Abducens nucleus and root (Nu. et rad. N. VI). The charac-
teristically dorsal position of this nucleus among sharks appears
to be also a primitive one, and in this situation the nucleus is
reflexly dominated by impulses from the acustico-lateral area
which reach the nucleus through the posterior longitudinal
bundle and also directly by dorsal arcuate fibers (64 and 66).

The position of the nucleus between the exit levels of the
motor VII and IX roots seems also to be a primitive character,
so that the phylogenetic sequence of emergent motor roots in
this region evidently should read VII, VI, IX. Indeed in the
primitive form Hexanchus the most caudal fascicle of the
abducens root emerges on the level of the motor glossopharyngeus
exit (vide supra, figs. 13 and 17 B).

Trigeminal nucleus and root (Nu. et rad. N.V.). In all selachi-
ans the motor trigeminal nucleus lies in a somewhat dorsal posi-
tion and extends some distance both caudal and rostral of its
root exit. In the latter respect and in its isolation from the
motor facial elements, the trigeminal nucleus of sharks differs
from that in cyclostomes. Indeed, with the exception of birds,
a greater proportion of the motor V nucleus is placed rostrad
of the level of its root exit in sharks than in any other group of
vertebrates.

In sharks the rostral position of the motor V nucleus, quite
removed from the caudal viscero-motor column, is of signifi-
cance in view of the peculiar arrangement of the respiratory
mechanism in these animals. It has been pointed out already
that in cyclostomes both inspiration and expiration takes place
through the gill openings when the animal is attached by its
mouth to any object. As a corollary to this the vascular gill
folds in these forms are fixed in such a manner that they can
act as efficient respiratory organs without regard to the direc-
tion of the flow of water through the gill openings. The gills in
sharks consist of closely approximated transverse lamellae which
also are firmly attached to the sides of the interbranchial septa.
In the latter respect the gills of sharks and cyclostomes resemble
one another and differ from those of all other fish.

MOTOR NUCLEI IN PHYLOGENY 499

It is an observation of very long standing (10) that among
sharks, during the dilation of the mouth cavity and pharynx
in inspiration, water may enter either through the mouth, the
spiracle (Gf present), or even through the gill slits themselves.
In sharks, as in cyclostomes, the arrangement of the gills per-
mits of the efficient action of these organs irrespective of the
direction of the respiratory flow.’

It becomes evident, therefore, that in sharks the alternate
opening and closure of the mouth in synchronism respectively
with the alternate dilation and constriction of the pharynx, is
not a sine qua non tor efficient breathing. It follows from this
that efferent impulses from the motor V nucleus are not neces-
sarily called forth during each reflex respiratory cycle in the
same way as are those from the various constituents of the
caudal viscero-motor column. ‘The rostral position of the motor
V nucleus in sharks may thus be said to express a function of
the negative influence of the communis center upon the reflex
action of the jaw musculature in these forms.

In this connection the work of Harman (33) on the innerva-
tion and development of the musculature of the nictitating
membrane and eye-lids in sharks should also be noted. The
musculature in question is innervated by the trigeminus and the
perfection of its development appears to vary inversely with the
size of the spiracle. Thus, according to this author, in rays where
the spiracle is of large size, the musculature is absent, while in
Galeus where the spiracle is absent or minute, the musculature
of the eye-lid is well developed. Scyllium stands intermediate in

? Baghoni (7 and 8) evidently does not consider that the inflow of water
through the external gill openings in selachians is a part of the normal respiratory
cycle in these animals. However, Couvreur (14) working on Torpedo marmo-
rata has confirmed this observation and the work of Hyde (48) demonstrates
beyond a doubt that respiration of a sufficiently efficient nature to prolong life
indefinitely can be maintained in the skate by the action of the last four gill
arches alone. This observer also expressly states that the ‘“‘motor nerves con-
cerned in respiration are the fifth, seventh, ninth and tenth nerves, of which
the fifth is least important and the tenth most important”’ (I. ¢., p. 240). The
relative independence of the trigeminal musculature during the respiratory
cycle has also been noted in Rhina by Darbishire (17).

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 4

500 DAVIDSON BLACK

this respect, having a medium sized or small spiracie and a well
developed nictitating membrane but no eyelid musculature.

On comparing the reconstruction charts of Raja (66, fig. 12),
Seyllium (66, fig. 11) and Galeus (64, map C) it appears that in
Galeus the motor V nucleus extends furthest rostral from the
frontal margin of the emergent motor VII root, Scyllium comes
next in order and in Raja the motor V nucleus reaches nearest
to the frontal margin VII root. (For details consult Kappers’
list of topographic relations, 64, pages 119-125).

There would thus appear to be some relationship existing
between the position of the motor V nucleus in sharks and the
degree of development of prespiracular trigeminal eye-lid mus-
culature, though at present no accurate comparison can be
made.

Though the bulk of the motor V nucleus lies in a dorsal posi-
tion, there is evidence of a tendency toward ventro-lateral dis-
placement throughout the whole length of the nucleus, for a
greater number of the large dendrites of its cells extend ventro-
laterally and the diameter of the nucleus itself is increased in
this direction (fig. 14).

The elongation of the dendrites and the increased ventro-
lateral diameter of the nucleus, bring the motor elements into
close relationship with the reticular formation surrounding
both the upper end of the descending sensory trigeminal root
and the relatively slightly developed secondary ascending
gustatory tract.

The influence of the trigeminal cutaneous impulses upon the
reflex action of the jaw musculature in elasmobranchs must be
great in view of the ventral position of the mouth. In manipu-
lating its food, before this can be swallowed, the animal must
rely almost entirely upon its sense of touch. The slight displace-
ment of some of the motor V elements and the extension of pro-
cesses in the direction of the reticular gray about the rostral
end of the descending sensory V nucleus thus illustrate further
the first concept of neurobiotaxis (vide supra).

Oculomotor and trochlearis nuclei and roots (Nu. et rad. Nn. IIT
and IV). Compared with petromyzonts, the trochlear nucleus

MOTOR NUCLEI IN PHYLOGENY 501

in sharks is much more rostrally placed and in all the latter
forms lies in a dorsal position in the Sylvian gray immediately
caudal to the oculomotor nucleus.

In Petromyzon the trochlear nucleus lies on the exit level of
the motor V root and caudad of its own root exit, while among
sharks this nucleus is always placed far rostrad of the exit level
of the motor V root and with few exceptions (e.g., Selache,
Hexanchus) among these forms lies wholly rostrad of its own
root exit.

The position of the trochlear nucleus just caudad of the oculo-
motor nucleus appears to be characteristic of all vertebrates
above cyclostomes in which the eyes are functionally well de-
veloped. Thus the close mutual association of these two nuclei
appears to have occurred phylogenetically at the same time as
the development of the midbrain roof to form the great terminal
nucleus for the optic tract.

On the other hand, the site at which the trochlear decussation
and exit occurs seems to be quite variable even among closely
allied forms (cf. teleosts) and it would appear that the root of
this nerve, unlike its nucleus, is subject to variable influences
of a purely mechanical character.

In conclusion, it is evident that the motor roots and nuclei in
Selache maxima are arranged to form a pattern which is in all
essentials similar to that obtaining elsewhere among selachians.

In certain respects this selachian pattern exhibits primitive
characters, i.e. (a), the emergence of the motor roots in phylo-
genetic sequence, viz, LIT, 1V, V, VIL, VI,.EX, X, sp. ece-.; (b),
the predominantly dorsal situation of both visceral and somatic
motor nuclei; (c) the direct continuity and dorsal position of
vagus and accessory nuclei, etc. In other respects unmistak-
able signs of specialization are evident, such, for example, as
the relations of the rostral end of the spino-occipital nucleus, the
caudad development of the accessory complex, the development
of the ascending motor VII and IX roots and the rostral position
of the trochlear nucleus in direct contiguity with that of the
oculomotor nerve.

Oo
=)
bo

DAVIDSON BLACK

GANOIDEI8
Motor nucler in Polyodon spathula

An excellent account of gross features of the brain and of the
superficial attachments of the cranial nerves in the Mississippi
spoon-bill, has been contributed by Garman (27). Up to the
present, however, no work has been published on the finer anat-
omy of the brain in this animal. Among ganoids, the motor
nuclei have already been studied and reconstruction charts have
been made in the following forms: Amia (22 and 66), Lepidosteus
and Acipenser (91). :

Lob.vag,
: Lob.vag.

Fun.et trig.sp, NuVilim.
RuVilimmasc.<

Ey ~Tr.sec.com.

\
Tr.sec.com.

“Fib.arc.ext. = Tr.te.b

18 19

Figs. 18 to 24 Transverse sections of the brain of Polyodon spathula; figures
19 to 24 drawn to the same scale.

Fig. 18 Transverse section of brain stem at the exit level of the first motor
spino-occipital rootlet.

Fig. 19 Transverse section of brain stem at the exit level of the motor
glossopharyngeus root. Abbreviations: C.c., crista cerebellaris; F.l.m., fascicu-
lus longitudinalis medialis; Fib.arc.ext., external arcuate fibers; Fib.arc.int.,
internal arcuate fibers; Fun.et trig.sp., funicular area; Lob.lin.lat., lobus lineae
lateralis; Lob.vag., visceral sensory column; N.U.1.X., nervus lineae lateralis;
Nu.sp.oce.m., motor spino-occipital nucleus; Nu.VJ7.m., motor facial nucleus;
Nu.X.m., motor vagus nucleus; ?.desc.V., tractus spinalis trigemini; R.sp.oce.m.,
first spino-occipital motor rootlet; R.VII.m.asc., ascending bundle of motor
facial root; R.JX.m., motor glossopharyngeal root; 7'r.sec.com., secondary gusta-
tory bundle; 7’r.tc.b., tractus teeto-bulbaris; Twb.ac., tuberculum acusticum.

8 This term is retained because of its great convenience and is employed here
in the sense defined by Jordan (55, p. 32). With regard to the literature on
Polyodon, I am much indebted to Professor C. J. Herrick both for the loan of
Garman’s paper (27) and for assistance during my vain search for any work
dealing with the finer anatomy of the brain stem of this animal.

MOTOR NUCLEI IN PHYLOGENY 503

Spino-occipital nuclei and roots (Nu. et rad. mot. Nn. spin. occ.).
The dorsal portion of the somatic motor column of the cord, is
continued upwards into the medulla in Polyodon in a manner in
many respects recalling the relations prevailing in this region
among selachians (figs. 10 and 18). The rostral end of this
column is, however, not sharply defined as in sharks, and it
is difficult to set a limit between the nucleus in question and the
reticular elements of higher levels. A similar condition was
noted in Bdellostoma. In figure 25 A, the level above which
none of the emergent motor rootlets can be traced is taken to
be the rostral limit of this motor column.

Like the spino-occipital column in Selache, this nucleus in
Polyodon les close to the ventricular gray, lateral to the fascicu-
lus longitudinalis medialis, and the cells toward its rostral end are
most dorsally placed. The emergent motor fibers pass very
obliquely ventrad and caudad from this nucleus to reach the
periphery of the bulb. In the latter respect these roots differ
from the emergent spino-occipital fibers in Selache which pass
almost directly ventrad (figs. 10 and 18). It should also be
noted as a corollary to this observation, that in none of the
selachians hitherto examined, does the spino-occipital nucleus
extend so far rostrad of the first spino-occipital rootlet as does
this column in Polyodon, above its first emergent fiber. Of the
ganoids examined, Acipenser only seems to be an exception to
this rule (figs. 17 and 25). The origin and relation of the spino-
occipital nerves in Amia and Lepidosteus, and in Acipenser
(except as noted above) closely correspond to those obtaining in
Polyodon.

Nucleus paramedianus (Nu. paramed.). On each side of the
raphé, on the level of the first spino-occipital rootlet and extend-
ing for some distance caudad, the nucleus paramedianus may be
distinguished in Polyodon. Many internal arcuate fibers course
across this ill defined nucleus, whose cells, as in Amia, are so
diffusely distributed that it was not possible to indicate its out-
line in the reconstruction chart. However, in both Acipenser
and the young Lepidosteus specimens which Theunissen exam-
ined, the nucleus paramedianus has been charted.

504 DAVIDSON BLACK

Motor facial, glossopharyngeal and vagal nuclei and roots (Nu.
et rad. mot. Nn. VII-IX—X). In Polyodon the caudal viscero-
motor column lies throughout within the ventricular gray and
extends from a level about midway between the emergent motor
roots of the facial and glossopharyngeal nerves, to a point some
distance caudad of the first spino-occipital motor rootlet (figs.
18,19 and 25 A). As in Selache, within the limits of this column
are included all the cells of origin of the motor VII-IX—X
roots.

R.VII.m.asc
vas

Fib.arc.int. 23

“er \R.desc.V
Trt. beet \R.ViLm
Tr.sec.com.
20 20 a

Fig. 20 Polyodon spathula. Transverse section of brain stem at the exit
level of motor facial root.

Fig. 20a Portion of transverse section of brain stem at somewhat more
caudal level than the preceding to illustrate the relations of the ascending motor
facial root. Abbreviations: N.1.1.VII., nervus lineae lateralis; R.VII.m., emer-
gent fibers of motor facial root; R.VII.m.asc., ascending bundle of motor facial
root; R.VII.s., sensory facial root. Other abbreviations as in figures 18 and 19.

The fibers of the motor VII root arise from cells occupying
the rostral end of the caudal visceral motor column. They
collect to form a compact bundle which courses rostrad for a
considerable distance in the ventricular gray beside the fasciculus
longitudinalis medialis, before they arch laterally and emerge
on the ventro-lateral periphery of the medulla (figs. 19, 20
and 20 A).

The glossopharyngeus motor root arises from cells of the
viscero-motor column situated immediately caudad of the motor

MOTOR NUCLEI IN PHYLOGENY 505

VII nucleus. The fibers pass rostrad a short distance and form
a root which courses laterad below the motor VII nucleus to
gain the ventro-lateral periphery (fig. 19).

The arrangement and relations of the constituents of the
caudal visceral motor column and their motor roots in Acipenser,
Amia, and Lepidosteus, are essentially similar to those described
above in Polyodon.

Abducens nucleus and roots (Nu. et rad. N. VI). In the teg-
mentum, medial and somewhat ventral to the rostral end of the
motor VII nucleus, a poorly defined and scattered collection of
cells forms the abducens nucleus. In Polyodon, as in Lepi-
dosteus, the presence of numerous reticular elements in this
neighborhood makes it difficult to define the exact limits of this
small nucleus. Thus, in Polyodon it is only possible to indicate
the approximate outline of the abducens nucleus by means of
dotted lines. Its rostro-caudal extent in figure 25 represents
the limits beyond which no emergent radicles could be followed
centrally. In studying Lepidosteus, Theunissen also experi-
enced this difficulty so that, though he described the nucleus in
question, he has not indicated it in his reconstruction chart
(fig. 25D). In Amia and Acipenser, however, the abducens
nucleus is somewhat less diffusely arranged and its position is
indicated in the reconstruction charts (fig. 25 C and B).

Three thread-like rootlets emerge in series from the abducens
nucleus in both Polyodon and Acipenser, while four such root-
lets can be identified in Lepidosteus and Amia. In each case
they course almost directly ventrad to the periphery of the
bulb.

The position of the emerging abducens rootlets in ganoids
midway between the exit levels of the motor VII and IX roots,
corresponds closely to the arrangement of these roots in sharks.
Also in regard to the position of the abducens nucleus with ref-
erence to the exit level of the motor glossopharyngeus, the two
groups closely resemble one another. In sharks, however, the
abducens nucleus lies upon a more dorsal plane than it does in
ganoids.

506 DAVIDSON BLACK

Motor trigeminal nucleus and roots (Nu. et rad. mot. V). In
Polyodon, the motor V nucleus is dorsally situated in the lateral
area of the ventricular gray, and it les wholly caudal to the
rostral border of its emerging root (fig. 25 A). In the rostral
third of its length the nucleus becomes thinned somewhat,
so that a partial division into two is evident. The fibers aris-
ing in this nucleus become collected to form a bundle which
ascends just laterad of the nuclear mass (fig. 21 A). Toward the

ica

_- Valyula ==

Fig. 21 Polyodon spathula. Transverse section of brain stem at the exit
level of the motor trigeminus root.

Fig. 214 Portion of transverse section at somewhat more caudal level than
the preceding to illustrate the relations of the ascending motor trigeminal root.

Fig. 22 Transverse section of brain stem at the exit level of the trochlear
nerve. Abbreviations: A.cb., auricular lobe of cerebellum; Cb., cerebellum;
N.IV., trochlear nerves decussating at their emergence; Nu.V.m., motor trigem-
inal nucleus; R.V.m., emergent motor trigeminal fibers collected into two root-
lets; R.V.m.asc., ascending fibers of motor trigeminal root. Other abbreviations
as before.

MOTOR NUCLEI IN PHYLOGENY 507

rostral end of the nucleus, the bundle curves ventro-laterad to
reach the periphery. In this part of its course the fibers become
arranged in the form of two distinct emergent bundles, one more
rostrally placed than the other. These rootlets are separated
only by a few sections, and it was not possible to demonstrate
any specific localized relationship between the two rootlets on
the one hand, and the two nuclear subdivisions on the other;
each rootlet apparently derives its fibers from the nucleus as a
whole, and not from any special portion of it.

Aside from the partial division of the motor V nucleus and the
double nature of its emergent root, the relations of this com-
plex in Polyodon correspond closely to those in Lepidosteus
(fig. 25 A and C). In all other ganoids examined, the nucleus
in question is dorsally situated and its chief bulk is placed
caudad of the level of its root entrance. The last mentioned
relation is especially evident in Amia, where the nucleus ex-
tends caudally well beyond the level of the emergent motor VII
root. In selachians, on the other hand, the motor V nucleus is
placed more nearly on the level of its root entrance and, while it
always extends a considerable distance rostrad of this point,
the nucleus never extends caudad of the exit level of the motor
VII root (fig. 17). Among cyclostomes, however, the motor V
nucleus in Petromyzon closely corresponds to that of Polyodon
both in its position and its extent. Similarly in Myxine and
Bdellostoma, the motor V nucleus agrees with that of Polyodon
both in its rostro-caudal extent and in its relation to the exit
levels of the motor V and VII roots, though their resemblance is
somewhat obscured owing to the very ventral situation of the
nucleus in question and its continuity with motor VII column
in the former animals (figs. 7, 17 and 25).

