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Bulletin of the Museum of Comparative Zoology

AUT! SHEAR OV AVR D= ClO BiG

VoLUME 124

MAMMALIAN HIBERNATION

PROCEEDINGS OF THE FIRST INTERNATIONAL SYMPOSIUM ON
NATURAL MAMMALIAN HIBERNATION, MAY 18-15, 1959

Supported by the Office of Naval Research and sponsored
by the American Institute of Biological Sciences.

Edited by

CHARLES P. LYMAN
and
ALBERT R. DAWE

MCZ

Ree Monee

Pic A TV
L i SBSRAR Y

CAMBRIDGE, MASS., U.S.A.
PR eNe TE BD) Ook, Tan Me Urs UM

NoveMBER, 1960

PUBLICATIONS ISSUED BY OR IN CONNECTION
WITH THE

MUSEUM OF COMPARATIVE ZOOLOGY
AT HARVARD COLLEGE

BULLETIN (octavo) 1863 — The current volume is Vol. 124.

BREVIORA (octavo) 1952 — No. 126 is current.

Memorrs (quarto) 1864-1938 — Publication was terminated with
Mole aa:

JOHNSONIA (quarto) 1941— A publication of the Department of
Mollusks. Vol. 3, no. 39 is current.

OcCASIONAL PAPERS OF THE DEPARTMENT OF MoLLusKs (octavo)
1945 — Vol. 2, no. 25 is current.

PROCEEDINGS OF THE NEW ENGLAND ZOoLoGicAL CLUB (octavo)
1899-1948 — Published in connection with the Museum. Publication
terminated with Vol. 24.

The continuing pubhecations are issued at irregular interva!s in num-
bers which may be purchased separately. Prices and lists may be
obtained on application to the Director of the Museum of Comparative
Zoology, Cambridge 38, Massachusetts.

Of the Peters ‘‘Cheek List of Birds of the World,’’ volumes 1-3 are
out of print; volumes 4 and 6 may be obtained from the Harvard Uni-

versity Press; volumes 5, 7 and 9 are sold by the Museum, and future
volumes will be published under Museum auspices.

Bulletin of the Museum of Comparative Zoology
AT HARVARD COLLEGE

VOLUME 124

MAMMALIAN HIBERNATION

PROCEEDINGS OF THE FIRST INTERNATIONAL SYMPOSIUM ON
NATURAL MAMMALIAN HIBERNATION, MAY 13-15, 1959

Supported by the Office of Naval Research and sponsored
by the American Institute of Biological Sciences.

Kdited by

CrHARLES P. LYMAN
and
AusBertr R. DAWE

CAMBRIDGE, MASS., U.S.A.
PRINTED FOR THE MUSEUM

NOVEMBER, 1960

“Think with eompassion on the furry
Where they dig their homesteads deep
And feed on the Summer of their bodies

Through the long Winter of their sleep.”’

Rh. P. T. Corrin

Preface ..

TABLE OF CONTENTS

POCO ANS 2 60a the aioe Re ee ees Bas

IT.

Tt,

WELT.

a BF

Hibernation versus hypothermia
Ravser. sare. cei soy ase ae oto oe

Heat regulation in primitive mammals and in
tropical species... Martin Hisentraut

Comparative ecology of hibernating mammals
.N. 1. Kalabukhov

Some interrelations between weight and hiber-
nation function . Peter Morrison

Torpidity in birds ... Oliver P. Pearson

Kndocrines in hibernation . Vojin Popovie

Histological changes during the hibernating
evele in the Aretie ground squirrel
Wilham V. Maver

Seasonal variations in physiologic functions of
Arctic ground squirrels and black bears...
Raymond J. Hock

Observations on a colony of captive ground
squirrels throughout the year Barbara
Rt. Landau and Albert R. Dawe

Aestivation in the Mohave ground squirrel,
Citellus mohavensis ... George A. Bartholo-

mew and Jack W..-Hudson 2.62.26 2.085
Day-night rhythms and hibernation .. . @.

Rdear JMolk, «tis sees once ete ae ete

Brown fat and its possible significance for

hibernation ... Benet Johansson

Some problems of reproduction in relation to
hibernation in bats... William A, Wimsatt

Stress and neurosecretion in the hibernating

hedgehog -aavo Suomalainen ........
Some physiological principles governing
hibernation in Citellus beecheyt Felix

Strumwasser

193

209

285

XVI.

a VIP,

XVIII.

XIX.

XXIT.

ANT.

AXIV.

XXV.

XAVI.

XXVIT.

XXVITI.

BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The mechanisms of hypoxic tolerance in hiber-
nating and non-hibernating mammals
Robert W. Bullard, George David and C.
Thomas Ni@ROIS oo 5.2% 2<n0 5 dda duce meres ae ee

On the cardiae response in hibernation and
induced hypothermia ... H. S. S. Sarajas

Circulatory changes in the thirteen-lined

eround squirrel during the hibernating cycle

Charles P. Lyman and Regina C.

OF BtIGU: “udcvgs cus wagaeee ween ees. coee

Vascular changes related to hibernation in
the vespertilionid bat Iyotis lucifugus .

Mrank -C. Wallen... o6<S.20wasseeee oes s ote

Peripheral nerve function and hibernation in
the thirteen-lined ground squirrel, Spermo-
philus tridecemlineatus . . . Theodore H.
Kehl and Peter Morrison. .< 02.6. ..0oe80

Tissue respiration and hibernation in_ the
thirteen-lined ground squirrel, Spermophi-
lus tridecemlineatus . . . Marion P. Mever
and Peter Ormisonm: hnius ds anddvecaeeaets

The internal environment during hibernation
a Alamein Toe. Wriediesel. gaes/% a 4 2h.asdmeharee

A study of the metabolism of liver, diaphragm
and kidney in cold-exposed and hibernating
hamsters... Arliss Denyes and Joan Hassett

Phosphates as related to intermediary metab-
olism in hibernators ... Marilyn L. Zimny

Some metabohe specializations im tissues. of
hibernating mammals ... Frank KE. South

The effects of ionizing radiation in hibernation
Douclas WS Sunthy hn oes ee eee

Panel discussion—Albert R. Dawe, Chairman,
IK. I. Adolph, George H. Bishop, Kenneth
(’. Fisher, Donald R. Griffin, Rev. Basile JJ.
Laryet, Cuadd:Prosser>. 2 oc... eee ee

General cdiseussiown, acer soe ices oe ee

PAGE

321

373

387

d07

1960 MAMMALIAN HIBERNATION 5

PREFACE

The papers and discussions collected here represent the pro-
ceedings of the First International Symposium on Natural Mam-
mahan Hibernation. As such, it is hoped this book will be a
milestone in the study of mammalhan hibernation. Anyone read-
ing through this volume will realize that there are great gaps
in our knowledge and that much of the critical work remains
to be done. It is to be hoped that such a realization will spur
the interest in this fascinating field.

In violating the primary principle of homeothermism, the
hibernators encounter problems which are unique in the mam-
malian and avian world. The mechanies of this almost purposeful
abandonment of the warm-blooded state are a challenge to the
investigator, and the means by which the tissues and organs are
able to function at such low temperatures are fruitful avenues
for further research. These questions cry out for more basic
ecology and more sophisticated physiological techniques. A good
beginning only has been made in the fundamental biochemistry
of the problems involved. These same challenges attracted
Claude Bernard and Raphael Dubois in the last century, but
after their time there was only sporadic interest in hibernation
until a reawakening which started in the European laboratories
about twenty years ago. The interest spread to the New World,
and the combined advance of our knowledge in this field during
the past decade has greatly accelerated. The purpose of the
conference was to bring this knowledge together.

To further this purpose, a series of 26 papers were presented
during the first two and one-half days of the Symposium, with
the senior editor as chairman. On the afternoon of the last day
a ‘‘Philosopher’s Panel’’ of seven scientists, with the junior
editor as moderator, discussed the problem of mammalian hiber-
nation as a whole. These men had attended the presentation
of all the papers, with two minor and unavoidable exceptions, but
had not concentrated in this precise field of research. Their
refreshing point of view is presented after the formal papers. A
final period of discussion, in which everyone could participate,
follows the Panel.

The Symposium was held at Massachusetts Institute of Tech-
nology’s Endicott House in Dedham, Massachusetts, from May
13 to 15, 1959. It was sponsored financially by the Office of
Naval Research, and held under the auspices of the American

6 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Institute of Biological Sciences (represented at the Symposium
by Irvin CC. Mohler). Smith, Kline and French Company of
Philadelphia generously supphed refreshments at the end of
each lone day, and we feel sure that all the participants are
evratetul to them.

There are a number of people to whom we would like to
express our personal thanks. Dr. Roger Reid and Captain Bruce
Carr encouraged our original idea for this conference, and have
been most helpful throughout. Mrs. Regina C. O’Brien co-
ordinated the arrangements for the members, took notes at
the meetings, and proof-read and checked the bibliographies of
all the papers. Miss Leola Hoffman of the Office of Naval Re-
search at Chicago took stenographie notes of all the discussions
and assisted in bringing the transcriptions of the discussions and
the panel meeting into their present readable condition. The
help of Mrs. O’Brien and Miss Hoffman has been invaluable.