Oculomotor and trochlear nuclei and roots (Nu. et rad. Nn. IIT
and [V. The oculomotor and trochlear nuclei are both small in
Polyodon, as in other ganoids. The oculomotor nucleus lies
upon the dorso-median aspect of the fasciculus longitudinalis
medialis in the Sylvian gray and is placed entirely rostrad of the
caudal border of its emergent root (fig. 25 A).

508 DAVIDSON BLACK

It is possible to distinguish two cell groups within the nucleus:
a dorsal group which is present throughout the nucleus, and a
group more ventrally situated which is present only in its caudal
portion. In figure 24, some cells of the latter group may be
seen lying ventrally, between the fasciculi longitudinales mediales
‘and below the small dorsal cell groups. The emergent rootlets
pass ventrad through the fine strands of the commissura
ansulata.

Tectum °

at ‘Ss SS Tas

Tectum

Pe eS \
\i
)

PZ] ~Telob.b.
fy

: N.IL.
\ ‘Nulll.vent.
Com.ans.

23 24

Fig. 23 Polyodon spathula. Transverse section of brain stem to illustrate
the relations of the trochlear nerve to the neighboring structures.

Fig. 24 Outlines of transverse section of brain stem at exit level of oculo-
motor root. Abbreviations: Coll., colliculus; Com.ans., commissura ansulata;
N.III., emergent bundles of oculomotor nerve; N.JV., trochlear nerve;
Nu.II1.dors., dorsal moiety of oculomotor nucleus; Nu.J1/.vent., ventral moiety
of the oculomotor nucleus; Tr.lob.b., tractus lobo-bulbaris; 7'’r.lob.cb., tractus
lobo-cerebellaris. Other abbreviations as before.

In both Amia and Acipenser, dorsal and ventral cell groups
may be distinguished in the oculomotor nucleus, though in the
young Lepidosteus specimens described by Theunissen this con-
dition was not apparent. Among these forms the oculomotor
complex occupies the most rostral position in Amia, where the
nucleus lies almost wholly in front of the rostral border of its
emergent root. In Acipenser the nucleus is somewhat more
caudally placed while in both Polyodon and Lepidosteus the

MOTOR NUCLEI IN PHYLOGENY 509

caudal end of the nucleus corresponds to the level of the caudal
border of the oculomotor root (fig. 25). In Polyodon the sub-
division of the elements of the small oculomotor complex into
dorsal and ventral cell groups is unmistakable and presents an
interesting contrast to the conditions obtaining in the large
undifferentiated oculomotor nuclei in Selache.

The trochlear nucleus in Polyodon is placed some distance
caudal to the oculomotor nucleus. Its relation to the fasciculus
longitudinalis medialis is essentially similar to that of the dorsal
cell group of the oculomotor complex to the same structure.
From the trochlear nucleus a very fine strand of fibers arises.
In its caudal course the root passes dorso-laterally through the
ventricular gray into the valvula. Immediately beneath the
floor of the crevice between the valvula and the cerebellum, the
nerves of either side decussate and emerge from the dorsal
surface of the brain in the mid-line. The peculiar transverse
band which the two trochlear roots form at their decussation
between the valvula and the cerebellum, is shown in figure 22.
Garman (I. ¢c.) has drawn attention to the similarity of the
trochlear exit in Acipenser and Polyodon. The nerve roots
course laterad after their exit and then pass rostrad between the
auricular lobes of the cerebellum and the tectum (fig. 23).

In Polyodon, Acipenser and Lepidosteus the position of the
trochlear nucleus with reference to its root exit and that of the
oculomotor nerve is almost identical. In Amia, however, in
correspondence with the more rostral position of the oculomotor
nucleus, that of the trochlear nerve is placed nearer the level
of the caudal border of the oculomotor root than in other
ganoids (fig. 25).

Discussion

Spino-occipital complex. In ganoids there are no epibranchial
spinal muscles as in sharks (97) and the pre-hyal hypobranchial
elements are represented by the musculus branchio-mandibu-
laris. Lepidosteus, however, would appear to be an exception
to the latter part of this statement for according to Edgeworth
(20) there is no trace of the m. branchio-mandibularis (m. genio-

510 DAVIDSON BLACK

ip
N
yy
N fe
N
S
N
N
N

LILLE LLL LLL LLL

0
OLD

.o.
+

>

2,

i

eee o28 os ©
e76e@8eedcns

Fig. 25 Reconstruction charts of motor roots and nuclei. VY. Polyodon,
B. Acipenser (after Theunissen, 91), C. Lepidosteus (after Theunissen, 91),
D. Amia (after Kappers, 66). Signs and abbreviations as before (vide p. 476).

MOTOR NUCLEI IN PHYLOGENY ona

hyoideus) to be found even in the embryo of this form. In
Amia according to Allis (1) this muscle, which is subject to great
variation in different individuals, ‘‘undoubtedly represents a
muscle in the process of deterioration and disappearance.”’
This author demonstrated that the nerve supply of the branchio-
mandibularis is derived from a terminal twig of the fused ven-
tral branches of the first, second and third occipital nerves
(equivalent to Furbringer’s nerves z, a and b). MeMurrich (83),
though unable to trace the innervation of this muscle in Amia,
pointed out that it was the probable forerunner of the muscles
of the tongue of higher forms, and that, as no trace of the
branchio-mandibularis is to be found in teleosts, Amia was
probably the last piscine form to possess it. This muscle was
found in Acipenser by Vetter (95), in Polypterus by Pollard
(86) and Edgeworth (1. c.) and in Polyodon by Danforth (16).
In the latter animal the branchio-mandibularis is a very small
muscle whose innervation was not actually determined but whose
relations, when compared with Amia, left no doubt as to its
complete homology with the similarly named muscle in that
form.

In correspondence with the evident reduction of these periph-
eral elements in ganoids as compared with sharks, a reduction in
the number of spino-occipital nerves has occurred in the former
eroup. The most rostral of these nerves found in the ganoids is
Furbringer’s occipital nerve x which occurs in individual cases
in Acipenser alone, while the occipital nerve z is the only one of
this sub-group of nerves which is characteristically retained in
all ganoids.

Within the central nervous system, one expression of these
peripheral changes 1s to be seen in the oblique caudal course which
the emergent spino-occipital rootlets take from their nucleus to
the periphery—as if the somatic motor column were pushed
rostrad, dragging with it the remaining motor rootlets. Acipen-
ser alone proves an exception to this rule and according to Fur-
bringer it is just this form among ganoids that shows the least
reduction in the number of occipital nerves. It is also signifi-
cant to note in this connection that alone among ganoids, coraco-

512 DAVIDSON BLACK

branchiales muscles (which, as is sharks, are innervated by
spino-occipital nerves), are developed from. all the branchial
myotomes in Acipenser (Edgeworth, Vetter, Furbringer).

That a rostral migration of the spino-occipital nucleus should
occur pari passu with the reduction and absorption into the
head of occipital elements, is rather to be expected and further
evidence of such a displacement of this nucleus is not lacking.
Thus, if the distance between the first emergent spino-occipital
root and the emergent motor IX nerve be measured in sharks
and ganoids (figs. 17 and 25), a comparison will show this dis-
tance to be the same in Lepidosteus and Selache and almost the
same in Acipenser, Amia and Hexanchus, though a greater dis-
crepancy is evident in the case of Polyodon. In other words,
though certain occipital nerves are known to have been lost: in
ganoids, those that remain have been displaced rostrad.

Rostral displacement of the spino-occipital column must
have been preceded first by the loss of its peripheral motor ele-
ments (motor perikaryons), followed by the reduction and modi-
fication of the remaining coordination elements of the nucleus.
This is indicated in ganoids as in Bdellostoma by the difficulty
with which the rostral end of this nucleus is defined. The
column passes over gradually into the inferior reticular nucleus
and thus appears to extend a considerable distance further ros-
trad of the exit level of its first root than is really the case (vide
supra).

It should also be noted that the peripheral pre-hyal hypo-
branchial musculature, at least in Amia, shows evidence of con-
siderable individual variation, so that a condition may obtain
here somewhat analogous to that already pointed out in Bdellos-
toma, where the individual variation was so great that a ques-
tion arose as to whether the extent of the somatic motor column
in the reconstruction chart in figure 7 could be taken as repre-
sentative of the species.

Aside from this difficulty of delimitation, the rostral end of
the somatic motor column in ganoids shows unmistakable evi-
dence of specialization, so that it differs considerably from the
anterior horn in the cervical region. The rostral end of the

MOTOR NUCLEI IN PHYLOGENY aile

spino-occipital nucleus is situated dorsally upon the fasciculus
longitudinalis medialis, and, as in sharks, is lacking in ventral
elements. This position is probably due to the same influences
that were noted in the discussion of this region in Selache, viz.,
to the action of reflex impulses by way of the well developed
dorsal arcuate fibers and posterior longitudinal bundles and to
the comparatively slight development of ventrally situated reflex
pathways.

Allis (1. ce.) has suggested that ‘‘the tongue of the adult Amia
may, therefore represent a condition of that organ in which its
partial muscularization has been lost, rather than not yet ac-
quired, as Gegenbauer (28) states to be the case for fishes in
general.’”’ However, within the central nervous system, no
evidence has yet been forthcoming to indicate that the spino-
occipital nucleus in Polyodon or other ganoid was at any time
more specialized than is this complex in modern sharks. But
it is interesting to note that: in both sharks and ganoids the
nucleus which innervates the forerunner of the tongue muscu-
lature is placed, as in higher forms, in close proximity to the
visceral motor center innervating the musculature of the
foregut.

Nucleus paramedianus (Nu. paramed.). The varying develop-
ment of the nucleus paramedianus among ganoids is worthy of
note in connection with the question of the homology of this
nucleus and the inferior olive of mammals. In Amia, which
probably represents the nearest relative of the modern teleost
group, there is no well circumscribed nucleus paramedianus and,
since this nucleus does not appear as a definitely circumscribed
gray mass in teleosts, amphibians or reptiles, it is possible and
even probable that the nucleus in question was represented in
the ganoid stock merely by an undifferentiated reticular area.
Thus, the nucleus paramedianus of selachians would appear
to be a structure independently specialized within the group
after its divergence from an ancestral type common to sharks
and ganoid stock.

In Polyodon, the acusticum and lobus lineae lateralis are di-
rectly continuous with the cerebellum, with which they corre-

514 DAVIDSON BLACK

spond in structural detail, as Johnston (49) long ago pointed out
to be the case in Acipenser. The acusticum and lobus lneae
lateralis in Polyodon are relatively even larger than in Acipenser
and together with the cerebellum form by far the greater bulk
of the rhombencephalon. It is possible that the poorly circum-
scribed but recognizable nucleus paramedianus in Polyodon and
in other ganoids has been differentiated under the influence of
these structures and does not represent the more or less degen-
erated remains of a once more highly developed nucleus. By
this it is implied that the acusticum, lobus lineae lateralis and
cerebellum together in ganoids bear the same causal relation-
ship to the differentiation of the paramedian area as does the
cerebellum to this area within the groups in which the latter
organ has independently gained a high state of relative speciali-
zation, viz., sharks, birds, and mammals.

Motor vagus nucleus (Nu. mot. N. X.). The trapezius muscle
in ganoids has become much reduced as compared to selachians,
a condition which is probably due in a large measure to the
development of the operculum and consequent general modifi-
eation of the musculature in the branchial region.

In Acipenser the trapezius is represented in the 11 mm. em-
bryo (Edgeworth) but, according to Vetter, disappears in the
adult. In Amia, Lepidosteus, and Polypterus, Edgeworth de-
scribes the development of the trapezius. In the first named
form this muscle, which is described by Allis (1. ¢., p. 669) as
the ‘fifth externus’ levator of the branchial arches, is inner-
vated by a branch of the vagus. In Polyodon also the trapezius is
present though not a large muscle, and is innervated by a long
branch of the vagus (Danforth).

It is to be expected from what has been said in the case of
selachians that a reduction of the trapezius musculature such as
obtains in ganoids would lead to some curtailment in the caudal
extent of the vagus nucleus. Judging from a comparison of the
extent of the overlap of the caudal viscero-motor column and
spino-occipital nucleus in the selachians and ganoids charted
(figs. 17 and 25), some shortening of the vagus nucleus in the
latter forms is apparent. However, as a greater overlap of these

MOTOR NUCLEI IN PHYLOGENY ies)

two nuclei obtains in the selachians charted in figure 17 than in
many other sharks hitherto examined (vide 66, 72, etc.) and since
it is known that ganoids have lost certain occipital nerves which
are characteristically present on sharks, but little if any reli-
ance can be placed upon the results of such a direct comparison
as that suggested above.

A comparison of this region among ganoids themselves (fig.
25) brings out the fact that the extent of the vagus column
caudad of the exit level of the first spino-occipital rootlet, as
well as the extent of overlap of the caudal vagus column and
spino-occipital nucleus, is greatest in Acipenser and least in
Polyodon, while in these respects Amia and Lepidosteus occupy
an intermediate position in the series. An examination of the
relative development of the musculature to which the vagus is
distributed shows that these central relations closely agree
with the peripheral conditions obtaining in these forms (ef.
discussion of spino-occipital complex).

Thus, though the trapezius be absent in Acipenser, the levatores
arcuum branchialium externi which are closely related to this
muscle ontogenetically, are more numerous in this form than in
Amja (MecMurrich, |. ¢.) and they are chiefly innervated from
the caudal vagus column. Further, in Amia (Allis) and in
Lepidosteus (Edgeworth) in both of which the trapezius is a
very small muscle, the only coraco-branchialis present in these
forms (pharyngo-clavicularis externus) receives its innervation
from the caudal part of the vagus (cf. supra, innervation of coraco-
branchiales in Acipenser). Finally in Polyodon, where a re-
duction of the caudal part of the vagus nucleus appears to be
most evident among ganoids, the pharyngo-clavicularis is inner-
vated not by the vagus but by the first spinal (Danforth), the
trapezius though present is small, and the levators of the bran-
chial arches are not so extensive or so numerous as in Acipenser.

°If the so-called pharyngo-claviculares muscles of Polyodon are homolo-
gous with the coraco-branchiales muscles of Acipenser, as their respective
nerve supply would suggest, then in this respect both these forms resemble
selachians, while in Amia and Lepidosteus, on the other hand, the pharyngo-

claviculares muscles receive their innervation in a manner similar to that
obtaining among most teleosts.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27; No. 4

516 DAVIDSON BLACK

From the above it becomes evident that, though the caudal
portion of the motor vagus nucleus is well formed in ganoids,
yet in these animals the elements homologous with those form-
ing the bulk of the accessory nucleus of higher forms must neces-
sarily be few. It is also evident that, apart from the question
of the homology of the muscles whose innervation has been noted
with similarly named muscles in other forms (Herrick, 34) the
variations in peripheral motor distribution in this region within
the ganoid group appear to be faithfully reflected in the arrange-
ment of the central motor nuclei.

Motor facial and glossopharyngeal nuclei and roots (Nu. et Bae
mot. Nn. VII-IX). The arrangement of the motor nuclei form-
ing the rostral part of the caudal viscero-motor column in gan-
oids is, inter se, typically selachian, consisting as it does of a nu-
clear mass from which arise the motor facial and glossopharyngeal
roots and which is continuous caudally with the motor vagus
nucleus. There is, however, a significant difference to be ob-
served in the relations to neighbouring structures of this part
of the caudal viscero-motor column as a whole in the two
groups.

In every case among ganoids the chief bulk of the elements
forming the motor facial nucleus, lies rostrad of the exit level
of the motor IX root. In selachians, on the other hand (at
least among the more primitive members of the group) the reverse
is true and the motor VII nucleus lies chiefly caudad of the exit
level of the motor LX root.

The different position of the motor VII nucleus with refer-
ence to the exit level of the motor glossopharyngeus in these
two groups might of course be due to a shifting of the motor
IX root. However, the relation of the nucleus in question to
that of the abducens nerve and to its root in both ganoids and
sharks would tend to negative such a supposition.

It has been pointed out already that the position of the
abducens nucleus with reference to the exit level of the motor
glossopharyngeus, is practically the same in both ganoids and
sharks, so that for purposes of comparison here it is reasonable
to consider this nucleus as a more or less fixed point.

MOTOR NUCLEI IN PHYLOGENY 517

The entire motor VII nucleus in all sharks lies characteristi-
cally some distance caudad of the abducens nucleus and the
rostral end of the motor facial nucleus is placed either caudal
to or at the level of the last emergent abducens root. On the
other hand, in all ganoids the rostral end of the motor VII
nucleus overlaps the abducens nucleus and its emergent root-
lets for a considerable distance (figs. 17 and 25). It thus be-
comes apparent that the motor VII nucleus of ganoids lies on a
more rostral level in the medulla than does this nucleus in
sharks. In this respect ganoids resemble teleosts (vide infra).

In the discussion of this region in selachians, the important
part played in the formation of the ascending motor root of the
facial and glossopharyngeal nerves by the development in these
animals of a caudally situated communis nucleus, has already
been indicated. In Polyodon the communis system seems not
to be so highly developed as in sharks but to be overshadowed
in relative importance by the large and extensive acusticum and
lateral line area (figs. 11 and 13 with 19 and 20). As a some-
what similar condition obtains in Lepidosteus and Acipenser,
this may account in part at least for the less caudal position of
the motor VII nucleus in these forms than in sharks. This ques-
tion is considered further in the discussion of the motor V nucleus
(vide infra).

In this connection it may be noted that the structure of the
brain stem of Polyodon furnishes no evidence indicative of a
gustatory function for the ‘primitive pore’ elements which are so
numerous on the head and bill of this animal (Kistler, 76; Col-
linge, 13). On the other hand, the highly specialized and
extensive acusticum and lobus lineae lateralis in Polyodon fur-
nish strong presumptive evidence in favor of these peripheral
organs being of the functional nature of neuromasts. As Wright
(102) originally employed this term to distinguish pear-shaped
hair cells from rod-shaped taste cells, and since the sense-cells
of Kistler’s figures strongly resemble the latter elements, the
term neuromast is here used in a strictly functional sense, i.e.,
to indicate organs centrally related by afferent nerves to the
special somatic area of the rhombencephalon.

518 DAVIDSON BLACK

Abducens nucleus,and roots (Nu. et rad. N. VI). The position
of the poorly defined abducens nucleus in all ganoids midway
between the exit levels of the motor VII and IX roots, is deter-
mined in all probability by the termination of the acoustic nerve
at this level. Kappers (64) has already pointed out the im-
portance of this factor as well as that of the dorsal arcuate
fibers of both the crossed and direct octavo-motor tracts in
determining the position of the abducens nucleus in sharks. The
nucleus in Polyodon is small in correspondence with the slight
functional development of the eye and lies somewhat ventrally
in the tegmentum between the tractus tecto-bulbaris and the
fasciculus longitudinalis medialis and is traversed by the arcuate
fibers of the tractus octavo-motorius.

In all ganoids the abducens is placed more ventrally in the
tegmentum than in sharks and appears to be characteristically
more closely associated with the tractus tecto-bulbaris in the
former animals. This apparently would indicate that the tractus
tecto-bulbaris was more highly developed in ganoid ancestors
than in the modern representatives of the group, for otherwise
it is difficult to account for this ventral position of the abducens
nucleus in the presence of such evidently well developed dorsal
acustico-motor tracts.

Such a conclusion is strengthened by the relations obtaining
in the oculomotor and trochlear nuclei in Polyodon to be noted
subsequently and also by the findings of Danforth (1. c.) in his
dissections of the eye-muscles in this form. According to this
author, considerable variation was met with in connection with
these muscles and in one case he records the complete suppres-
sion of the external rectus on the left side as evidence of a retro-
grade tendency in this area. Further, Allis (Il. ¢.) regards the
type of arrangement of the eye-muscles in ganoids as more
specialized than in selachians, and, though this generalization
may be subject to some question, yet the fact remains that,
like Polyodon, in Amia the external rectus muscle also varies in
its development.

Motor trigeminal nucleus and root (Nu. et rad. mot. N. V). In
all ganoids the motor V nucleus occupies a dorsal position in the

MOTOR NUCLEI IN PHYLOGENY 519

ventricular gray and is placed wholly caudad of the rostral
border of its emergent root. In its dorsal position this nucleus
resembles that of sharks, but in its extent caudad of its emergent
root the nucleus recalls the condition obtaining in cyclostomes
and, in a manner of speaking, it foreshadows the more com-
plicated relations which are characteristic of the motor V nucleus
among teleosts.

It is an unfortunate fact that the mechanism of branchial
respiration has not been investigated in ganoids, although the
air-breathing propensities of these animals have been described
and recorded by many observers.'° However, a study of the
arrangement of the musculature of the branchial area among
ganoids and teleosts makes it evident that, though an operculum
is characteristically developed in both groups, yet in the rela-
tive specialization of this apparatus as a respiratory organ, the
two groups present important differences.

Among most teleosts the operculum acts in conjunction with
the maxillary, mandibular and branchiostegal valves as a highly
efficient pumping mechanism which maintains the flow of water
from the oral cavity outwards over the gills through the oper-
cular cleft. In consequence of this, the intrinsic branchial
musculature among teleosts is relatively poorly developed, while
the respiratory action of the m. adductor mandibularis (closure
of the mouth during expiration) is largely abolished (vide infra).

In ganoids, on the other hand, though rudiments of the maxil-
lary and mandibular valves may be represented (Allis, 2), yet
their efficiency as such is negligible, so that during expiration the
mouth must be closed to direct the respiratory flow backwards
over the gills.

10 The only observations on the respiratory movements of the.gills in gan-
oids that I have been able to find in the literature are those of Kouliabko (77).
This author carried out a series of experiments upon the isolated heads of various
fish, among which were specimens of Acipenser ruthenus and another closely
related form which is referred to as the ‘costeur’ and probably was the great
Russian sturgeon, Acipenser husio. No exact study of the mechanism of bran-
chial respiration was made, but Kouliabko brought to light the interesting fact
that ganoids are more tolerant of CO» than are teleosts. The interest of this

observation is increased in view of Baglioni’s finding that selachians resemble
ganoids in this respect (7).

520 DAVIDSON BLACK *

Further, in Acipenser, Polyodon and Amia the expiratory
branchial muscles are relatively more highly developed as com-
pared with the expiratory opercular muscles than is the case
among teleosts. Also, the m. adductor hyomandibularis (ex-
piratory opercular muscle) in Acipenser, Polyodon, and Amia
is less highly differentiated than among teleosts (Vetter, 95;
Danforth, 16; MeMurrich, 83).

Thus on purely anatomical grounds, it would appear that the
respiratory cycle in ganoids must present a curious admixture of
selachian and teJeostean characters. In inspiration these char-
acters are chiefly teleostean, viz., contraction of the m. sterno-
hyoideus and the opercular dilator muscle, together with the
valvular action of the free border of the operculum. In expira-
tion, on the other hand, selachian characters predominate, viz.,
the action of the relatively well developed branchial levator and
adductor muscles in conjunction with that of the adductor
hyomandibularis, and the necessarily coordinate action of the
m. adductor mandibularis."!

Turning now to a consideration of the arrangement of the
motor nucleus in the light of these observations, it becomes evi-
dent that the caudal extent of the motor V nucleus, together
with the rostral position of the motor VII nucleus, brings the
two nuclei innervating the opercular (and jaw) musculature into
closer relation to one another than is the case among sharks
where ap operculum is lacking. In this respect these nuclei
present teleostean characters.”

On the other hand, the selachian arrangement of the motor
VITI-IX—X nuclei to form the caudal viscero-motor column ap-
pears to be directly correlated with the relative importance of
the intrinsic branchial muscles among ganoids and with the es-

11 The closure of the mouth during expiration is much more important in
ganoids than in sharks owing to the teleostean arrangement of the branchial
lamellae in the former animals.

Tt is of interest in the present connection to refer to the peculiar arrange-
ment of the motor nuclei in Chimaera (Kappers, 66, fig. 20). In this animal, in
which the operculum is also developed, the isolated rostral portion of the motor
VII nucleus charted by Kappers bears a relation to the caudal motor V ele-
ments in all essentials identical with that obtaining in ganoids.

MOTOR NUCLEI IN PHYLOGENY 521

sentially selachian character of the communis area in these
forms. Further, the simplicity of the arrangement of the
elements composing the caudal viscero-motor column is in itself
an indication of the absence of any great specialization of the
derivatives of the hyo-branchial myomeres in ganoids.

Oculomotor and trochlearis nuclei and roots (Nu. et rad. N. III
and IV). The small size of the oculomotor and trochlear nuclei
and their roots in all ganoids is indicative of the relatively slight
functional importance of the eye in these forms as compared with
sharks. In discussing the abducens nerve, mention was made
of the evidence furnished by the structure of the oculomotor
and trochlear nuclei in support of the view that ganoid ancestors
were possessed of a more highly developed optic apparatus than
the modern representatives of this group. This evidence rests
upon the following facts:—Though these nuclei and their roots
are both relatively and absolutely smaller than in sharks, yet
the oculomotor nucleus presents distinct signs of specialization,
so that within it may be distinguished two secondary nuclei
such as elsewhere are found among fish only in teleosts. Fur-
ther, the definite gap which exists between the oculomotor and
trochlear nuclei in all ganoids, is a condition which also obtains
elsewhere among fishes only in certain teleosts (e.g., Rhombus,
Hippoglossus, ete.) in which the visual apparatus is highly
developed.

In conclusion, it may again be emphasized that the arrange-
ment of the motor nuclei in Polyodon furnishes additional evi-
dence of the importance of neurobiotactic influences in the de-
termination of nuclear pattern. The predominantly dorsal
position occupied by the motor nuclei in this form and in other
ganoids is in correspondence with the relative importance of the
dorsal reflex pathways (posterior longitudinal bundle, octavo-
motor tracts, ete.). In this respect ganoids resemble sharks.
The more ventral position of the abducens nucleus in ganoids
than in sharks is explained on the hypothesis that the tecto-
bulbar paths were functionally more highly developed in the
ganoid stock than in the modern representatives of the group.
This hypothesis is borne out by the evidence furnished by the

522 DAVIDSON BLACK

relations obtaining in the oculomotor and trochlear nuclei. The
rostral position of the motor VII nucleus, together with the
caudal extent of the motor V nucleus, point to the probability
that these characters are dependent upon the development of a
modified type of opercular respiration and the consequent more
close association of the motor nuclei upon whose coordinate
action certain respiratory movements depend.

Finally, it would appear that further evidence for the grouping
of modern ganoids together and apart from teleosts is furnished
by the close correspondence of the motor nuclear pattern in all
ganoids examined and by the characteristic differences of this
pattern on the one hand from that of sharks and on the other
from that of teleosts.

TELEOSTEI
Motor nucler in Ameiurus nebulosus and Solea vulgaris

Among teleosts, two forms whose life habits present a very
marked contrast, have been selected for study: the flat-fish Solea
vulgaris and the siluroid Ameiurus nebulosus.

Occipito-spinal nuclei and roots (Nu. et rad. mot Nn. occ. spin.).
In Solea and also in Ameiurus the rostral end of the somatic
motor column of the cord is continued for some distance unin-
terruptedly into the medulla, and there forms the nucleus of
origin of certain precervical motor rootlets.

The general term ‘spino-occipital’ has been made use of in
describing this region in selachians and ganoids to distinguish
the rostral portion of the somatic motor column of the trunk and
its associated motor rootlets. Since the reduction of the spino-
occipital elements among teleosts is evidently more extensive
than is the case in lower forms (e.g., selachians and ganoids) as
well as in many higher forms (e.g., amphibians), it seems desira-
ble to indicate this fact by the use of some term other than
spino-occipital in describing this region in teleosts, especially as
the arrangement of the elements in the rostral end of the somatic
motor column among Teleostei presents certain characteristics
distinctive of the group. For this reason in the present de-

MOTOR NUCLEI IN PHYLOGENY a5)

scription, Furbringer’s term ‘occipito-spinal’ has been used in
its broadest sense.'’

In teleosts, unlike selachians, the rostral termination of the
occipito-spinal motor column, is rounded and blunt, so that in
transverse section the distribution of the motor cells in this
nucleus closely resembles the arrangement obtaining in the
anterior horn of the cord (figs. 26, 29, 40 A and 41 A).

Gals, - ,Lobwvag.
Re i aa

OE gs St

SoS 3 a Nu.Xm.
rr y : 2 - 4

aS QS se

Nu.occ.sp.m. dors.. 59, *38

Nu.occ.sp.m.vent.--*e» 2+
Soe a |
3G

__.-Fib.arc.dors.

Fig. 26 Solea vulgaris. Transverse section of brain stem just caudad of
the calamus.

Fig. 27. Transverse section of brain stem somewhat rostrad of the preceding
at the exit level of a motor vagus rootlet. Abbreviations: C.c., crista cerebel-
laris; Fib.arc.dors., dorsal arcuate fibers; Fib.arc.int., internal arcuate fibers
from funicular area, vagal lobes and motor vagal nuclei; F.d.l., fasciculus dorso-
lateralis; F.I.l., fasciculus longitudinalis lateralis; F.l.m., fasciculus longitu-
dinalis medialis; Lob.vag., visceral sensory column; Nu.fun., nucleus of funicular
area; Nu.occ.sp.m.dors., dorsal moiety of motor occipito-spinal nucleus;
Nu.oce.sp.m.vent., ventral moiety of motor occipito-spinal nucleus; Nu.X.m.,
motor vagal nucleus; R.desc.V., tractus spinalis trigemini; R.X., vagus root;
R.X.m., motor vagus rootlet; T'r.tc.b.cruc., tractus tecto-bulbaris cruciatus;
Tr.tc.b.rec., tractus tecto-bulbaris rectus; Tr.sec.com., secondary gustatory
bundle; Tub.ac., tuberculum acusticum.

13 In his earlier works Dr. Kappers has used the term spino-occipital to de-
scribe this region in teleosts, but the present change in usage has been under-
taken on the advice of this author. It is to be recalled that Furbringer applied
the term ‘occipito-spinal’ to those nerves (viz., a, b, and c) which had only in-
completely lost their spinal character and which issued from the cranio-spinal
canal rostral to the first true spinal (Furbringer’s spinal nerve 4) and caudal
to the protometameric portion of the cranium.

524 DAVIDSON BLACK

In Solea, the fibers of the first motor rootlet which arises from
the occipito-spinal nucleus, pass obliquely caudad and ventrad
and become collected upon the periphery to form a small bundle
which courses some sections caudad in this location before leav-
ing the brain. A similar arrangement obtains in Ameiurus and
in other teleosts (cf. Van der Sprenkel, 1. c., fig. 4).

The caudal viscero-motor column (Col. visc. mot. caud.). The
caudal viscero-motor column in Solea, unlike that of selachians

\ 4
Nu.occ.sp.m.

28

Fig. 28 Ameiurus nebulosus. Transverse section of brain stem at the exit
level of the second occipito-spinal rootlet.

Fig. 29 Transverse section of brain stem somewhat rostrad of the preceding
at the exit level of motor vagus rootlets. Abbreviations: Nu.f.l., lateral funicu-
lar nucleus; Nw.occ.sp.m., motor occipito-spinal nucleus; F.occ.sp.m., second
motor occipito-spinal rootlet; R.X.8., sensory root bundle of the vagus. Other
abbreviations as in figures 26 and 27.

and ganoids, is divided into two distinet portions:—a rostral
part formed by the rostral motor VII nucleus and a long caudal
cell column comprising the motor nuclei of the vagus and
glossopharyngeus together with the caudal motor VII nucleus.
A similar subdivision of the caudal viscero-motor column occurs
in Ameiurus, but with this characteristic difference, that the
rostral subdivision contains all the cells of origin of the motor
facial root, while the caudal subdivision comprises only the
motor vagus and glossopharyngeus nuclei.

MOTOR NUCLEI IN PHYLOGENY SPAR

As in ganoids, the vagus column in teleosts extends caudally a
considerable distance beyond the rostral end of the occipito-
spinal motor column, and lies dorsal to this nucleus (figs. 26, 29
and 18).

It will be seen from the reconstruction charts in figure 40 that
the extent of this overlap may be variable even in closely allied
forms. In Solea, the motor X nucleus ends rostrad of the exit
level of the first occipito-spinal rootlet, while in Hippoglossus, it
extends a considerable distance caudad of the corresponding
point. It is to be noted, however, that there is a much smaller
amount of variation to be observed when the total length of the
VII-IX—X column in these forms is compared. A similar con-
dition is to be seen jn a comparison of the reconstruction charts
of the siluroids Ameiurus and Silurus, though of course in this
case it is the IX—X columns that are to be compared one with
the other (figs. 41 A and 42 A).

In Solea, the position of the motor X nucleus in transverse
section is illustrated in figures 26 and 27. The motor rootlets
of the vagus pass laterally, and emerge ventral to the descending
trigeminal root and below their corresponding sensory root. In
Amelurus, the relations of the motor X rootlets and of their
nucleus of origin are illustrated in figures 28 and 29.

It is of interest at this point to compare Bartelmez’ reconstruc-
tion of the motor nuclei in Ameiurus melas (9, fig. 2) with my
own chart of these relations in Ameiurus nebulosus (fig. 41 A).
The only essential difference in the sagittal relations recorded
in the reconstructions, lies in the position of the caudal end
of the vagus nucleus with reference to the occipito-spinal motor
column. In the specimen of Ameiurus nebulosus from which
figure 41 A was prepared, the overlap of the columns in question
was considerable; but as no such overlap is indicated by Bartel-
mez in Ameturus melas, it is probable that there is a certain
amount of ‘specific’ variation in the extent of this overlap—
just as there is a considerable ‘generic’ variation in this rela-
tion among closely allied forms (vide supra Pleuronectidae).
If, however, the total length of the IX—X nucleus in A. melas and
A. nebulosus be measured and then divided into the distance

526 DAVIDSON BLACK

between the caudal end of this nucleus and the rostral end of the
motor V nucleus, the quotient is expressed by 1.8 in both re-
constructions. Thus, the relative length of the nucleus in ques-
tion in the two species (Ameiurus nebulosus and Ameiurus
melas) is exactly the same. It is probable that the nucleus
which Bartelmez terms “‘nucleus motorius nervi cervicalis I’
corresponds to the nucleus of origin of the first occipito-spinal
motor rootlet of my description and not to the motor nucleus
of the first true cervical nerve of this form.

Motor glossopharyngeal nucleus and root (Nu. et rad. mot. N.
IX). In Solea, the motor glossopharyngeal root, in passing
from its nucleus to its point of exit, takes the peculiar round-
about course that appears to be characteristic of this nerve in
teleosts. The motor IX root arises from cells situated in the
rostra] part of the VII-IX—X column and passes rostrad for a
considerable distance alongside the most dorsal bundle of the
fasciculus longitudinalis medialis. At the level of the caudal
end of the rostral motor VII nucleus, the fibers of the LX nerve
pass through this nucleus laterad and ventrad, but do not re-
ceive any fibers from it. The root then courses caudad and
laterad to emerge, ventral to the descending trigeminal tract,
at the level indicated in the chart, figure 40 A. This level
is somewhat rostrad of the entrance of the sensory radix of
glossopharyngeus.

The course of the motor LX root in Ameiurus closely corvre-
sponds to that obtaining in Silurus (Van der Sprenkel, |. c., fig. 4)
and differs in no essential way from the course of this root in
Solea. It would appear, however, that among siluroids the
‘genu’ of the motor [X root, tends to enclose within its concavity
a considerable part of the whole motor VII nucleus, while among
the pleuronectids, a relatively smal] part an of the rostral motor
VII nucleus lies in this relation.

This peculiar geniculate course of the motor LX root has been
described also by Mayser in certain cyprinoids (79) by C. J.
Herrick in Menidia (34) and in Hippoglossus, Rhombus, Pleuro-
nectes and Tinca by Kappers, who recently has restudied the

MOTOR NUCLEI IN PHYLOGENY a2%

question of the course of the motor glossopharyngeus and its
relation to the motor VII nucleus in these forms (72).

Motor facialis nucleus and root (Nu. et rad. mot. N. VII). In
Solea, the rostral motor VII nucleus is placed for the most part in
the ventricular gray, Jateral to the most dorsal fibers of the

NuVil.m.(1)

¢
Nr ee
. ae. ¢ R.VIlm.asc
re AAS m. <5
Sa a, ‘

SS
, R.desc.

AT r.tc.b.cruc.

\Tr.tc.b.rec.

30

Fig. 30 Solea vulgaris. Transverse section of brain stem to illustrate the
relations of the ascending motor glossopharyngeal root.