Captain Trent Ruebush of the Office of Naval Research aided
in planning the conference, and attended the meetings. His
quiet encouragement and sound advice were a never-failing
source of help to us. His untimely death in Cairo is a grea
shock to all of those who knew and admired him.

Ch Pood,
Aah. D.-

PARTICIPANTS

Dr. KE. le. Apontpi, Department of Physiology, School of Medicine
and Dentistry, University of Rochester, Rochester, New York.

Dr. Erie G. Bau, Department of Biochemistry, Harvard Medi-
cal School, Boston, Massachusetts.

Dr. Grorce A. BarTnolomEew, Department of Zoology, Univer-
sity of California, Los Angeles, California.

Dr. Grorce Il. Brsuop, Department of Psychiatry and Neurol-
ogy, Washington University School of Medicine, Saint Louis,
Missouri.

Dr. Bayarp Il. Brarrsrrom, Department of Biology, Adelphi
College, Garden City, New York.

Dr. Mary ANNE Brock, Department of Medicine, Harvard Medi-
cal School, Boston, Massachusetts.

Dr. Roperr W. BuLuarp, Department of Physiology, Indiana
University Medical Center, Indianapolis, Indiana.

Dr. AnBerr R. Dawe, Office of Naval Research, Department of
the Navy, 86 East Randolph Street, Chicago, Illinois.

1960 MAMMALIAN HIBERNATION ‘L

Dr. WinuiAmM Dawson, Department of Zoology, University of
Michigan, Ann Arbor, Michigan.

Dr. H. Aruiss Denyrs, Department of Biology, Queen’s Uni-
versity, Kineston, Ontario, Canada.

Dr. Martin Ersentraur, Zoologisches Forschunegsinstitut und
Museum Alexander Koenig, Bonn, Germany.

Dr. Kennetu C. Fisuer, Department of Zoology, University of
Toronto, Toronto, Canada.

Dr. EUGENE FLAMBOE, Department of Physiology, Colorado State

University, Fort Collins, Colorado.

Dr. G. Epaar Foux, Jr., Department of Physiology, College of
Medicine, State University of Iowa, Iowa City, Iowa.

Dr. Donaup R. Grirrin, Department of Biology, Harvard Uni-
versity, Cambridge, Massachusetts.

Dr. RaymMonp J. Hock, Aretic Aeromedical Laboratory, Fair-
banks, Alaska. Present address: White Mountain Research
Station, University of California, Big Pine, California.

Dr. Benar JOHANSSON, Cardiological Laboratory, Department
of Medicine, Malm6 General Hospital, Malmo, Sweden.

Dr. Nuxonar IT. KauasuKHov*, Institute of Microbiology and
Epidemiology of the South-East of the U.S.S.R.. Saratov,
U.S.8S.R. Present address: Chief Post Office, Post Box n.
10, Astrakhan, U.S.S.R.

Mr. FraNK C. Kantenx, Department of Zoology, Cornell Uni-
versity, Ithaca, New York.

Dr. CHARLES Kayser, Institut de Physiologie, Faculté de Méde-
cine, Université de Strasbourg, Strasbourg, France.

Mr. THropore H. Keun, Department of Zoology, University of
Wisconsin, Madison, Wisconsin.

Dr. BarBara R. LANDAU, Department of Physiology, Saint Louis
University School of Medicine, Saint Lows, Missouri. Present
address: Department of Physiology, University of Wisconsin,
Madison, Wisconsin.

Dr. Basite J. Luyer, Research Laboratory, Department of Bio-
physies, American Foundation for Biological Research, Madi-
son, Wisconsin.

Dr. Cuarues P. Lyman, Department of Anatomy, Harvard Medi-
eal School, Boston, Massachusetts, and Museum of Compara-
tive Zoology. Harvard University, Cambridge, Massachusetts.

Dr. WiturAmM V. Mayer, Department of Biology, Wayne State
University, Detroit, Michigan.

* Not present.

s BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Mr. Micuacnn Menaker, Department of Biology, Princeton Uni-
versity, Princeton, New Jersey. Present address: Department
of Biology, Harvard University, Cambridge, Massachusetts.

Mrs. Marion P. Meyer, Department of Zoology, University of
Wisconsin, Madison, Wisconsin.

Dr. Perer Morrison, Departments of Physiology and otf Zool-
ogy, University of Wisconsin, Madison, Wisconsin.

Dr. X. Josepn Musaccnta, Department of Biology, Saint Louis
University, Saint Louis, Missouri.