Fig. 31 Solea vulgaris. Transverse section of brain stem at the exit level
of the motor facial root. Abbreviations: Cb., cerebellum; Com.ac., decussating |
fibers of acusticum; Nuw.ret., nucleus reticularis; Nu.VII.m.(1)., rostral moiety
of motor facial nucleus; Nu.VJ., abducens nucleus; R.V/J., abducens rootlet;
R.VII.m., emergent motor facial root; R.VII.m.asc., ascending motor facial
root; R.VII.s., descending sensory facial root; R.IX.m., emergent fibers of motor
glossopharyngeus passing laterad through motor facial’ nucleus; R.JX.m.asc.,
ascending motor glossopharyngeus root; VJJIJ., lateralis nerve below which the
sensory facial root is seen. Other abbreviations as before.

fasciculus Jongitudinalis medialis, and resting upon the bundles
of the fasciculus longitudinalis lateralis (figs. 30 and 40 A). The
size of the nucleus increases somewhat towards its caudal end,
where many of the motor cells together with their larger den-
drites lie in between the bundles of the fasciculus longitudinalis
lateralis, and thus occupy a more ventral position than those

528 ; DAVIDSON BLACK

at the rostral end of the nucleus. The nucleus is traversed by
many thick bundles of the dorsal arcuate fibers.

The motor VII fibers which arise from cells in the rostral end
of the VII-IX—X column (caudal motor VII nucleus) pass up-
wards through the medulla as a small compact bundle, situated
just above the most dorsal fibers of the fasciculus longitudinalis
medialis (fig. 30). At the level of the rostral motor VII nucleus,
fibers from this center join the ascending root. In this way at the
level of the upper border of the rostral motor VII nucleus, a
large compact bundle is formed which derives its fibers from
both rostral and caudal motor VII nuclei. The VII motor
root then passes rostrad for some distance and finally curves
outwards over the radix descendens V and reaches the lateral
surface of the medulla some sections rostrad of the sensory VII
root (fig. 31).

In Ameiurus the motor VII nucleus consists of a large mass of
cells situated in the tegmentum below the acoustic commissure,
resting medially upon the fasciculus longitudinalis lateralis and
laterally upon the reticular substance bordering the ascending
secondary gustatory tract of Herrick. These relations are
brought out in figure 32 and are essentially similar to those
obtaining in Siluris glanis. A caudal motor VII nucleus, in
continuity with the [IX—X motor column, is not present in either
Silurus or Ameriurus, so that in these forms all the motor VII
fibers arise in a nucleus corresponding to the rostral motor VII
nucleus of Solea (fig. 41 A). ;

The motor VII fibers collect to form a large compact bundle
which arches dorsally over the massive acoustic commissure and
courses rostrad in the ventricular gray. At the level of the
caudal portion of the motor V nucleus, the root curves laterad
through the bundles of dorsal arcuate fibers and pierces the sec-
ondary gustatory tract to gain the lateral periphery of the
bulb (fig. 33).

Abducens nucleus and roots (Nu. et rad. N. VI). In Solea, in
the ventral tegmentum, at the exit level of the motor VII root, a
poorly circumscribed nucleus is found, from which arises the
most rostral of the two abducens rootlets (fig. 31). The emer-

MOTOR NUCLEI IN PHYLOGENY 529

“— A
Pa >

ence. Seti
/ Lob.fac \., \

Nibvil.®

30

Fig. 32. Ameiurus nebulosus. Transverse section of brain stem at the level
of the caudal end of the motor facial nucleus.

Fig. 33 Ameiurus nebulosus. Transverse section of the brain stem at the exit
level of the motor facial root and rostral abducens rootlet. Compare with
Hexanchus (fig. 13), where an abducens rootlet emerges on the exit level of the
motor glossopharyngeus. Abbreviations: Lob.fac., facial lobe of visceral sensory
column; N.U.1.VII., nervus lateralis; Nw.intermed., nucleus intermedius;
Nu.V.m.(2)., caudo-ventral moiety of motor trigeminus nucleus; Nu.VII.m.,
motor facial nucleus; R.VI/.s., entering sensory facial root. Other abbrevia-
tions as before.

530 DAVIDSON BLACK

gent fibers from this nucleus pass ventrad and rostrad and make
their exit from the ventro-lateral surface of the medulla on a
level with the rostral border of the motor VII root. A second
abducens nucleus can be made out caudal to the first, and sepa-
rated from it by a few cell-free sections. The second abducens
rootlet, which arises from the latter nucleus, emerges three sec-
tions behind the caudal border of the motor VII root. The
presence in this region of numerous diffusely arranged reticular
elements, has made it difficult to draw a sharp line of demarca-
tion about the limits of the abducens nuclei, and for this reason
they are surrounded by dotted lines in figure 40 A.

The arrangement of the abducens nuclei in Ameiurus is essen-
tially similar to that in Solea, though the caudal abducens root
is not separated from the emerging motor IX root by so great a
distance in Ameiurus as in the former animal. These relations
will be evident on comparing figure 40 A and figure 41 A (also
figs. 31 and 33).

It is also of interest to note that the abducens nuclei in Bartel-
mez’ reconstruction of Ameiurus melas (1. ¢.) bear exactly the
same sagittal relations to the motor VII and V nuclei that they
do to these structures in Ameiurus nebulosus.

Motor trigeminal nucleus and root (Nu. et rad. mot. N. V). In
Solea the motor V root arises from two closely associated cell
groups, which from their general relations may be termed re-
spectively dorsal and ventral. The dorsal motor V nucleus occu-
pies a position in the ventricular gray just dorsal to the fascicu-
lus longitudinalis lateralis, and extends in this situation, from a
short distance in front of the motor V root, to the level of its
caudal border (figs. 35 and 40 A). Numerous large dendrites
of the motor cells of this nucleus can be seen passing. ventrad
between the bundles of the fasciculus longitudinalis lateralis,
and occupying a position in the tegmentum analogous to that
occupied by the ventral nucleus itself at more caudal levels.

The ventral motor V nucleus begins a few sections caudad of
the dorsal nucleus, and extends to within a short distance of the
rostral border of the emergent motor VII root. In Solea, un-
like Hippoglossus, it is somewhat larger than the dorsal nucleus.

MOTOR NUCLEI IN PHYLOGENY 531

The nucleus intrudes its cells and their large dendrites between
the bundles of the fasciculus longitudinalis lateralis for some
distance into the tegmentum. ‘They pass in the direction of the
substantia reticularis grisea which lies below and medial to the
descending trigeminus root, and in the neighborhood of the

.

Grx22, ool rtc.b.cnie.

Fig. 34 Solea vulgaris. Transverse section of brain stem on the level of
the caudo-ventral moiety of the motor trigeminus nucleus.

Fig. 35 Solea vulgaris. Transverse section of brain stem on the level of
the rostro-dorsal moiety of the motor trigeminus nucleus. Abbreviations:
Nu.V.m.(1)., rostro-dorsal moiety of motor trigeminus nucleus; Nwu.V.m.(2).,
caudo-ventral moiety of motor trigeminus nucleus; R.V.m., emergent fibers of
motor trigeminus root; R.V.s., entering fibers of sensory trigeminus root. Other
abbreviations as before.

small secondary ascending gustatory tract (fig. 34). Like the
rostral motor VII nucleus, the most caudal cells of the ventral
motor V nucleus lie deepest in the tegmentum. The motor V
root passes laterad in a curved course and emerges from the
medulla dorsal to the sensory root as in Lophius (64).

In Ameiurus, the motor V root emerges in two bundles, as in
Silurus, though in the latter form the bundles are not so definitely

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 4

532 DAVIDSON BLACK

separated. The two bundles arise respectively from the rostral
and the caudal end of the motor V nucleus. The cells of this
nucleus are divided into two closely related groups, so that it is
possible to differentiate rostral and caudal motor V nuclei. A
similar arrangement of the elements of this nucleus obtains in
Amelurus melas (Bartelmez, |. c.).

Both rostral and caudal motor V nuclei are placed ventrally
in the tegmentum. The caudal nucleus extends over the longer

‘ ea ee ie
a "ar ? ,
a ANu Fetoe ues

- Rag
> Nuvret.

Ne

Fig. 36 Ameiurus nebulosus. Transverse section of brain stem on the
level of the emergent trigeminus root. Abbreviations: Nuw.sec.com., secondary
gustatory nucleus; Nu.V.m.(1)., rostral moiety of motor trigeminus nucleus;
R.V.m., emergent fibers of motor trigeminus root. Other abbreviations as

before.

distance sagittally, and is closely related along its dorso-lateral
surface to the secondary ascending gustatory tract (fig. 33).
The rostral nucleus is somewhat more dorsally placed, and its
relations in transverse section are indicated in figure 36. The
emerging motor V roots pass almost directly laterad to gain the
surface of the medulla, and in their course, they pass through the
secondary ascending gustatory tract.

Oculomotor and trochlear nuclei and roots (Nu. et rad. Nn. IIL
et IV). The nuclei of the oculomotor and trochlear nerves are

MOTOR NUCLEI IN PHYLOGENY 533

closely associated in Solea, so that the nucleus of the latter nerve
is separated by but a slight interval from the most dorsal part
of the oculomotor complex. The oculomotor nucleus is situ-
ated in the mesencephalic ventricular gray, and at its rostral
end is made up of a quite compact cell group, lying dorsal to the
fasciculus longitudinalis medialis. As this nucleus is_ traced
caudad, its dorsal part becomes enlarged, and in addition along
the raphé, an extensive ventro-median cell group appears,
whose elements, though separated into bilaterally symmetrical
groups, give rise apparently to fibers passing to both right and
left oculomotor nerves. The major portion of this nuclear com-
plex lies frontal to the rostral border of the emergent III nerve
but a small part of the ventro-median division of the nucleus
extends a short distance caudad of this level. The emergent
radicles converge in the raphé to form several stout bundles,
which in turn pierce the commissura ansulata in a ventro-lateral
direction to reach the surface of the midbrain (fig. 37).

The trochlear nucleus in Solea, is almost a caudal continuation
of the dorsal part of the oculomotor nucleus, there being but
two cell-free sections separating the two. The IV nucleus is a
compact cell group lying upon the fasciculus longitudinalis
medialis, and extending in this situation approximately over
the length of the superficial attachment of the oculomotor nerve
(fig. 40 A). Thus, in figure 37, it is possible to show the emerg-
ing radicles of N. III, together with the ventral part of nucleus
N. III and nucleus N. IV, all in one transverse section.

The course of the trochlear roots caudad to their points of
emergence is most complicated in Solea. The rootlets become
separated shortly after their origin into two distinct bundles,
one of which after bending around the tractus cerebello-mesen-
cephalicus, decussates with its fellow of the opposite side and
emerges through the valvula. The second rootlet passes rostrad
a short distance in the substance of the valvula, and, after de-
cussating with its fellow at this level, descends between the
valvula and the tectum, to join the rootlet first described at its
point of emergence from the brain.

534 DAVIDSON BLACK

The conditions which bring about this complicated course of
the IV root have been thoroughly investigated, independently by
V. Franz (23) and by Huet (1. c.).. The latter author has shown
that the division of the trochlear root into two bundles and their
subsequent decussation at different levels, only occurs among
those teleosts having a highly complicated valvula cerebelli

( im

Valvula jo 6
a) |
an J

Fig. 37 Solea vulgaris.- Transverse section of brain stem on the exit level
of the oculomotor root. Abbreviations: Coll., colliculus; Com.ans., commissura
ansulata; Fib.prof.tc., fibrae profundae tecti; N.JIJ., oculomotor nerve;
Nu.III.vent., ventral moiety of oculomotor nucleus; Nu.JV., nucleus troch-
learis; Tor.long., torus longitudinalis; Tr.mes.cb.s., tractus mesencephalo-
cerebellaris superior. Other abbreviations as before.

(e.g., Gadidae and Pleuronectidae). In selachians, where no
valvula is developed, in ganoids and in teleosts having a relatively
small and simple valvula (e.g., Lophius), no such complicated
arrangement of trochlear roots obtains. Thus, from the evidence
at his disposal, Huet concludes that the action of the valvula
cerebelli, in producing this split in the decussating trechlear
roots, is purely a mechanical one due to its growth and the
consequent folding of the part.

MOTOR NUCLEI IN PHYLOGENY 535

The oculomotor nucleus and root are both relatively and
absolutely smaller in Ameiurus than in Solea. It is possible,
however, to distinguish dorsal and ventral parts in the oculo-
motor nucleus of,the former animal, though there is but little
difference in the rostro-caudal extent of these subsidiary cell
groups, such as was so obvious in Solea. No definite decussation
of the fibers of the oculomotor nerves at their origin could be
made out in Ameiurus, and the whole nucleus lies on a more dor-
sal plane than in Solea. The emergent roots of the oculomotor
nerve pierce the commissura ansulata on their -way to the
ventral periphery of the midbrain (fig. 39).

The trochlear nucleus in Ameiurus is almost directly contin-
uous with that of the oculomotor nerve but lies on a somewhat
more dorsal plane. The emergent trochlear fibers pass dorsally
in the Sylvian gray (Franz, |. c.) to decussate and emerge through
the valvula on the same level as their nucleus (fig. 38). Though
the trochlear roots in Ameiurus have not been divided into two
parts as in Solea, yet they make their exit from the brain stem
at an unusually rostral level. A similar condition has been -
recorded by Van der Sprenkel in Silurus glanis, though in this
form on account of the very slight functional development of
the eyes, both oculomotor and trochlear nuclei were much
reduced (fig. 42 A). It would appear that the unusually rostral
place of exit of the trochlear roots in both Ameiurus and Silurus,
like the splitting of the rootlets in Solea has also been due in a
large measure to the mechanical action of the valvula and
cerebellum during the development of these parts.

Discussion

Occipito-spinal-complex. In teleosts no pre-hyal hypobranchial
spinal muscles are present and according to Furbringer the most
rostral spino-occipital nerves represented in these animals are
the occipito-spinal roots b and c. In Siluridae according to this
investigator both dorsal and ventral roots are represented in the
occipito-spinal nerve b, the nerve ¢ is missing, while caudal to
and including the first cervical nerve (Furbringer’s nerve 4)

536 . DAVIDSON BLACK

€e : eee
aay PF esta x fe

—

Fig. 38 Ameiurus nebulosus. Transverse section of brain stem on the level
of the trochlear nuclei and roots.

= NAy:
ta,
~

EN
PAA CaN SNE
ee

Fig. 39 Ameiurus nebulosus. Transverse section of brain stem on the
level of the oculomotor nuclei and roots. Abbreviations: Dec.dors., decussating
fibers of fasciculus longitudinalis medialis; Dec.vent., commissura ansulata;
Ginter pedunc., ganglion interpedunculare; Lob.inf., lobus inferior; Nu. III. dors.,
dorsal moiety of oculomotor nucleus. Other abbreviations as before.

037

PHYLOGENY

MOTOR NUCLEI IN

a
Cu
YO
oO
O
rom
“
oO
oO
fo)
x< ><
>

Fig. 40 Reconstruction charts of motor roots and nuclei. A. Solea, B.
Signs

Pleuronectis (after Kappers, 72), C. Hippoglossus (after Kappers, 72).

and abbreviations as before (page 476).

538 DAVIDSON BLACK

¢.

N
N
N
XK
\
\
N
N
\
N

¢,
6
1%

WOW LLL LLL LLL

=

Fig. 41 Reconstruction charts of motor roots and nuclei. A. Ameiurus, B.
Tinea (after Kappers, 72), C. Gadus (after Kappers, 72). Signs and abbrevia-
tions as before (page 476).

MOTOR NUCLEI IN PHYLOGENY 539

A. Silurus (en-

Fig. 42 Reconstruction charts of motor roots and nuclei.
larged from Van der Sprenkel, 92), B. Mormyrus (enlarged from Van der

The charts A and B have been

Sprenkel, 92), C. Lophius (after Kappers, 72).
Signs and

enlarged to the same scale as that used throughout this paper.

* abbreviations as before (page 476).

540 DAVIDSON BLACK

dorsal and ventral roots are regularly represented. In Pleuro-
nectidae the dorsal root of nerve b may be missing and both the
dorsal and. ventral roots of nerve ¢ are fused with nerve b as
they make their exit from the skull but the arrangement of the
nerves from this point caudad is similar to that obtaining among
siluroids.

The peripheral relations of the cervical nerves in Ameiurus
appear to conform closely with Furbringer’s generalized scheme
for the family outlined above. Thus, according to Wright
(1. ¢.), the first spinal nerve (occipito-spinal b) possesses both
dorsal and ventral roots, which unite however, and emerge by a
common foramen through the exoccipital bone (auximetameric
cranium). ‘The second spinal nerve of this author (Furbringer’s
nerve 4) is separated from the first by a considerable distance—
more than twice the distance intervenes between the exit levels
_of these two nerves than between those of the second and third.
This apparent hiatus in the series would seem to indicate the
disappearance of the nerve c. The dorsal and ventral roots of
the second nerve emerge through separate dural foramina, as do
those of the third nerve, but owing to the modification of the
anterior vertebrae in connection with the auditory organ, they
pass directly through the membranous wall of the neural canal.
The dorsal and ventral roots of the fourth and following cervical
nerves emerge through separate foramina in the arches of the
corresponding vertebrae.

Within the central nervous system also there are many evi-
dences that both reduction and rostral displacement have
occurred in the precervical somatic motor column in Ameiurus
as compared with more primitive forms. As these characteris-
tics are typical of all teleosts to a greater or less extent, a gen-
eral summary of the evidences furnished by the group as a
whole will be of value in the present connection.

Tvidences of reduction. 1) With the exception of Hippo-
glossus, in all teleosts examined the distance between the exit
level of the first precervical motor root and that of the motor
glossopharyngeus is greater than in selachians. 2) Except in the
Pleuronectidae, the distance between the exit level of the first

MOTOR NUCLEI IN PHYLOGENY 541

ptecervical motor root and that of the motor IX is usually
greater in teleosts than in ganoids and always greater in teleosts
than in certain ganoids (e.g. Lepidosteus). 3) In teleosts a
sharp line of demarcation between the motor occipito-spinal
nucleus and the coordinating elements of the nucleus motorius
tegmenti cannot be drawn. In this respect ganoids stand in an
intermediate position between selachians and teleosts.. 4) The
elements composing the nucleus motorius tegmenti are arranged
within the caudal reticular area on-.a plan almost exactly like
that of the rostral occipito-spinal motor nucleus—the only dif-
ference between the two lies in the absence of neurones of the
peripheral efferent type in the former nucleus, the coordination
neurones alone being represented there. 5) The occipito-spinal
motor nucleus of teleosts resembles the caudal portion of the
spino-occipital nucleus of selachians in the arrangement of its
constituent neurones.