Dr. Onrver P. Pearson, Museum of Vertebrate Zoology, Um-
versity of California, Berkeley, California.

Mr. E. T. PENGELLEY, Department of Zoology, University of
Toronto, Toronto, Canada. Present address: Department of
Zoology, University of California, Davis, California.

Dr. Vostxn Popovic, Division of Applied Biology, National Re-
search Couneil, Ottawa, Canada.

Dr. C. Lapp Prosser, Department of Physiology, University of
Illinois, Urbana, Hlinois.

Dr. Marvin L. Rrepesen, Department of Occupational Health,
Graduate Sehool of Pubhe Health, University of Pittsburgh,
Pittsburgh, Pennsylvania. Present address: Department of
Biology, University of New Mexico, Albuquerque, New Mexico.

Capr. TrentTON K. Rurnusnu, U.S.N., Physiology Branch, Office
of Naval Researeh, Department of the Navy, Washineton, D.C.
(Now Deceased ).

Dr. H. 8S. Samutt Sarasas, Institute of Physiology, University
of Helsinta, Helsinki, finland.

Dr. Epwarp ScuoNBAUM, Department of Sureery, Banting In-
stitute, University of Toronto, Toronto, Canada.

Dr. Doveuas EK. Smrrx, Division of Biological and Medieal Re-
seareh, Argonne National Laboratory, Lemont, [linois.

Dr. FraNK KE. SourH, Department of Physiology, University
of Illinois College of Medicine, Chicago, THhnois.

Mr. JOuUN STEEN, Department of Biology, Harvard University,
Cambridee, Massachusetts.

Dr. FeLrx StruMWaAsSER, National Institute of Mental Health.
National Institutes of Health, Bethesda, Marvland.

Dr. PAAVO SUOMALAINEN, Zoological Laboratory, University of
Helsinki, Helsinki, Finland.

Mr. JoHN S. Wiuuis, Department of Biology, Harvard Un1-
versity, Cambridge, Massachusetts.

Dr. WitLtiam A. Wimsatt, Department of Zoology, Cornell Uni-
versity, Ithaca, New York.

Dr. Marityn LL. Zimny, Department of Anatomy, School of
Medicine, Louisiana State University, New Orleans, Louisiana.

I
HIBERNATION VERSUS HYPOTHERMIA

By CHARLES KAYSER
Institut de Physiologie, Faculté de Médecine
Université de Strasbourg
Strasbourg, France

If we compare physiological hibernation of mammals with
experimental hypothermia of homeothermic mammals, three
points appear to be essential :

(1) The difference between homeothermic mammals and active
hibernators in summer as to their response to artificial
cooling ;

(2) The difference between active hibernators in summer and
hibernating hibernators ;

(3) Despite the differences appearing in these comparisons,
a more careful study shows that there are many inter-
mediate states between these extremes. The intermediate
states are shown by the study of the effect of climatic
factors on certain active hibernators in summer, and by the
study of the incomplete homeotherms, especially those
from the Southern Hemisphere.

This special point will not be considered, Dr. Morrison being

more informed than I.
First Point

A) Since Walther (1865) and his pupil Horvath (1876),
we know the essential differences which contrast experimental
hypothermia in active hibernators in summer with that in homeo-
thermie rodents. These differences are:

(1) Artificial cooling (immersion in cold water) produces
death by respiratory arrest in homeothermic animals
when the central temperature falls to 19°C; the same
method fails to stop the respiration of hibernators down
to central temperatures of about 5°C.

(2) The use of artificial respiration allows the lowering of
the central temperature of homeothermic mammals down
to about 10°C.

(3) The speed of cooling, in cold water, is much faster for
the ground squirrel than for the rabbit.

(4) The rabbit’s death in hypothermia is to be ascribed to some
encephalic vascular trouble, as the eye fundus appears
slate-colored.

10 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

These four observations show the importance of the special
resistanee of the nervous system of hibernators towards deep
hypothermia. Hall (1852), Dubois (1896), Merzbacher (1904),
and Uiberall (1934) all emphasized the importance of the
nervous system in hibernation. Lyman and Chatfield (1950,
1953), Kayser et al. (1951), and recently Strumwasser (1959a,b)
also pointed it out. Chatfield ef al. (1948) could show the per-
sistence of nervous conductivity down to 38°C in in vitro experi-
ments,

The question arises as to whether it is possible to show a special
cellular property which would explain this resistance. We hoped
to obtain such evidence by the investigation of the effect of
temperature on the respiration of brain shees with Warbure’s
method; we had observed (1954a,b) that the critical heat inere-
ment of the respiration of kidney slices was significantly lower
(11,150 em. eal.) in the European hamster than in the albino
rat (13,320 em. cal.), the hamsters having been killed either in
the hibernating or in the active state.

We could, however, find no difference in the eritical heat mere-
ment (studied between 388°C and 5°C) for the respiration of
brain slices between rats and European ground squirrels or
European hamsters (Table I).

TABLE |
Critical heat increment of the oxygen consumption of brain
cortex slices of albino rat, albino mouse, European hamster
and European ground squirrel
(Suspension liquid: Krebs [I + glucose, without lactate,
pyruvate or fumarate).

Mean

Critical ;
Number of heat Correlation body
Species Number of measure- increment coefiicient weight
Physiological state animals ments — (gm. cal.) (®t) (gm) ©

Albino rat 18 27 13,485 0.979 199

Albino mouse 12 12 12,205 0.985 24
Muropean hamster

active (summer ) 12 13 13,61] 0.986 2638
European hamster

active (winter ) 13 16 13,051 0.914 285
uropean hamster

hibernating (winter) 13 19 13,326 0.972 270

European ground
squirrel, hibernating
(winter) 5 a) 13,722 0.985 57

1960 MAMMALIAN HIBERNATION 1]

This observation led us to study systematically the critical heat
increment for the oxygen consumption of all the tissue slices we
eould obtain, without any major difficulty, in hibernators and
homeothermie mammals (Table II], A and B).

TABLE ITA
Critical heat Increment of the oxygen consumption of slices of
various tissues of three homeothermie rodents (guinea pig,
albino rat, albino mouse).
(Suspension liquid: Krebs II + glucose, without lactate,
pyruvate or fumarate).

Mean Critical
body Number of heat
weight Number ot measure- increment Correlation
Species (gm.) animals ments Tissue (gm. cal.) coefficient
Guinea pig 846 7 15 Kidney 14,292 0.961
Albino rat 193 13 26 kidney 13,319 0.984
ee oe 199 18 27~—Ss Brain cortex 13,483 0.979
ag oe 193 21 39 Liver 13,918 0.974
ge ~ 178 13 Lf Spieen 14,345 0.906
a et 191 16 17 Heart muscle 8,853 0.878
Albino mouse 19.6 12 20 Kidney 12,668 0.959
ee oe 24.0 12 12 Brain cortex 12,205 0.985
os es 22.9 10 15 Liver 12,068 0.961
os i 21.0 9 Hla Spleen 14,586 0.973
us ms 20.0 8 8 Lung 11,467 0.961
me os 21.7 13 16 Heart muscle 8,861 0.978

He we 24.0 26 26 Diaphragm

7,165 0.968

TABLE IIB

Critical heat increment of the oxygen consumption of slices of
various tissues of three hibernators (Kuropean hamster,
European ground squirrel, marmot) in different physiological
states.

(Suspension liquid: Krebs II + glucose, without lactate,
pyruvate or fumarate ).

Mean Number Critical
Species body Number of _ heat ;
Physiological weight of measure- increment Correlation
state (gm.) animals ments Tissue (gm. cal.) coefficient
European
hamster
uetive

(summer ) 954 10 20 Kidney 11,051 0.991

eae 263 12 13. Cerebral cortex 13,611 0.986

a ral 9 i) Liver 15,981 0.958

es 270 6 11 Heart muscle 7,878 0.943

MUSEUM OF COMPARATIVE ZOOLOGY

TaB_e I]p (Continued )

Vol. 124

12 BULLETIN :
Mean
Species body
Physiological welght
(gm.)

state

uropean
hamster
netive

293

(winter )

285
BS 308
oe 260

Huropean

hamster
hibernating 249
a 270
et 298
« 226
me 241
Muropean
ground
squirrel,
active
(summer ) 154
ae 156
Muropean
ground
squirrel,
hibernating
(winter) 151
- 157
ee 154
ee 159
ee 159
Marmot,
active
(sumnier ) 3,000
oe 3,000
Marmot,
hibernating 2.357
mie 2.357

Number
Number of
of measure-
animals ments ‘Tissue

9 20 I<idney

3 16 Cerebral cortex
13 PA Liver

6 6 Heart muscle
12 25 Kidney
13 19 Cerebral cortex
3 26 Liver

8 8 Spleen
10 17 Heart muscle

5 5 Kidney

D 5 Liver

1] 1] Kidney

5 5 Cerebral cortex
12 23 Liver

6 6 Spleen

6 10 Lung

2 7 Kidney

2 7 Liver

3 ala Kidney

3 lal Liver

Critical
heat
Increment

13,302
13,051
11,168

8,414

11,727
13,501
13,326
11,216
10,792

14,125
10,498

11,084
13,722
11,113
12,588

9,901

14,448
11,388

pe
12,227

11,658

Correlation
(gm. cal.) coefficient

0.982
0.914
0.902
0.900

0.988
0.972
0.945
0.947
0.927

0.959
0.985
0.939
0.984
0.949

0.9835
0.976

0.999
0.995

1960 MAMMALIAN HIBERNATION 13

There appeared to be no systematic difference as to the effect
of temperature on tissue respiration between homeotherms
(albino rat, albino mouse, guinea pig) and hibernators (marmot,
ground squirrel, hamster).