Evidence of rostral migration. 1) Though it is known that
at least four of the most rostral spino-occipital nerves are not
represented in the Pleuronectidae, yet in Hippoglossus the
occipito-spinal’ nerve b is placed as near the exit level of the
motor IX nerve as the most rostral occipital nerve is in selachians,
(and even nearer than is the case in most of the latter animals).
2) In most Pleuronectidae the distance between the exit level
of the motor IX nerve and that of the first precervical motor
root is less than in Amia, Acipenser, or Polyodon. 3) The
somatic motor nucleus projects further rostrad of its first emer-
gent root in teleosts than is the case in either sharks or ganoids.
4) The most positive evidence of rostral migrations, however,
is to be seen among teleosts in the very oblique caudal] course
taken by the emergent occipito-spinal roots (especially those of
the nerve b)—as if the rostral displacement of their peripheral
attachments had not kept pace with that of their motor nucleus.

Thus in view of Kapper’s earlier work (1. c.) it may be said that,
in the absence of manifest mechanical influence, positive evi-
dence of nuclear displacement may safely be adduced when a
motor root takes a more or less indirect emergent course, and
that the direction of this displacement is indicated by the direc-

542 DAVIDSON BLACK

tion taken by the emergent fibers. On the other hand, rostral
or caudal nuclear migration and emergent root displacement
may keep pace so that the direct emergence of a motor root
from its nucleus does not altogether exclude the possibility of
nuclear displacement.

The presence of both dorsal and ventral cell groups in the
occipito-spina] motor column in teleosts in contrast to selachians
and ganoids has already been pointed out by Kappers (66 and
74). This author has shown that the more ventral situation of
certain of the motor e’ements of this nucleus in teleosts is pos-
sibly one of the direct expressions of the strong reflex influence
of the tractus octavo-motorius cruciatus which courses together
with the tecto-bulbar fibers along the ventro lateral periphery
of the bulb at this level (see also Wallenberg, 96).

In the occipito-spinal nucleus of teleosts the ventral cell group
differs in many histological details from the dorsal cells of this
nucleus (Kappers, 66). Some light is shed on the different
functions of these two cell groups when the peripheral distribu-
tion of the first two precervical motor roots in ganoids is
compared with that in teleosts.

In Polyodon, where the ventral cell group is lacking’ in the
most rostral portion of the precervical motor column, the first
somatic motor nerve of this series does not furnish branches to
any lateral (pectoral fin) muscles but is distributed solely to
ventral (hypobranchial) spinal musculature (Danforth). The
second nerve may also be entirely restricted in its motor dis-
tribution to ventral musculature in some individuals (Fur-
bringer). Indeed it is a general rule for ganoids that the first
spino-occipital nerve never participates in the formation of the
brachial plexus, though the second nerve usually does so. Simi-
larly in selachians the upper members of the motor spino-
occipital series take no part in the innervation of pectoral fin
musculature (brachial plexus)—and no ventral cell group is
present in the rostral end of the spino-occipital column in
these forms.

In Ameiurus (McMurrich and Wright, l. ¢.), however, the
first precervical somatic motor root is distributed not only to

MOTOR NUCLEI IN PHYLOGENY 543

ventral musculature (hypopectoralis) but also to lateral trunk
musculature (abductors and adductors of pectoral fin). In this
animal as in other teleosts a ventral cell group is present in the
rostral part and throughout the occipito-spinal nucleus.

In Menidia a similar condition obtains only in addition to
motor fibers to the ventral and lateral musculature the first
precervical nerve sends out motor fibers to the dorsal trunk
muscles of its own segment (Herrick, 1. c.).

The relations obtaining in Ameiurus and Menidia may in a
broad way be said to hold for all teleosts. At least it is evi-
dent that the first precervical motor root in all teleosts becomes
distributed to both ventral and lateral musculature, i.e., par-
ticipates in the formation of both the so-called cervical plexus
and the brachial plexus (Furbringer, |. c.).

From this short review it becomes clear that the ventral cell
group of the precervical somatic motor column first makes its
appearance as a constituent of the most rostral portion of the
nucleus in those forms in which the first motor root habitually
participates in the formation of both cervical and brachial plex-
uses. Conversely it is evident that among all forms in which the
first precervical somatic motor rootlet is distributed entirely
through the branches of the cervical plexus, a ventral cell group
is lacking in that part of the nucleus from which the first motor
root arises.

It thus emerges that the ventral cell group of the motor
occipito-spinal column of teleosts is apparently a nucleus of
origin for motor roots distributed through the brachial plexus to
dorsal or lateral derivatives of trunk musculature. Further, it
would appear that the presence of this ventral cell group in the
most rostral part of the precervical somatic motor nucleus in
teleosts is due to the phylogenetic loss of the rostral, more
specialized, dorsal cell group concerned in other forms in the
innervation of epibranchial and pre-hyal hypobranchial spinal
musculature, and is probably not the result of a forward migra-
tion of ventral elements beneath a phylogenetically older dorsal
cell group; for the evidence at our disposal points to the rostral
migration of the constituent elements of the occipito-spinal
nucleus of teleosts as a whole.

544 DAVIDSON BLACK

With regard to the cause of this rostral displacement, Kappers
(1. ec.) has noted that in teleosts the occipito-spinal nucleus, like
that of the abducens nerve, occupies its most rostral position
among those forms in which the optic reflex systems are most
highly developed (e.g., Pleuronectidae). It would appear prob-
able, however, that the rostral migration of the occipito-spinal
motor complex in teleosts has been determined by influences
quite independent of those acting similarly upon the abducens
nucleus.

In teleosts the general cutaneous components of the trigeminus
and vagus nerves pass caudad in the radix descendens trigemini
to their chief nucleus of termination in the immediate vicinity
of the funicular nuclei (Herrick, 38, 40 and 41). Though in
Ameiurus some of the trigeminal fibers end in relation to the
deeper layers of the facial lobe (39), by far the greater number
end in the terminal nucleus in the funicular region. The rostral
portion of the funicular region in all bony fishes becomes a center
of the highest importance for the correlation of the tactile im-
pressions originating in the head and trunk. The long conduc-
tion paths of the dorso-lateral fasciculus arise in this center from
which also emerge numerous ventral arcuate fibers to establish
immediate reflex connection with the subjacent somatic motor
nucleus.

In the discussion of the funicular region in Prionotus (1. ¢.)
Herrick draws attention to Sherrington’s observation: ‘‘ Broadly
speaking, the degree of reflex spinal intimacy between afferent
and efferent spinal root varies directly as their segmental prox-
imity”’ (89, p. 158). At the rostral end of the cord, however,
owing to the changes brought about by the phylogenetic re-
duction of the precervical segments, the dominant reflex influ-
ence of the first sensory segmental nerves upon the motor column
has been to a large extent suppressed and in its place is substi-
tuted that of the funiculo-trigeminal area. Thus it would ap-
pear that the position of the rostral end of the occipito-spinal
nucleus is determined primarily by that of the nucleus of the
descending trigeminal root and the associated funicular nucleus.

MOTOR NUCLEI IN PHYLOGENY 545

Motor vagal nucleus (Nu. mot. N. X). The position of the ros-
tral end of the motor occipito-spinal nucleus. relative to the cau-
dal end of the motor vagus column and to the exit level of the
first occipito-spinal rootlet is one which is subject to a con-
siderable amount of variation in teleosts, even among closely
related forms (vide supra). Consequently the degree of overlap
of the occipito-spinal nucleus and the motor vagus column in
teleosts is not an accurate measure of the caudal extent of the
latter nucleus. However, to estimate the relative development
of the caudal end of the motor vagus nucleus it becomes only
necessary to compare the total length of this structure in dif-
ferent forms regarding the rostral end in all cases as a fixed
point. The reason for this becomes evident when it is recalled
that the musculature receiving its innervation from the first
three branchial trunks of the vagus among teleosts is subject
to but little variation in its relative development, while on the
other hand the pharyngo-clavicularis muscles and especially
the trapezius muscle may vary considerably both in their
relative development and in the character of their innervation.

A trapezius muscle which is innervated solely by the vagus
nerve and whose homology with the muscle of this name among
selachians can hardiy be doubted, has been demonstrated in the
following teleosts, Salmo (Edgeworth, 20), Menidia (Herrick,
34), Silurus (Juge, 56), Lophius (Guitel, 31) and Ameiurus
(Herrick, 36). A trapezius muscle is absent or represented by a
muscle innervated by spinal nerves, in the following teleosts:
Esox, Cyprinus, Perea (Vetter, 95), and Gadus (Herrick, 35). The
pharyngo-claviculares muscles are innervated by the vagus in
an essentially similar manner in each of the following forms:
Menidia, Gadus, and Ameiurus (Herrick) and in Silurus (Juge).

Of these forms, the motor nuclei have been studied and re-
constructed in Silurus, Ameiurus, Lophius, and Gadus, so that
jt is possible to institute comparisons here with a considerable
degree of accuracy (vide figs. 41 and 42). In the case of Gadus
and Lophius, the motor vagus nucleus is completely isolated
from the other constituents of the caudal viscero-motor column
and it becomes at once evident that the length of the nucleus

546 DAVIDSON BLACK

in question is considerably greater in Lophius, where the trape-
zius is present, than in Gadus, where the representative of this
muscle is innervated by spinal nerves. The motor vagus nucleus
in both Silurus and Ameiurus is continuous with that of the
glossopharyngeus, but the line of demarcation between these
two nuclei has been determined as the level beyond which no
motor IX fibers can be traced. The total length of the motor
vagus nucleus determined in this way is approximately equal in
the two forms and is evidently greater than that of the isolated
nucleus in Lophius.

From this it would appear that the caudal end of the vagus
nucleus in Silurus, Ameiurus, and Lophius, represents a true
nucleus accessorius, homologous in all respects with that of
selachians, while in Gadus this nucleus is wanting.

In view of the fact that the trapezius muscle of higher forms is
habitually innervated from two distinct sources, it seems highly
significant that a muscle is present in certain teleosts (e.g.,
Gadus) which by reason of its connection with the cranium and
shoulder girdle must functionate as a trapezius, even though its
innervation by spinal nerves must preclude its homology with
the trapezius of selachians.

In the case of Gadus Herrick (35, p. 298) notes: ‘‘The muscle
running from the cranium to the pectoral girdle in Gadus is
innervated from the spinals and not from the vagus. It is
therefore, merely a detached portion of the general dorsal mus-
culature.”’ If, however, this muscle in Gadus be considered in
comparison with conditions obtaining in higher instead of lower
forms, the peculiarity of its innervation would not exclude it
entirely from homology with the cucularis.

Evidence that the cucularis of higher forms has been derived
in phylogeny from two different sources is furnished by its double
nerve supply. As the presence of definite somatic components
inextricably mixed within this muscle complex seems limited to
higher vertebrates (Sauropsida and Mammalia), this pecu-
liar condition of fusion must be a comparatively recent phylo-
genetic acquisition. If this be so, both components may be

MOTOR NUCLEI IN PHYLOGENY DAT

looked for among lower vertebrates as muscles distinct from one
another, though closely associated and synergic in their action.

Among elasmobranchs the well developed trapezius is wholly
a specialized visceral muscle, but it is closely associated in its
action with somatic muscles innervated by cervical motor nerves.
It would thus appear that in these animals a prostadium of the
cucularis complex might be recognized in these two distinct
though synergic sets of muscles.

In teleosts, however, the elements of the trapezius musculature
have necessarily been much reduced owing to the development
of the bony operculum. It is probably on this account that both
components of the cucularis complex of higher forms are ap-
parently never represented in one individual among bony fishes,
but either the visceral element (e.g., Ameiurus, Lophius) or the
somatic element (e.g., Gadus) is alone retained. In either case,
however, this levator musculature of the shoulder girdle in these
forms would appear to be homologous with one or other of the
components of the cucularis complex of mammals.

This concept accords well with the facts of embryology and
is in harmony with the further phylogenetic history of the acces-
sory nucleus, as well as with its ontogenetic history in mammals.

Motor facialis and glossopharyngeal nuclei and roots (Nu. et rad.
mot. Nn. VII and IX). In contrast to the condition obtaining
in ganoids and selachians, among all teleosts thus far examined a
large portion of the VII motor nucleus is separated from the
caudal viscero-motor column and lies more ventrally than the
latter in the tegmentum. This sequestration of motor facial
elements may affect the whole nucleus (e.g., Silurus, Ameiurus,
Lophius), or only its rostral portion (e.g., Tinea, Pleuronectidae).

The relations of the motor LX nucleus to the other constituents
of the more primitive caudal viscero-motor column are also
highly variable among the different species of teleosts. Thus,
the motor IX nucleus together with the elements of the caudal
motor VII nucleus may be in direct continuity with the motor
X nucleus (e.g., Tinea, Pleuronectidae), or the motor IX nucleus
alone may form the rostral end of the motor vagus column
(e.g., Silurus, Ameiurus). Further, the motor IX nucleus may

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. 27, NO. 4

548 DAVIDSON BLACK

be situated on the level of its root exit intermingled with the
elements of the caudal motor VII nucleus but completely sepa-
rated from the motor X nucleus (e.g., Gadus), or again it may be
intimately associated with the motor VII nucleus and form
with the latter one large nuclear complex entirely apart from the
motor vagus column (e.g., Lophius).

The possible factors operating to produce the characteristic
ventral displacement of the motor VII elements in teleosts have
been discussed in detail by Kappers (62, 64, 72 and 73). This
author has definitely excluded the possibility of the production
of this displacement through purely mechanical influences.
In addition he has shown that the ventral sequestration of the
motor VII nucleus has undoubtedly been strongly influenced
by the anterior gustatory tract among forms in which this
pathway is well developed; while among those animals in which
the anterior taste tract is relatively small, the ventral optic and
tactile systems are most probably chiefly responsible for this
displacement.

Kappers has also shown that the characteristic geniculate
course of the emergent motor IX root in teleosts has been pro-
duced largely through the more or less mechanical traction exer-
cised upon it during the ventral migration of the motor VII
elements (72). However, with regard to the migration of the
perikaryons of the motor [IX nucleus, no such direct mechanical
influence can be demonstrated and there can be no doubt that
the peculiarly variable relations of this nucleus among teleosts
are due to the action of neurobiotactic forces of a nature similar
to those which have brought about the ventral displacement
of the motor VII nucleus in these forms.

In teleosts the arrangement of the musculature innervated by
the nerves V, VII, and IX has been strongly influenced in its
development by the perfection among these forms of two char-
acteristic organ complexes, viz., (1) the respiratory opercular
mechanism, and (2) the pharyngeal tooth-bearing apparatus.
The important effect of the development of the opercular type
of respiration in ganoids upon the motor V and VII nuclear
pattern has already been alluded to and will be discussed again

MOTOR NUCLEI IN PHYLOGENY 549

subsequently. The influence exercised upon the feeding re-
flexes, and consequently upon the pattern of the motor nuclei
involved, by the development of a tooth-bearing apparatus in
the pharynx must also be great among teleosts. Especially
must this be so among those forms in which this structure
becomes functionally of much more importance than the buccal
teeth.

In Ameiurus the mm. levatores branchiales from the fourth
to the seventh are innervated by branchial branches of the vagus
and become inserted into the superior pharyngeal bone to which
they “impart a rocking motion . . . . which must be
very effective in grinding the food against the inferior pharyn-
geal’ (McMurrich, 82). The nicety of the reflex adjustment
of the pharyngeal ‘masticatory’ apparatus in this animal has
been aptly described by Macallum in part as follows: ‘‘When the
epipharyngeal pads are touched . . . . the pads are thrust
down, and at the same time those of the floor are elevated in
opposition. This is for the purpose of comminuting the food
as it passes into the oesophagus, mere contact of food or other
matter serving to bring the pads into action” (80, p. 388).

In Gadus (39) the muscles moving the pharyngeal bones
appear to be arranged on quite another and more simple plan,
and they are also differently innervated. From Herrick’s de-
scription I gather that the condition in Gadus closely resembles
that in Menidia, in which the pharyngo-branchial bones are
moved by but two muscles, viz., the first and second internal
levators of the branchial arches. The first levator, which is the
smaller, is innervated by the IX nerve and acts both as a levator
and a protractor, while the large second muscle receives its
nerve supply from the vagus and serves simply as a protractor.

Such variation in the relative complexity of this musculature,
as well as its nerve supply, is to be expected in view of the fact
that the tooth bearing plates in question are not developed in
connection with definite branchial arches but only become
attached to them secondarily (McMurrich, 81).

From such scant data as the above it is only possible to point
out that in Ameiurus and in Gadus when different muscle com-

550 DAVIDSON BLACK

plexes have been elaborated to produce movement of the pharyn-
geal plates, a most striking difference in the arrangement of the
caudal viscero-motor nuclei (especially the glossopharyngeus)
is to be observed. It is significant, also, that the motor IX
nucleus is subject to such wide variations in its relations (and
consequently in its reflex connections) among teleosts, while in
no other group of the vertebrate series does this nucleus display
such a lack of conformity to type pattern.

Abducens nucleus and roots (Nu. et rad. N. VI). Among
teleosts the abducens nucleus is evidently composed of two more
or less separated subnuclei which are situated ventrally in the
tegmentum and from each of which emerges a single rootlet.
The more rostral rootlet usually emerges on the exit level of the
motor VII root or a very short distance caudad of this structure
though among the Pleuronectidae it characteristically emerges
rostrad of the motor VII root. The more caudal rootlet usually
emerges at a level which is relatively more rostrad than that
of the frontal abducens rootlet of selachians (figs. 17 and 41).
In exceptional cases the abducens nerve may arise from the
brain stem from three rootlets as in Lophius.

The most rostrad of the abducens sub-nuclei lies either on
the level of the emergent motor VII root or but a short distance
caudal of this structure and the second sub-nucleus is situated
slightly caudad of the exit level of the second abducens rootlet.
In Tinca Kappers has described four abducens sub-nuclei,
three of which are represented in his chart of the motor nuclei
(fig. 41 B), while the fourth part, which lies mediad of the ros-
tral ventral sub-nucleus and on the same level, could not be
represented in such a reconstruction.

The relations of the abducens nucleus and of its emergent root-
lets noted here have already been fully described and discussed
by Kappers (63, 64, 66). This author has shown that the fronto-
ventral position of the abducens nucleus in teleosts as compared
with selachians stands in direct relation with the great develop-
ment of the ventral tecto-bulbar paths in the former animals.
Among flat-fish the relatively enormous development of the
tecto-bulbar tract is to be directly correlated with the fact

MOTOR NUCLEI IN PHYLOGENY 551

that among these forms abducens nuclear elements occupy a
more rostral position than obtains in any other group of verte-
brates yet investigated.!!

Motor trigeminus nucleus and root (Nu. et rad. mot. N.V). The
motor V nucleus among teleosts is, with few exceptions, divisible
into two quite definite sub-nuclei. Of these, one is rostro-dorsal
in position, while the other is more caudo-ventrally placed.
Kappers has demonstrated that the chief factor responsible for
the ventral position of trigeminal elements in teleosts, in con-
trast to selachians and ganoids, is the neurobiotactic influence
of the secondary ascending gustatory tract (64, 66). This author
has also shown that the caudo-ventral cell group of this nucleus
is even more intimately associated with the secondary gustatory
tract than are the elements of the dorsal cell group, and is largest
and most ventrally placed among those forms in which this tract
is best developed (I. c.).

A study of the mechanics of respiration among teleosts brings
out the fact that the different muscles innervated by the motor
fibers of the trigeminus are by no means equally concerned in
the respiratory act. In view of the direct bearing which such
considerations must have upon the question of the arrange-
ment of the reflex nuclear pattern in these forms, it becomes
desirable before further discussion to review briefly the recent
results of investigations in this field.

The respiratory current in most teleosts is produced chiefly
by the action of the opercular apparatus in conjunction with the
maxillary, mandibular and branchiostegal valves in a manner
to which reference has already been made (Dahlgren, 15;
Baglioni, 8).

According to Deganello (18) the principal muscles concerned
in these movements are the following. In inspiration: (a) m.
sterno-hyoideus (spinal nerves 1 and 2); (b) m. dilator operculae

4 It is worthy of note in view of the small size and the lack of compactness of
the abducens nucleus in Pleuronectidae, despite the relatively great importance
of the oculomotor apparatus in these forms, that according to Harman (33) the
external rectus muscle is peculiarly subject to variation among flat-fish and in
most of these forms showed evidence of considerable reduction.

552 DAVIDSON BLACK

(R. mand. V); (c) m. levator arcus palatini (R. mand. V); (d)
m. levator operculi (R. opere. VII). In expiration:® (a) m.
adductor operculi (R. opere. VII); (b) m. adductor arcus pala-
tini (R. mand. V); (c) m. adductor hyomandibularis (R. opere.
VII); (d) m. geniohyoideus (R. mand. V.+R. opere. VII).

The action of the opercular apparatus is reinforced to a vari-
able though usually slight extent by the intrinsic branchial
musculature as follows. In inspiration: (a) mm. interarcuales
ventrales (both oblique and transverse group) (Rr. branch. [X—
X); mm. interarcuales dorsales (Rr. pharyng. X). In expira-
tion: (a) mm. levatores arcum branchialium externi et interni
(Rr. branch. [X—X); (b) m. hyohyoideus (R. opere. VII).

In addition to the opercular and branchial respiratory mechan-
ism, the branchiostegal apparatus may play a most important
role in the production of the respiratory current in certain marine
forms (e.g., Lophius). Borcea (11) has pointed out that the
development of the branchiostegal apparatus and its muscula-
ture (m. hyohyoideus and special adductors and abductors)
occurs in inverse ratio to the development of the opercular
mechanism. Baglioni (8), carrying this investigation further,
has arranged teleosts into groups depending upon the type of
their respiratory mechanism. Of these groups it will only be
necessary to mention three in the present connection, viz.,
(1) those in which the branchiostegal apparatus is practically
wanting (e.g., Conger); (2) those in which both branchiostegal
and opercular mechanisms are more or less equally well repre-
sented (e.g., Pleuronectidae); and (3) those in which practically

15 The m. geniohyoideus, whose action was considered to be inspiratory by
Deganello and others, has been shown by Holmquist (46) to be chiefly an ex-
piratory muscle. On account of its action, as well as for other reasons, the
muscle has been termed by this investigator the m. protractor hyoidei.

16 The extent to which the fibers of the facial nerve contribute to the innerva-
tion of this muscle is subject to some question. In Ameiurus few. motor VII
fibers can reach the geniohyoideus owing to the very slight anastomosis between
the mandibular V and opercular VII branches (Herrick, 36). The anastomosis
between these nerves seems to be more extensive, however, in Gadus (35) where
the condition resembles that obtaining in Amia (1), but in Menidia (34) the m.
geniohyoideus is supplied wholly by motor trigeminal branches.

MOTOR NUCLEI IN PHYLOGENY 553

the whole respiratory current is maintained through the action
of the branchiostegal apparatus (e.g., Lophius).

This review makes it evident that among teleosts the m.
adductor mandibulae does not play any important part in
respiration. Indeed, it is concerned almost wholly either in
adduction or protraction of the lower jaw or in producing traction
upon. the maxilla.

The musculature innervated by the elements of the motor V
‘nucleus is thus divisible into two quite distinct functional com-
plexes: one whose action is necessarily of a rhythmic character
and intimately associated with that of the opercular muscles
innervated by the motor VII; and another complex, which is
concerned almost wholly with movements necessary for the
primary ingestion of food.

The importance of gustatory stimuli in the reflex activity of
the respiratory musculature has already been pointed by Kap-
pers. In many forms, however, the gustatory sense plays but
little, if any part in initiating the reflex action of the jaw mus-
culature (e.g., Lophius and the Pleuronectidae) and in such
forms the influence of visual impressions upon this reflex is
great.

The subdivision of the motor V nucleus in most teleosts into
two groups has already been described but the significance of the
arrangement becomes increasingly evident when correlated with
the peripheral conditions outlined above. Thus in Lophius, in
which respiration is carried on chiefly through the action of the
branchiostegal apparatus while the opercular musculature is
reduced to a minimum, the motor VII nucleus is strikingly
specialized both as to size and position, while the component
groups of the motor V nucleus are also peculiarly modified.
The small ventral nucleus in relation to the slightly developed
secondary gustatory tract evidently may be correlated with the
slight development of the effector organ with which it appears
to be associated, viz., the trigeminus opercular musculature.
The large dorsal cell group, on the other hand, lies in close rela-
tion to the tecto-bulbar tracts in the laqueus, and in size and

554 DAVIDSON BLACK

importance corresponds to that of the jaw musculature which
in this form is exceedingly well developed.

A comparison of the reconstruction charts brings out the addi-
tional fact that the independence of the rostro-dorsal motor V
nucleus from the caudo-ventral moiety varies directly with the
development of the visual apparatus, being greatest in Lophius
and the Pleuronectidae and least in Ameiurus and Tinea.
Further, this independence of the cell groups of the motor V
nucleus varies indirectly with the development of the gusta-
tory apparatus, being greatest in Lophius and the Pleuronec-
tidae, where the secondary ascending gustatory tract is relatively
small, and least in Ameiurus and Tinca, in which this system is
highly developed. In regard to the functional development of
the neurone systems contrasted above, Gadus occupies an inter-
mediate place, a condition which is accurately reflected in the
arrangement of the elements of the motor V nucleus in this
form.

‘Though many further examples might be cited of the variations
of the moieties of the motor V nucleus in conformity to periph-
eral development, especially when the relations of this nucleus
are compared to this end among the Pleuronectidae, sufficient
has been said to indicate that the dorsal group of motor tri-
geminal elements is most probably concerned in the innervation
of the musculature of the jaw, while the ventral group fune-
tionates chiefly in the supply of the trigeminal opercular muscles.

Oculomotor and trochlear nuclei and roots (Nu. et rad. Nn. III
et IV). The peculiar and complex relations of the emergent
trochlear root fibers have already been discussed in connection
with the description of this nerve in Solea. However, the posi-
tion, make up and mutual relations of the oculomotor and
trochlear nuclei among teleosts present certain points of in-
terest and will require some further consideration here.

Kappers has already pointed out that the oculomotor nucleus
as a general rule occupies a more rostral position among tele-
osts than among selachians and broadly speaking this teleostean
characteristic may be correlated with the increased relative im-
portance of the tectum opticum and its efferent pathways among

MOTOR NUCLEI IN PHYLOGENY 555

these forms. Certain it is that the dorsal and ventral cell
‘groups of the oculomotor nucleus are definitely placed in rela-
tion respectively to the dorsal and ventral decussations of the
tecto-bulbar tracts (66).

The differentiation of the elements of the oculomotor nucleus
into dorsal and ventral moieties reaches its maximum develop-
ment in fish, among members of the teleostean group. The
probable significance of the presence of dorsal and ventral oculo-
motor cell groups among ganoids has already been alluded to
and it remains only to point out that this differentiation, which
apparently must have been already forecast if not completed
in the ancestral teleostome stock, would seem to be correlated
rather with specialization in intrinsic effectors than in the
extrinsic oculomotor apparatus. The reason for such.a supposi-
tion becomes evident when it is recalled that, among modern
fishes, in the general arrangement of the extrinsic oculomotor
apparatus there are no characters of a truly fundamental nature
distinguishing teleostomes from selachians (Harman, 33; Herrick,
34; Workman, 98).

In the unusually small size of its trochlear nucteus Solea pre-
sents a marked contrast to the condition obtaining among other
members of the flat-fish group in which, as in Lophius, this
nucleus is well developed. In this connection the observations
of Harman (I. c.) are of further interest. This investigator has
pointed out that among flat-fishes the superior oblique muscle
is of unusually large size relative to the other eye muscles and
that in general, in fish when the visual axes are capable of con-
vergence, there occurs a specialization of the m. obliquus su-
perior. In view of this, it is possible that further investigation
may show that the small size of the trochlear nucleus in the speci-
men of Solea here studied is merely an individual variation.

CONCLUSION

Among myxinoids the most important information concerning
environment must reach the central nervous system through
olfactory or tactile channels. The restrictions imposed upon
these forms by the absence of a functional visual apparatus

556 DAVIDSON BLACK

together with the relatively slight development of gustatory
and vestibular organs have necessitated a corresponding read-
justment of their life habits and a consequent limitation of their
range and simplification of their motor reactions. In order to
exist at all these animals must seek and occupy a favorable
environment where adequate protection may be had and where
food may be obtained successfully despite the limitation of their
sensory equipment.

Success under these circumstances means specialization and
the degree of success with which myxinoids have attained this
end is admirably exemplified both by the survival of the type
under modern conditions and by the highly specialized motor
nuclear pattern already described.

That the motor nuclear pattern is specialized in myxinoids
is but another way of stating that the neurones in question
occupy a position most favourable for the reception of reflex
impulses from the dominant afferent nuclei of these animals.

Among elasmobranchs the vestibular, visual and lateral line
sense organs are all functionally well developed and with the
aid of gustatory and tactile sensibility provide ample means
whereby these forms may receive information of a most varied
character concerning their environment. Within the brain
stem in these forms the terminal sensory nuclei are developed in
correspondence with this receptor equipment so that, in con-
trast to myxinoids, no one constituent of either somatic or
visceral areas can be said to dominate the anatomical arrange-
ment of the medulla. On this account the organization of the
afferent divisions of the nervous system within the medulla is
of a more generalized type among elasmobranchs than in
myxinoids.

One of the chief differences between the motor nuclear pat-
tern of cyclostomes and that of elasmobranchs les in the asso-
ciation of the motor VII elements with those of the vagus and
glossopharyngeus to form the caudal viscero-motor column
among all members of the latter group. This arrangement has
undoubtedly taken place under the influence of the elements
within the well developed communis area and has resulted in

MOTOR NUCLEI IN PHYLOGENY Ros

placing the motor nuclei of the nerves to the hyobranchial
musculature in intimate association with the chief afferent center
acting upon them reflexly, in conformity with the first concept
of neurobiotaxis.

The most striking characteristic of the motor nuclear pattern
of elasmobranchs is its relative fixity in all members of the
group. Thus, as regards their nuclear pattern, the most primi-
tive shark resembles the most specialized ray much more closely
than such allied teleostean forms as Tinea and Gadus resemble
one another.

However, that this reflex organization within the elasmobranch
brain stem has been entirely adequate for the needs of these
animals is indicated by the successful manner in which the
members of this group have competed with other more specialized
and modern types in practically every variety of marine
environment.

The reduction of the visual apparatus among ganoids has
apparently been followed by no marked compensatory develop-
ment of other of the special senses and has been accompanied
indeed by a great reduction of the functional development of
the cerebellum as compared with sharks. Apart, however, from
the relatively small tectum opticum and the reduced cerebellum
the general organization of the afferent functional divisions of
the ganoid brain stem is on the whole more selachian than
teleostean in character.

The selachian arrangement of the communis area among
ganoids has necessarily affected the distribution of the elements
of the caudal viscero-motor column. Thus, except for the gen-
erally more rostral position of the motor facial elements, the
viscero-motor nuclear pattern of these animals is essentially
similar to that of sharks.

On the other hand, the effector organs of the head region in
ganoids are modified in general away from the selachian type,
so that especially in the arrangement of the gill laminae and
operculum these forms present important teleostean resemblances.

The peculiar combination of central selachian and peripheral
telesotean characters noted above is apparently an evidence of

558 DAVIDSON BLACK

the inability of the central nervous system in ganoids to specialize
part passu with changed peripheral conditions. Definite evi-
dence of this loss within the central nervous system of the
capacity for unlimited specialization (suppression of neurobio-
tactic activities) is to be seen in the restricted distribution of
modern ganoids.

Among teleosts the capacity for apparently unlimited varia-
tions in the reflex pattern of the brain stem nuclei reaches its
acme among the vertebrate series. Within this group the ex-
treme specialization of any of the organs of special sense is
followed by a corresponding amplification of the primary afferent
nucleus or nuclei involved, together with a modification of the
motor nuclear pattern in perfect harmony with reflex needs of
the animal. Further, the specialization of effectors of whatever
nature is also followed by an adequate corresponding adjust-
ment of the reflex connections of the sensory and motor neurones
involved.

Thus, within the teleost group it is difficult to construct any
generalized scheme of motor nuclear arrangement owing to the
characteristic ability of the elements of the primary nuclei to
react to neurobiotactic influences and to develop a pattern based
chiefly upon peculiar specific requirements.

_

bo

10

18

19

MOTOR NUCLEI IN PHYLOGENY 559

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PRIMARY AND SECONDARY FINDINGS IN A SERIES
OF ATTEMPTS TO TRANSPLANT CEREBRAL
CORTEX IN THE ALBINO RAT

ELIZABETH HOPKINS DUNN

From the Marine Biological Laboratory, Woods Hole, Mass., and the Anatomical

Laboratory of the University of Chicago
EIGHT FIGURES
INTRODUCTION

This paper is to report the findings of a somewhat extended
attempt to transplant cerebral cortex. In two instances, at
least, the attempt to keep alive the neurons within the tissue
seemed to meet with success, although not in the manner de-
sired, since the tissue transferred from one animal to a second
animal became adherent in a position such that the extending
axons could not grow into adjacent nervous tissue.

The secondary findings have some interest also, and the en-
tire problem has a value because of its bearing on the question
of the vitality of nervous tissues. Continuation of the life and
growth of nervous tissue in vitro has been accomplished suc-
cessfully, Harrison, ’07, Burrows, 711, Lewis, ’12, but the per-
petuation of the vitality of nervous tissue transferred from one
region of the nervous system to another region has met with
greater difficulties.

In the earlier attempts at transplantation it was found that
the transplanted mass did not disintegrate entirely and disap-
pear, but that the neurons died, leaving the supporting struc-
tures to represent the original transplanted portion. Of the earlier
attempts at transplantation those of W. Gilman Thompson,
90, of Saltykow, ’05, and of Del Conte, ’07, may be cited. In

565

566 ELIZABETH HOPKINS DUNN

1909, Ranson reported successful transplantation of the spinal
ganglion into the brain. Marinesco and Bethe had earlier
transplanted ganglia to a position adjacent to the sciatic nerve.
In these cases of successful transplantation the neurons remained
alive.

In 1904, while research assistant in the Neurological Labora-
tory of the University of Chicago in charge of Professor H. H.
Donaldson, I began some experiments in the transplantation of
cortical cerebral tissue in young albino rats.

MATERIAL SELECTED

The albino rat was selected because of its adaptability as a
laboratory animal. In contrast to the adult animals which had
earlier been selected for attempted transplantation, a more 1m-
mature animal was utilized. At the ninth or tenth day from
birth the cerebral tissue of the albino rat is not mature, the
hairy coat is not sufficiently developed to interfere with opera-
tion, and although the young must be left in the mother’s care
after the operation, they are not given that assiduous care
which renders operation on very young animals so difficult.
It is possible to return the operated young to the nest without
fear of attack upon them from the mother, if the precautions
mentioned under the discussion of methods of operation are
observed.

The records show that in all forty-six rats were operated.
Two of these rats died soon after operation. Nine other brains
showed nothing of interest on macroscopical examination.
Thirty-five brains were sectioned and studied.