This observation seems normal: we know today that tempera-
tures near O°C do not kill the tissues of homeotherms.

34 _ 182 days old animals Adult animals
3,2 0
30
28
26
24
22
20
18
1.6
1,4
1.2
1,0
eke)

| | — Dinitrophenol

0, mm?/mg Fresh tissue weight /hour

Weight (gm)
w
O

O
13 5 7 O91 1315 17 19 212325272931 | 360 Days

Fig. 1. Effect of Na malonate (0.01 M) and dinitrophenol (10-6)
on the oxygen consumption of brain slices of growing rats (Kayser and
Lucot, 1959).

After this failure, we approached the same problem in another
way; like Tyler (1942), Chesler and Himwich (1944), and
Locker (1958), we decided to make use of sodium malonate and
dinitrophenol in respiratory experiments 7m vitro. We were able
to confirm the observations of Tyler: malonate depresses the
oxygen consumption of brain shees from new-born rats less than
it does in the ease of adult ones (Fig. 1).

14 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

We observed no variation in respect to age in the effect of
dinitrophenol on the oxygen consumption of brain slices from

rats.

35 Cricetus cricetus

’

Dinitrophenol @2)
b =3292
r =0,99

30
©)
25 Na Malonate (26)
: b =3151
r= 0,95
Q)
)
20
35 Rattus norvegicus

Dinitrophenol
b #3111
r 0,99

@3)
b= 3275
r=0,98

Control

Nc Malonate @9)
b=3124
r=098

Log Oo x 10° mm*?/me fresh tissue weight/hour
ine)
(6)

210 250 300 350 400 450

AZT #AG?

Fig. 2. Critical heat increment for the respiration of brain slices from
European hamsters and albino rats under the action of Na malonate
(0.01 M) and dinitrophenol (10-8M) (Kayser, 1959a). Encirecled numbers =

number of animals used,

The use of the same poisons in respiratory experiments on
brain slices from rats or hamsters shows that the depressing

1960 MAMMALIAN HIBERNATION 15

effect of Na malonate is less in hamsters than in rats, and that
dinitrophenol increases the oxygen consumption more (120 per
cent) in hamsters than in rats (70 per cent). The differences
observed are statistically valid (Fie. 2).

We think that the observations of Edwards (1824), Britton
and Kline (1945), Adolph (1948, 1951), Adolph and Lawrow
(1951) and Iliestand ef al. (1950) on the resistance of young
mammals and hibernators to anoxia and hypothermia could be
explained by the fact that glycolysis plays a more important role
in the energetics of the brain of young homeothermie mammals
and libernators than in adult homeotherms.

>) It is well-known that the variations of nervous excitability
with temperature depend to a large extent on the Ca/K ratio:
if IX inereases in the suspension liquid, the nerve block appears
at a higher temperature and if K decreases the block appears
at a lower temperature. If there is an increase in Ca concentra-
tion, the nerve block appears at 0—2°C instead of 10°C.

Studying the leneth of the different phases of the EKG we
had observed (Kayser, 1957a) that the effect of temperature
on the distance between the S inflection and the summit of the
Osborn-wave (Osborn, 1953) in cooled homeotherms was nearly
null (critical heat increment of 1,000 e@m.eal. approximately ).
The effect of temperature on the same distance in cooled hiber-
nators showed the same heat increment as for the oxygen con-
sumption of heart muscle slices (7,000-8,000 @m.eal.). The value
of 1,000 could not be statistically ensured. We were then unable
to assert that the effect of temperature on the repolarization was
different in cooled homeotherms and in cooled hibernators.