For purposes of tabulation as appears in table 1, the forty-
six operations are divided into four series made up of an irregu-
lar number of groups. The term group had a definite connota-
tion and marked the number of animals operated at one time.
A group usually consisted of the young of one litter, the trans-
plantation being made from one to another young rat of one
litter. The brains were thus equally mature at time of opera-
tion and the rats of the closest consanguinity. It was hoped
that these two points might have weight in the preservation of

TRANSPLANTATION OF CEREBRAL CORTEX

Qn
ey)
<r

TABLE 1
» | ao
a Ee
SEX BORN OPERATED KILLED Z S %
Balhae
Series I
Group east alae & 2? | 12/16/1903 | 12/28/1903 4/ 5/1904 | 111) 99
Group II, Rat 1... 2? | 12/23/1903 1/ 4/1904 1/12/1904 20 8
Group III, Rat 1.. of 1/ 4/1904 1/14/1904 | 4/ 9/1904 | 96) 86
iat, 22: fof 1/ 4/1904 1/14/1904 4/ 9/1904 96) 86
Group IV, Rat 1.. ie) 1/12/1904 1/23/1904 4/ 7/1904 86} 75
IREY Bs. 2 1/12/1904 1/23/1904 4/ 8/1904 87) 76
IRENG Bsc je) 1/12/1904 1/23/1904 4/ 9/1904 88] 77
Series II
Group) heat Q 5/30/1904 | 6/ 9/1904 | 7/12/1904 | 43) 33
Raite2eee ? 5/30/1904 6/ 9/1904 7/18/1904 49; 39
Group) Ui Rat 1 of 6/12/1904 6/22/1904 8/10/1904 57| 49
Rast Zee 2 6/12/1904 6/22/1904 8/10/1904 57| 49
Group III, Rat 1.. fof 7/10/1904 7/20/1904 | 10/26/1904 | 108) 98
IRENH 6.0 on 7/10/1904 7/20/1904 | 10/26/1904 | 108) 98
Series III
Giron Ih, Ieye ile. of 4/ 7/1965 | 4/17/1905
Rate. Q 4/ 7/1905 | 4/17/1905
Raiteouee Q 4/ 7/1905 | 4/17/1905
Gieouyo JOG IRA Iss coe 4 4/17/1905 4/27/1905 6/ 7/1905 51} 41
iaite2eee 2 4/17/1905 4/27/1905 6/ 7/1905 51} 41
IRAN Boo. ot 4/17/1905 4/27/1905 6/ 8/1905 52) 42
Group III, Rat 1... of 5/10/1905 5/20/1905 9/14/1905 | 127) 117
JEN eae of 5/10/1905 5/20/1905 9/14/1905 | 127) 117
Rises oO 5/10/1905 5/20/1905 9/14/1905 | 127} 117
Croup LVe eats: re 9/11/1905 9/21/1905 | 11/ 6/1905 | 54) 44
Raita. 2 9/11/1905 9/21/1905 | 11/11/1905 59} 49
altho see 2 9/11/1905 9/21/1905 | 11/21/1905 69| 59
Rat 4... ? 9/11/1905 9/21/1905
Series IV
Group I, Ie Ilse ? | 11/24/1906 | 12/ 4/1906 2/27/1907 95) 85
ial See ? | 11/24/1906 | 12/ 4/1906 3/20/1907 | 116) 106
Croupr lie Ratelen. Q 2/24/1907 | 3/ 5/1907 | 5/ 3/1907 | 68] 59
TRIE Ps of 2/24/1907 3/ 5/1907 5/ 9/1907 7A| 65
Groupe lice vate aes 2? | 4/14/1907 | 4/22/1907 | 4/29/1907 > | dh
Rat 2. fof 4/14/1907 4/22/1907 6/26/1907 (ay) (65
Girouye . OY, IRettllsccs: - Q 4/17/1907 4/26/1907 7/ 1/1907 75) 66
Rian Q 4/17/1967 4/26/1907 7/ 1/1907 75| 66
Riaitnouere (of 4/17/1907 4/26/1907 7/ 1/1907 75| 66

568 ELIZABETH HOPKINS DUNN

TABLE 1—Concluded

Se | sete

ae

SEX BORN OPERATED KILLED é é es

2 | 56

Giroyigoy We, Ite Wo. 2S 5/14/1907 5/23/1907 | 10/ 4/1907 | 143) 134
Raitie2e eee eee 5/14/1907 5/23/1907 | 10/ 4/1907 | 143) 1384

IRAE Bs 5 Snell Gt 5/14/1907 5/23/1907 | 10/ 4/1907 | 143) 134

Rv eS Gere olf or 5/14/1907 5/23/1907 | 10/ 4/1907 | 143) 134

Group VI, Rat 1 o 5/23/1907 6/ 1/1907 9/24/1907 | 125) 116
IRAN Ze oo aal| 6 5/23/1907 6/ 1/1907 | 11/28/1907 | 185} 176

IREMi Bosco of 5/23/1907 6/ 1/1907 | 12/28/1907 | 220) 211

Rat 4... fe} 5/23/1907 6/ 1/1907 | 12/28/1907.) 220) 211

Group) Vil, Rat i..: ot 6/16/1907 6/27/1907 | 11/30/1907 | 167) 156
IRENE 25 oc of 6/16/1907 6/27/1907 | 12/28/1907 | 195) 184

Ratnomer y 6/16/1907 6/27/1907 | 12/28/1907 | 195) 184

vitality in the transplanted material. The consanguinity may
have particular value if closely related individuals have a simi-
lar metabolism and hence a like chemical constitution of the
body tissues.

METHOD OF OPERATING

For the convenience of the operator, the left hemisphere of
the brain was chosen for operation in each case. A portion of
the cerebral cortex was selected for removal, the loss of which
would least interfere with the nutrition of the operated animal
The skin was first opened near the median line of the head by
an incision carried from the region of the eyes to the nape of
the neck, an incision of not excessive length in the young albino
rat. The flap on the left side was retracted by pulling on the
skin at some little distance from the line of incision. Then with
fresh sterile scissors a cartilaginous flap was made in the parie-
tal region of the skull. This was accomplished by a crescent-
shaped incision with the attached base just above the ear. This
procedure was repeated on a second rat. Then with a thin
knife a triangular portion of the thin cortex was removed from
the first rat and replaced by a similar portion from the second
rat. The ineision was usually made in such a way that the

TRANSPLANTATION OF CEREBRAL CORTEX 569

apex of the triangle extended downward. The time consumed
in the transfer of the cortex was made as short as possible. In
the earlier operations the lateral ventricle was often accident-
ally opened, although the intention had been to remove and
replace a thin superficial portion of cortex only. The appar-
ently successful cases were found to be those in which the
ventricle had been opened and a bit of cortex had become ad-
herent to the choroid plexus. During and after the operation
the dura was preserved if possible but in rats of ten days the
cerebral membranes are very delicate and difficult to differ-
entiate from one another. Sometimes the dura remained con-
veniently attached to the cartilaginous flap. After trans-
ference of the cortical mass the cartilaginous flap was freed,
the skin drawn over it and the edges of the skin retained in
proximity by a collodion dressing.

Attention to a number of details was found to be advanta-
geous. Complete anaesthesia before and during the operation
was found necessary, otherwise the struggles of the animal
caused protrusion of the cerebral substance through the incision.
Asepsis was secured by using successive sets of sterile instru-
ments. No antiseptic was used other than the ether of the
collodion dressing. Maintenance of the body warmth both dur-
ing and after the operation was essential. This eare for the
maintenance of the body heat and that for the exclusion of anti-
septics were the very helpful suggestions of Prof. C. 8. Sherring-
ton, who was a visitor to the laboratory during the early experi-
mental period. Care was exercised not to injure adjacent cere-
bral structures. Transplanted material was handled rapidly
and with a warm knife. An almost insuperable difficulty ap-
peared to be that of retaining the transferred material in the
desired place. At ten days of age the cerebral cortex of the
albino rat is soft and plastic. The removal of tissue leaves an
irregular and almost imperceptible cavity, so that the trans-
ferred tissue is displaced almost immediately from the con-
vexity of the cerebral hemisphere. In the later series an at-
tempt was made to permit the formation of a thin blood clot
lying over the transferred bit of tissue and extending to the

570 ELIZABETH HOPKINS DUNN

adjacent parts ot the hemisphere. This splint would be, I
believe, an important factor in ultimate success.

The operation was done under ether anaesthesia and the
animal kept warm, both during the operation and for two or
three hours after the operation, until it had thoroughly recov-
ered from the anaesthetic, and the odor of the ether from the
anaesthetic and from the collodion dressing had disappeared.
Then the young rats were returned to the nest and met with no
interference from the mother other than futile attempts to re-
move the collodion dressing. The quiet and seclusion of the
nest during the few days after operation aided convalescence.
Young rats are not inclined to stray from the nest until the
eyes open, about the fourteenth day of life.

EXAMINATION OF THE MATERIAL

No microscopical studies of the early conditions of the trans-
planted material were attempted, as the attention was cen-
tered on an effort to ascertain whether such transplanted ma-
terial would later contain mature neurons with medullated
axons. The brains of a few rats which died soon after operation
gave no suggestion of the survival of the transplanted tissue.

A few of the operated brains especially from the rats of the
earlier operations, showed some inflammatory changes, with
disintegration of the cerebral substance, about the region of the
incision.

The rats upon whom these experiments were carried out gave
the appearance of normal rats. No convulsions or paralyses
were noted in the operated animals. Control rats were studied
during the course of the first operations but, when no sequellee
of the operative procedure were noted, the observation of
control rats was abandoned.

The examination of the material removed post-mortem was
of two kinds. The first of these was the gross examination at
the time of autopsy, when notes were made regarding the con-
dition of the skull, of the meninges, and of the cerebral substance
as to the superficial extent of the wound, location of the cica-
trix, et cetera. The brains were then removed and fixed in

TRANSPLANTATION OF CEREBRAL CORTEX Di

ten per cent formalin. Some weeks before the brains were to
be embedded for cutting, they were mordanted in toto in
Miiller’s Fluid. The blocked material was cut serially either in
thirty or forty-five micra sections and stained by the Weigert-
Pal method. Alternate sections were counter-stained with
Upson’s Carmine.

The sections were studied individually under low and high
power.

I am indebted for the drawings to Miss Katherine Hill and
Mr. A. B. Stredain.

The completion of this study was made possible through the
courtesy of Dr. R. R. Bensley and Dr. C. J. Herrick, who granted
me an additional amount of free time for the microscopical
study of the sections.

The rats used were bred in the laboratory, with the exception
of Series IV, Group I, for which I am indebted to Dr. J. B.
Watson.

THE FINDINGS FOR CEREBRAL TRANSPLANTATION

The investigation now reported was undertaken for the pur-
pose of determining the possibility of maintaining the life of
nerve cells in bits of transplanted cerebral cortex. This con-
tinuity of vitality has been found possible and growth has gone
on in the neurons transplanted. The neurons which have sur-
vived have assumed their morphological relations to other neu-
rons within the transplanted bit. The growth changes within
the transplants are very similar to those of normal material of
about the same age. Medullation is fully accomplished. ‘The
number of medullated fibers is however relatively smaller than
in normal material but this is probably due to the absence of
such fibers as grow into any cerebral region from other parts
of the brain. The growth of the individual neurons has been
very considerable. Watson, 03, found that the cortex of the
brain of the albino rat is but slightly developed at the tenth
day of life, the age at which transplantation was attempted,
and that medullation appears much later. The transplanted
neurons must therefore have been very immature.

BV? ELIZABETH HOPKINS DUNN

The possible relations of these transplanted neurons with
neurons outside of the transplanted portions have not been de-
termined by the results of these experiments. In no brain of
the four, with successful transplants, did the transplanted bit
so attach itself that fibers could cross the line of attachment to
unite functionally with adjacent neuron masses.

The two points of chief importance in successful cerebral
transplantation are first, the retention in place of the material
transferred, and second, the furnishing to it of an adequate
blood supply. Apparently the death of neurons in blocks of
transplanted cerebral cortex has been due to some factor which
has not affected the vitality of other tissues. The supporting
tissues of the cortex have lived and retained the mass form of
the transplanted bit. This may suggest the lack of sufficient
nourishment for the nervous elements. In my own successful
operations, the transplanted portions have remained adherent
to the denuded portions of the cortex but have taken some
position near the choroid plexus of the lateral ventricle and have
apparently received their blood supply from that source. Dr.
Ranson permits me to mention that in carrying on some further
(unreported) studies in the transplantation of nerve ganglia
into the brain he found the most nearly normal conditions in
those ganglia which were within or adjacent to the choroid
plexus. This may have been due to the more complete anchor-
age of the material or to a more adequate nourishment, and
my own experience would put emphasis on the latter reason.
It would seem then that after the mechanical difficulties of
securing juxtaposition have been solved, the viability of the
transplanted tissues will be secured by guaranteeing sufficient
nourishment.

My chief reason for believing these four to be true trans-
plantations of cerebral cortex is the finding in each instance a
line of cicatricial tissue about the mass of cortex in question.
To follow the enclosing cicatrix it was necessary to study serial
sections, and to assure oneself that the tissue mass in ques-
tion was not partly separated from the remainder of the brain,
or a bit which had been twisted out of its original position in the

TRANSPLANTATION OF CEREBRAL CORTEX Sia

course of the operation and had retained its vitality because of
its ability to draw nourishment from its original blood supply.

To illustrate the conditions found in successful transplants,
drawings have been made from sections of the brain of Rat 2
of Series IV, Group IV Figures 1 and 2 are drawn from sec-
tions 119-120 of this brain. Figure 2 is a detail from the region

Fig. 1 Showing at A a bit of transplanted cerebral cortex in the albino rat.
From sections 119-120, Series IV, Group IV, Rat 2. X 7.5.
marked A in figure 1. This is by chance the first true trans-
plantation to be noticed as all the material was carried through
before detailed studies were made upon the completed slides.
Later in the course of reéxamination, other true transplants were
observed.

On the discovery of the transplant, figures 1 and 2, it was
thought possible that it might be a portion of tissue pinched
off from the hippocampus, to which it hes adjacent. However
a rather wide band of cicatricial tissue could be seen in the
double stained sections, separating the mass from the adjoining

574 ELIZABETH HOPKINS DUNN

structures. The position of the perikarya, also, and the rela-
tions of the medullated nerve fibers are those of a bit of inverted
cerebral cortex. The capillary blood supply appears to be
derived through the cicatricial adhesion to the plexus choroideus

of the lateral ventricle.

AOD yee

v 8 &@

TSE’ @ &, 4

Showing attachment of transplanted portion (A)

Fig. 2. Detail of figure 1.
The cortex is inverted and adherent to the

to hippocampus (//) at the left.
choroid plexus (C. P.) from which it seems to derive its blood supply.

While the general type of cerebral cortex prevails in the por-
tions transplanted, certain differences from normal cortex can
be detected. Such areas have a slightly different reaction to
staining agents than have surrounding areas. The colors vary
slightly in intensity and in shade from those of the rest of the

section. After fixation and staining the neurons appear some-

TRANSPLANTATION OF CEREBRAL CORTEX SID

what fragmented, especially the free endings of the medullated
nerve fibers which easily fray. The blood supply is less ample
in such regions, the capillaries being more slender and less well

filled.
THE MASSING OF CORTICAL FIBERS

In addition to what we may regard as true transplantation of
cerebral cortex, other interesting results of the operations were
noted. One of these was reported at a joint meeting of the
Chicago Neurological Society and the Biological Club of the
University of Chicago, March 30, 1909, under the title “‘On the
course of cortical tangential fibers developing after ablation of
encephalic cortical substance.’ Perhaps the use of the term
‘tangential’ in this connection is misleading. The fibers to
which the report refers were parallel to the surface of the brain
and located at various depths throughout the cortex. They
were not tangential in the narrower sense of the term as it is
applied to the fibers lying near the surface of the cortex. Ata
later time it was noted in other brains that vertical fibers were
also apparently increased in number. The materials in which
these conditions were noted were produced in the following way.
When the operator accidentally opened into the lateral ventricle
in the course of operation, there was a tendency for the sub-
ventricular substance to protrude through and to widen the
original opening. It was while studying serial sections of a brain
in which this had occurred that the apparent increase of fibers
about the open space was noted. In the normal cerebral cor-
tex of the albino rat many scattered medullated nerve fibers
may be found at various depths running parallel to the surface
of the cortex. In those brains in which considerable openings
occurred, there appeared, in transverse serial sections, to be a
massing of fibers parallel to the cortex. These bands of fibers
could be traced from outlying cortex and were found to merge
into cerebral tissue which had about the normal number of fibers
which were parallel to the surface. It seemed at the time that
neuron processes which in their growth were not able to follow
the path usual to them had been deflected by the wall of the

576 ELIZABETH HOPKINS DUNN

open space and formed a band along the margin. It appeared
that the massing was more noticeable when the margin of the
opening was near the center of the antero-posterior diameter of
the hemisphere than when it was near the frontal or occipital

Fig. 3. Showing margin of wound with a massing (M/) of cortical fibers.
These fibers can be traced in successive sections, to the surrounding margin of
the open space produced by the ablation. Zeiss microscope, camera lucida.
Outline on table level with Ocular 2, Objective 16.0 mm.

pole of the hemisphere and that this was correlated with the
marked inerease in the number of such fibers in the corre-
sponding region. It seemed possible then to interpret these fibers
as association fibers because they could be found at various

TRANSPLANTATION OF CEREBRAL CORTEX a7

levels in the cortex, could not be traced to projection fibers,
and extended some distance through the cortex. Fibers of
this kind can be found at M in figures 3 and 4 and in figures
5 and 6.

Fig. 4 Detail of figure 3. Showing the upper part of figure 3. Some small
neurons (V) may be seen among the massed fibers. Zeiss microscope, camera
lucida, outline on table level with Ocular 2, Objective 8.0 mm.

The more recent work of Greenman, 716, suggests a satis-
factory interpretation of the number of medullated nerve fibers
found circling the gap in the cortex. Dr. Greenman found in
studies on the regeneration of peripheral nerves that the num-
ber of nerve fibers on the proximal side of the lesion in the re-
generating nerve was enormously increased over the normal.

578 ELIZABETH HOPKINS DUNN

This increase apparently depended on the amount of obstruction
offered by the connective tissue through which the nerve fibers
must grow to reach their peripheral terminations. This inter-
pretation may well account for the large number of fibers found
in such sections as those shown in figures 3 and 4 and figures 5
and 6. The growth of tissue around a gap in central nerve sub-

Fig. 5 Showing massing of cortical fibers along the open wound (W). Fibers
apparently extend into the internal capsule (J. C.). Freehand drawing from Sec-
tion 121, Series I, Group IV, Rat 3.

stance is comparable to their growth through connective tissue
in peripheral nerves and may be governed by the same laws.

THE GROWTH OF MEDULLATED NERVE FIBERS ACROSS
CICATRICIAL TISSUE

Ranson, ’03, demonstrated that processes of neurons could
grow across cicatricial tissue and develop their medullary
sheaths. These findings were on stab wounds of the corpus
callosum in the albino rat. The cerebral cortex of operated

TRANSPLANTATION OF CEREBRAL CORTEX 079

rats, in my experiments, yielded similar nerve fibers in such
instances as the incision had extended for some distance into
otherwise normal tissue and the coapted edges had united by the
formation of cicatricial tissue. The fibers traversing the cica-
tricial tissue are no more numerous in my material than they
were in that of Dr. Ranson, but are distinctly to be seen and,
in favorable sections, may be traced for some distance.

Fig. 6 Detail from figure 5. Freehand drawing. Magnification unknown.

THE JOURNAL OF COMPARATIVE NEUROLOGY, VOL. PAs No. 4

580 ELIZABETH HOPKINS DUNN

Ranson interpreted these fibers as processes of neurons which
were immature at the time of operation. Their perikarya may
be located at some distance from the cicatrix. At F’,, in figures
7 and 8, may be noted such medullated nerve fibers crossing
cicatricial tissue in the cerebral cortex of an albino rat.

Fig. 7 Showing a section of the brain of the albino rat from a region near
the posterior pole. In the dorsal part of the left hemisphere is a light line (L)
marking the line of incision, with a few medullated nerve fibers crossing it (F).
From sections 81-82, Series IV, Group IV, Rat 3. X 6.

SUMMARY

By the use of immature nervous tissue from the brain of the
albino rat the life of the constituent neurons in the cerebral
cortex has been maintained after transplantation.

After many unsuccessful attempts this result was obtained by
utilizing a thin covering blood clot to retain the graft in position.

The best nourished grafts were those which lay near the plexus
choroideus of the lateral ventricle.

In the neurons of the transplanted cortex certain differences
from those of normal tissue were detected. These differences
were in the staining intensity and morphology of the perikarya
and medullated fibers. The blood supply was less ample.

TRANSPLANTATION OF CEREBRAL CORTEX DS 1

A massing of tangentially placed medullated nerve fibers

was found about the open spaces produced by accidental abla-
These tangential fibers probably connect dif-

tions of cortex.
This aberrance of nerve

ferent parts of the cerebral cortex.
fibers shows that a new path may be routed when the usual

path has been permanently blocked.

a ==-T

7 3

\

\ ee k ue

Fig. 8 Detail from figure 7, showing the line (Z) of cicatricial tissue with
several medullated nerve fibers (Ff) crossing it. X 28.

Similar bands of projection fibers were noted along the margins

of incised wounds.
Corroborative evidence was noted for Ranson’s finding re-

garding the growth of medullated nerve fibers across cicatricial

tissue in the nervous system.

582 ELIZABETH HOPKINS DUNN

LITERATURE CITED

Burrows, Montrose T. 1911 The growth of tissues of the chick embryo out-
side the animal body, with special reference to the nervous system.
Jour. Exp. Zoél., vol. 10, no. 1, pp. 63-83.

Det Conte, G. 1907 Einpfanzungen von Embryonalem Gewebe im Gehirn.
Ziegler’s Beitrage zur pathol. Anat. u. z. allg. Pathol., Bd. 42, H. 1,
S. 193-201.

GREENMAN, M. J. 1916 Regeneration of peripheral nerves. Proceed. Phila.
Neurolog. Soc., Journal of Nervous and Mental Diseases, vol. 43, no.
1, pp. 62-67.

Harrison, Ross GRANVILLE 1907 Observations on the living developing nerve
fiber. Anat. Rec., vol. 1, no. 5, pp. 116-118.

Lewis, WARREN H. anp Lewis, MarGaret Reep 1912 The cultivation of
sympathetic nerves from the intestine of chick embryos in saline
solutions. Anat. Rec., vol. 6, no. 1, pp. 7-32.

Ranson, 8S. WaLtTeR 1903 On the medullated nerve fibers crossing the site of
lesions in the brain of the white rat. Jour. Comp. Neur., vol. 13,
no. 3, pp. 185-207.

1909 Transplantation of the spinal ganglion into the brain. Quar-
terly Bull. Northwestern Univ. Med. School, pp. 1-4.

Sauttykow, 8. 1905 Versuche tber Gehirnplantation. Archiv f. Psychiat. u.
Nervenkrankh. Bd. 40, H. 2, S. 329-388.

THompson, W. GitMAN 1890 Successful brain grafting. New York Medical
Journal.

SUBJECT

CANTHIAS. The cerebral ganglia and
_ early nerves of Squalus

Albino rat. A revision of the arenes of
water in the brain and in the spinal cord
OMENS are CET ECU ais seme ciaciae oe:

Albino rat—in two parts. I. Effect of a de-
fective diet and of exercise. II. Number

of cells in bulb. Studies on the olfactory
bulbs of the

Albino rat.
in a series of attempts to transplant cere-
hralecortexaimnthew smelt nr. felsic eres ie

Albino rat—under normal conditions, in dis-
ease and after stimulation. The number,
size and axis-sheath relation of the large
myelinated fibers in the peroneal nerve
Ofthesinioned ayer eerie

Alligator mississippiensis. The forebrain of.

Axis-sheath relation of the large myelinated
fibers in the peroneal nerve of the inbred
albino rat—under normal conditions, in
disease and after stimulation. The num-
ber, size and...

EE during the life cycle. Nuclear size in

the nerve cells of the. ve
Behavior of frog embryos. Studies on regen-
eration in the spinal cord. II. The ef-
fect of reversal of a portion of the spinal
cord at the stage of the closed neural
folds on the healing of the cord wounds,
on the polarity of the elements of the cord
and on the
Biography, Susanna Phelps Gage, Ph.B.....
Buiack, Davipson. The motor nuclei of the
cerebral nerves in phylogeny; a study of
the phenomena of neurobiotaxis. Part
I. Cyclostomi and Pisces. .
Brain and in the spinal cord of the albino rat.
etme of the percentage of water in
ee oe Reet TA ee SEE MAS
Bulbs of the albino rat—in two parts. I.
Effect of a defective diet and of exercise.
Il. Number of cells in bulb. Sturies on
the olfactory.

ELL bodies by the use of the polar planim-
eter. Further verification of functional
size changes in nerve. .
Cells of the bee during the life cycle.
clear size in the nerve
Cells in bulb. Studies on the olfactor y bulbs
of the albino rat—in two parts. I. Ef-
fect of a defective diet and of exercise.
Il. Number of
Changes in nerve cell bodies by the use of the
polar planimeter. Further verifications
OfmuNcthional Size... weet ate ae ee eee
Cord of the albino rat. A revision of the per-
centage of water in the brain and in the
SDMA pene ee ck rt
Cord. IJ. Effeet of reversal of a portion ‘of
the spinal cord at the stage of the closed
neural folds on the healing of the cord
wounds, on the polarity of the elements
of the cord and on the behavior of frog
embryos. Studies on regeneration in the
spinal

ares ee

Primary and secondary findings

405

325

403

69

201

299

69

201

299

AND AUTHOR INDEX

Cord wounds, on the polarity of the-elements
of the cord and on the behavior of frog
embryos. Studies on regeneration in the
spinal cord. II. The effect of reversal
of a portion of the spinal cord.at the stage
of the closed neural folds on the healing
of the..... ane IN ee ayo arnyae

Cortex in the albino rat. Primary and sec-
ondary findings in a series of attempts to
transplant cerebral See

Crossy, ELizABETH C \ROLINE The
brain of Alligator mississippiensis. Ae

Cycle. Nuclear size in the nerve cells of the
bee dumine: the lifes:.2-2-.....-.--

Cyclostomi and Pisces. The motor nuclei of
the cerebral nerves in phyloweny: a study
of the phenomena of neurobiotaxis. Part

fore-

Bees

Diet and of exercise. II. Number of cells
in bulb. Studies on the olfactory bulbs
of the albino rat—in two parts. I. Ef-
fect of a defective :

Douiey, Davin H. Further verification of
functional size changes in nerve cell
bodies by the use of the polar planimeter 2

Donaupson, Henry H. A revision of the
percentage of water in the brain and in
the spinal cord of the albino rat..........

Dunn, ExizanetH HopkKIns. Primary and
secondar y findings in a series of attempts
to transplant cerebral cortex in the albino

EN eo Rene COSCO ERGs vy SODEoDS Mar
Dynamic ‘polariz zation of the neurone. “Fur-
ther contributions on neurobiotaxis. IX.

An attempt to compare the pheonmena
of neurobiotaxis with other phenomena
of taxis and tropism. The..

MBRYOS. Studies on regeneration in
the spinal cord. Il. The effect of re-
versal of a portion of the spinal cord at
the stage of the closed neural folds on the
healing of the cord wounds, on the polar-
ity of the elements of the cord and on the
DehaviOnOliTO peer eee talserr anes
Exercise. If. Number of cells in bulb.
Studies on the olfactory bulbs of the al-
bino rat—in two parts. I. Effect of a de-
fective ciebiandlOtesereee seer:

IBER. Some experiments on the nature
and function of Reissner’s

Fibers in the peroneal nerve of the fbr al-

bino rat—under normal conditions, in
disease and after stimulation. The
number, size and axis-sheath relation of

the large myelinated
Folds on the healing of the cord wounds, on
the polarity of the elements of the cord
and on the behavior of frog embryos.
Studies on regeneration in the spinal cord.
Il. The effect of reversal of a portion of
the spinal cord at the stage of the closed
neural

261

421

403

584

Forebrain of Alligator mississippiensis. The.
Frog embryos. Studies on regeneration in
the spinal cord. II. The effect of re-
versal of a portion of the spinal cord at
the stage of the closed neural folds on the
healing of the cord wounds, on the polar-
ity of the elements of the cord and on the

INDEX

325

behavior Ole «tes ere ee hee ee nee cia 421
Function of Reissner’s fiber. Some experi-
ments on the nature and................. 117
Functional size changes in nerve cell bodies
by the use of the polar planimeter. Fur-
theraveritication Ota.ca-pe emer ecient 299
AGE, Stmon H. Glycogen in the nerv-
ous system of vertebrates.. . 451
Gace, Susanna Puerps, PH.B. SRinenehy, 5
Ganglia and early nerves of Squalus acan-
thiasseehoercerebral crys. ereerteleion cara ore 19
Glycogen in the nervous system of verte-
IDTLOS sete eoeecerese ore ole ecole ere tee satate oiencte Chess a0 451

GREENMAN, M. J. The number, size and
axis-sheath relation of the large myeli-
nated fibers in the peroneal nerve of the
inbred albino rat—under normal condi-
tions, in disease and after stimulation.....

EALING of the cord wounds, on the
polarity of the elements of the cord and

on the behavior of frog embryos.
Studies on regeneration in the spinal
cord. II. The effect of reversal of a por-

tion of the spinal cord at the stage of the
closed neural folds on the. .

Hour, Carouine M. Studies on the olfac-
tory bulbs of the albino rat—in two
parts. I. Effect of a defective diet and
of exercise. IT.

Hooker, DaveNpoRT. Studies on regenera-
tion in the spinal cord. Il. The effect
of reversal of a portion of the spinal cord
at the stage of the closed neural folds on
the healing of the cord wounds, on the
polarity of the elements of the cord and
on the behavior of frog embryos......

NBRED albino rat—under normal condi-
iG tions, in disease and after stimulation.
The number, size and axis-sheath rela-
tion of the large myelinated fibers in the
peroneal nerve of the.

APPERS, C.U.

Arténs. Further con-
tributions on neurobiotaxis. IX. An
attempt to compare the phenomena
of neurobiotaxis with other phenomena
of taxis and tropism. The dynamic
nolerization of the neurone

ANDACRE, F. I The cerebral ganglia
and early nerves of Squalus acanthias..
Life cycle. Nuclear size in the nerve cells of
thewbee dunner tthe. .teceeee eee re
OTOR nuelei of the cerebral nerves in
phylogeny: a study of the phenomena
of neurobiotaxis. Part I. Cyclostomi

and Pisces. The
Myelinated fibers in the: perone al nerve of the
inbred albino rat—under normal condi-
tions, in disease and after stimulation.
The number, size and axis-sheath relation
OL CH ONATEO ss tee, Me ae tee eee
TERVE cell bodies by the use of the polar
planimeter. Further verification of
functional size changes in....
Nerve cells of the bee during the

¢ f th cycle.
Nuclear size in the

life

Number of cells in bulb £

403

421

. 421

403

261

19

69

467

403

. 299

Nerve of the inbred albino rat—under normal
conditions, in disease and after stimula-
tion. The number, size and axis-sheath
relation of the large myelinated fibers in
the peronealli-s 75a ee oeeneereotee

Nerves in phylogeny: a study of the phenom-
ena of neurobiotaxis. Part I. Cyclo-
stomi and Pisces. The motor nuclei of
ithe.cerebrallisi 5 wae ee ken Sener

Nerves of Squalus acanthias. The cerebral
gangliajandreankya-eec es eee eee

Nervous system of vertebrates.
PNUCHO Raf Acres ole ote Solarian ereh aieye ea ee ciae

Neural folds on the healing of the cord
wounds, on the polarity of the elements
of the cord and on the behavior of frog
embryos. Studies on regeneration in the
spinal cord. II. The effect of reversal
of a portion of the spinal cord at the stage

of itheclosedt sence ae OBC ee
Neurobiotaxis. Part I. Cyclostomi and
Pisces. The motor nuclei of the cerebral

nerves in phylogeny: a study of the phe-
Momens! Of. Wels ee eee e eee
Neurobiotaxis. IX. An attempt to com-
pare the phenomena of neurobiotaxis
with other phenomena of taxis and tro-
pism. The dynamic polarization of the
neurone. Further contributions on......
Neurone. Further contributions on neuro-
biotaxis. IX. An attempt to compare
the phenomena of neurobiotaxis with
other phenomena of taxis and tropism.
The dynamic polarization of the..........
Nicuoutrs, GrorGe E. Some experiments
on the nature and function of Reissner’s

0S ae er i a one onin ca enn
Nuclear size in the nerve cells of the bee dur-
ine-sthelifekeycless ae eae neem eee ee
Nuclei of the cerebral nerves in phylogeny: a
study of the phenomena of neurobio-

taxis. Part I. Cyclostomi and Pisces.
Phevmotorsetiec nse eae eee ee

Oo bulbs of the albino rat—in
two parts. I. Effect of a defective diet
and of exercise. II. Number of cells in
bulbs istudiesionitihe:- sae neeeeaoe

ERONEAT,. nerve of the inbred albino
rat—under normal conditions, in disease
and after stimulation. The number, size

and axis-sheath relation of the large mye-

linated hibersamighes eee eee
Puiuiuies, RutH I.., SMatLwoop, W. M. and.
Nuclear size in the nerve cells of the bee
Guringytheriieicyclessssm.s eee ee meee
Phylogeny: a study of the phenomena of
neurobiotaxis. Part I. Cyclostomi and
Pisces. The motor nuclei of the cerebral
MEV. SSH yj ery ees «a/R Ee
Pisces. The motor nuclei of the cerebral
nerves in phylogeny: a study of the phe-
nomena of neurobiotaxis. Part I. Cy-

Clostomlikand years. 0 sone

Planimeter. Further verification of func-
tional size changes in nerve cell bodies by
ion bte(oi (haleMolENOnnan pA noounacoasr nes

Polarity of the elements of the cord and on
the behavior of frog embryos. Studies
on regeneration in the spinal Cordsaul

The effect of reversal of a portion of the

spinal cord at the stage of the closed

neural folds on the healing of the cord
wounds oni thes. aes eerie

Polarization of the neurone. Further con-
tributions on neurobiotaxis. IX. An
attempt to compare the phenomena of
neurobiotaxis with other phenomena of
taxis and tropism. The dynamic

403

467
19
451

421

467

261

261

liv

69

201

467

_ 299

421

AT. A revision of the percentage of
water in the brain and in the spinal cord
ofgtheralbinowvrc coe eceeee ee eis

Rat—in two parts. I. Effect of a defective
diet and of exercise. II. Number” of
cells in bulb. Studies on the olfactory
bulbstofctheral binossssee ase erie

Rat. Primary and secondary findings in a
series of attempts to transplant cerebral
cCortexsin“bheyal binom-ereeeere eee cies

Rat—under normal conditions, in disease and
after stimulation. The number, size and
axis-sheath relation of the large myeli-
nated fibers in the peroneal nerve of the
riMfayneoleM UNO nasa arsnern doagananacweoppnedc

Regeneration in the spinal cord. II.
effect of reversal of a portion of the spinal
cord at the stage of the closed neural folds
on the healing of the cord wounds, on the
polarity of the elements of the cord and

on the behavior of frog embryos.
ASIAUGES Oils sanssesosanaaes Pabe “Ue Ae tnie
Reissner’s fiber. Some experiments on the

nature and function of..

Reversal of a portion of the spi inal cord at the
stage of the closed neural folds on the
healing of the cord wounds, on the po-
larity of the elements of the cord and on
the behavior of frog embryos. Studies
on regeneration in the spinal cord. II.
Elche eliectiOlancecen tesco sateen ees

HEATH relation of the large myelinated
fibers in the peroneal nerve of the inbred
albino rat—under normal conditions, in
disease and after stimulation. The
number, size and axis-..

Size and axis-sheath relation of the ‘large
myelinated fibers in the peroneal nerve of
the inbred albino rat—under normal con-
ditions, in disease and after stimulation.
Bovey T iis bel OD Pha eae A REL IRE 8 Geen

Size changes in nerve cell bodies by the use of
the polar planimeter. Further verifica-
tion of functional...

Size in the nerve cells of the bee during ‘the
life cycle. Nuclear...........

INDEX

77

201

565

403

421
117

421

403

403

299

SmaLitwoop, W. M. and Puiuips, Rurs L.
Nuclear size in the nerve cells of the bee
durmpitheilitercycles ee eae eee

Spinal cord of the albino rat. A revision of
the percentage of water in the brain and
HV] (Ce Geena RHE eres een

Spinal cord. II. The effect of reversal of a
portion of the spinal cord at the stage
of the closed neural folds on the healing
of the cord wounds, on the polarity of
the elements of the cord and on the be-
havior of frog embryos. Studies on re-
reSyaleyeinopalshay (ed aasou sons aoebce Geedos Godos

Squalus acanthias. The cerebral ganglia and
OCArlysNeEviesiOlemecrst sae eee en

System of vertebrates. Glycogen in the
DEL VOUS ee cseeeetot ee aoe

AXIS and tropism. The dynamic polar-
ization of the neurone. Further contri-
butions on neurobiotaxis. IX. An at-

tempt to compare the phenomena of neu-

robiotaxis with other phenomena of...... 2

Transplant cerebral cortex in the albino rat.
Primary and secondary findings in a

Senies ofattemptaston nena oe eens é

Tropism. The dynamic polarization of the
neurone. Further contributions on neu-
robiotaxis. IX. An attempt to compare
the phenomena of neurobiotaxis with

other phenomena of taxis and............. 3

ERTEBRATES. Glycogen in the nerv-
OUsSsystemyrOlaisac ce eee

ATER in the brain and in the spinal

W cord of the albino rat. A revision
ol theypercentaroolaseme eee
Wounds, on the polarity of the elements of
the cord and on the behavior of frog em-
bryos. Studies on regeneration in the
spinal cord. II. The effect of reversal of

a portion of the spinal cord at the stage

of the closed neural folds on the healirg
ofthe i cord Masa seec see a eee

585

69

421
19
451

77

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