Recently, G. Bach (personal communication, 1959) repeated
the same experiments with cooled dogs and observed the same
value for the critical heat increment for S-O distance (7,000-
8,000 em.cal.) as we had observed in cooled hibernators (‘Table

U0 ip ie

TasuE Il

Critical heat increment of the quick repolarization phase of
the heart muscle

Critical

heat Number of
increment Correlation measure- Number of
Species (gm. cal.) Author coethicient ments animals
Dog 8,145 Bach 0.64 43 D
European hamster 7,843 Kayser 0.74 D7 5

European ground

squirrel 7,093 Kayser 0.81 39 4

16 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Second Point

The difference between active hibernators in summer and hiber-
nating hibernators appears with much sharpness if we compare
the basal metabole rate of active hibernators in summer with the
minimal heat production of the same species during deep hiber-
nation (Fig. 3).

Tot .Cal./24h Tot .Cal./24h

100 10.000
o 5000
e

Cal/24h - 636 P0862
20 Basal Metabolism

Summer 2,000
iG 1,000
5 ‘e 0500

Cal/24h = 2,04 pose

Hibernation

M-Arctomys Marmota 0100

E - Erinaceus europaeus

Cr- titi cricetus

Ci- Citellus citellus

G - Glis glis 0050
El- Eliomys quercinus

Mu-Muscardinus avellanarius

V -Vesperugo noctula

F “Pipistrellis pipistrellus 0020

10 50 100 5001000 5000 gm
Weight

Fig. 3. Basal metabolie rate of some active hibernators in summer

and hibernating hibernators (Ixayser, 1959b).

1960 MAMMALIAN HIBERNATION 17

In the active state, the surface law accounts for the heat pro-
duction but in the hibernating state the surface law disappears
and the heat production is the same per unit of body weight for
the marmot of 2.5 kg. and the bat of 5 gm.

The disappearance of the surface law in the hibernating
state is to be related to the special endocrine syndrome in hiber-

100 Ground hog Nr1 ®
. == = Nee
90 [= —_ Nr3 ©

— — Nr4°
80

70
60
50
40

Respiratory rate (movements /hourJ

4 @)

10 20 30 ml/kg/hr
Oxygen consumption

Fig. 4. Respiratory frequency versus oxygen consumption in hibernat-
ing marmots (Kayser, 1940a).

nation and the special functioning of the nervous system in this
state: Galvao (1947, 1948-49, 1950-51) shows the disappearance
of the surface law in dogs and men acclimatized to tropical ch-
mate, and Brendel and Usinger (personal communication, 1959 )
find this also in the deep hypothermia of narcotized dogs, cats
and rabbits.

18 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The special functioning of the nervous system in hibernation
is shown if we study the respiratory frequency versus the oxygen
consumption im six-hour experiments on hibernating marmots
(Kayser, 1940a) (Fig. 4+), or the heart frequency versus the
respiratory frequency in hibernating European ground squirrels
(Kayser, 1957b) (Fig. 5).

movements /nin.

5
4

oO 3

2 2

ea
1

012 3 4 5 6 7 8 J beats/min.
Heart rate

Fig. 5. Heart frequency versus respiratory frequeney in hibernating
Kuropean ground squirrels (Kayser, L957b),

igure + shows that there is no correlation between respiratory
frequency and oxygen consumption, and Figure 5 shows that
there is a negative correlation between heart frequeney and
respiratory frequeney in deep hibernation. Both funetions, res-
piratory and cardiac, remain under the influence of the nervous
system but there is no longer integration of these two functions
in respect to the energetic needs in hibernation.

Third Point

Despite these enormous differences which contrast a hibernat-
ine hibernator to itself in the aetive state in summer, and whieh

1960 MAMMALIAN HIBERNATION 19

contrast it in artificial cooling experiments to a cooled homeo-
therm, there are as many intermediate states between hibernators
like bats and poikilotherms.

All the authors who, as Shaw (1921, 1925a,b), Johnson (1930),
Ismagilov (1955) and others, assert that climatic factors inter-
vene in the entrance into hibernation, implicitly emphasize that
hibernation —a true regulation to a minnnal energetic expendi-
ture — is also the effect of the overstraining of the animal. We
arrive thus at the conception of Suomalamen and Nyholm
(1956) that hibernation is also an adaptative syndrome of
Selye.

In 1958 we detected in summer, in a small hibernator, the
garden dormouse (50 em), states of hypothermia very near to
true hibernation (Kayser ef al., 1958; Lachiver and Kayser,
1958). These states were obtained by maintaining the animals
at 5-7°C with or without nest-buildine material and food. We
obtained in this way states of acclimatization with increased heat
production. As in our experiments in 1939 (Kayser, 1939b), the
oxygen consumption measured in the afternoon in experiments
at 5°C was above 5,000 ml/kg/hr. But we also observe states of
deep hypothermia (central temperature below 10°C) in_ the
morning (Table IV).

TABLE IV

Frequency of deep hypothermia in garden dormice, in August.
The animals remained at +5°C environmental temperature.

Animals Animals having starved for
fed 24 hr 48 hr 72 hr
Number of hypothermic
animals 6 17 V7 10
Number of normothermic
animals 62 2 0 0

From Lachiver and Kayser (1958).

After 48 hours starvation, one hundred per cent of the animals
are in deep hypothermia. The combined effect of cold and starva-
tion induces hypothermia. This hypothermia is only an accentua-
tion of the diurnal rhythm: in the evening the animals arouse ;
if they have the opportunity to feed, they keep their normal
body weight; if they are starved, the body weight decreases by
9 per cent during the first 24 hour starvation period, 5 per
cent from the second to the third day, and 2 per cent the last day.

20 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

This weight loss is very important, but in two dormice starved in
June for 8 days (the animals remaining untouched for this time)
the weight loss was 0.7 per cent per day, a value very near to that
observed in deep hibernation (0.6 per cent).

The oxygen consumption and the heart frequency in deeply
hypothermic dormice were very inconstant (Table V).

TABLE V
Oxygen consumption, heart frequency and colonic temperature
of 5 garden dormice staying at +5°C in August.

Mean heart

Mean Mean colonic frequency at
Number length of Mean temperature the end of
of Number measure body at theend of — the measure-
measure- of ment weight Ov consumption the measure- ments
ments animals’ (min.) (gm. ) “(ml/kg/hr) ments (°C) (beats/min. )
3 3 76 (+32) a See) 641 ee 12) D: 7 (+0.6) DieiGs= 73")
2 2 87 (#47) 50.2 (£0.7 150.8 (= 34) hg me eh (0!)
8 4 59 (= 24) 52.1 aoe 627.8 (+ 99) ied (EOL9) 65 C= .8)
6 3 57 (==20:) 56.3 (253.3) 1,528: (25256) 9 23) ¢225:0) 94 (+18)
2 | 46 (10) N4.5 2,840 (£506) 12:0) C2205.) 330 (one

measure-
ment )

From Kayser, “Lac hiver and Rietse h (1958).

If we study the animals from August to December (Table
V1), we see that the minimal oxygen consumption falls from
64.1 (August) down to 39 (October), 28.5 (November) and 31.1
(December) (Tables V, VIT)

TABLE VI

Hypothermia and hibernation in garden dormice staying at

9-7°C from August to January (animals in individual cages, fed

and with nest-building material at their disposal).

Number Number Number

Environmental Number of of of
Month temperature of obser- active hypothermic Torpidity
(°C) animals vations states states (% )

August 5 6 68 62 6 8.8
September i 4 S4 79 5 53.9
October 5 4 LT 3D 77 68.6
November 5 5 116 13 103 88.7

) 92.7

December 6 5 55 4 5] 92,

From Lachiver and Kayser (1958).

1960

MAMMALIAN HIBERNATION

The frequency of the hypothermic states in fed animals in-
creases sharply in October: we pass from hypothermia to hiber-

nation

(Table VIT).

TABLE VII

Oxygen consumption of 4 garden dormice, staying at +5°C from
September to January (animals in individual cages, fed, and

with nest-building material at their disposal).

Number

ot Number Body Oxygen Length of
Month measure- of weight consumption measurement
ments animals (gm) (ml/kg/hr) (min. )

October 14.~—~CS~tC‘(‘« HCCC) 89.0(E 4.7) 201
ve 6 2 55.3(+ 4.8) 55.8(+ 4.1) 200

“s 3 2 70.2°C211.1) 280.8 (+ 97.8) 225
November 5D 2 88.9(= 1.3) 28.3:( =. 5.1) 193
“ 6 2 95.7(= 8.9) 79.3(4 8.6) 194

ee » 2 95.0(= 7.2 354.5 (77.7) 194
December 8 3 72.8(= 6.1) S11 Se: 49!) 194
as 5) 3 75.8(+ 8.9) 59.3(= 5.9) 183

From Kayser, Lachiver and Rietsch (1958). :

The study of the thyroid glands, removed in August or in
December or January on the first day of the experiment, shows
a hyperactivity in August, and a reduced activity in January or
December, but the degree of involution is very variable (Plate).

The conelusion is evident: staying at a low temperature pro-
duces, in small hibernators, states very near to true hibernation
in spite of very active thyroid glands. These deep hypothermic
states are, at the beginning, only an accentuation of the normal
diurnal rhythm of temperature ; the ‘‘internal clock’’ still works :
in the evening the animal arouses. In deep hibernation we no
longer observe a difference between the oxygen consumption from
9 a.m. to noon and that measured from 3 p.m. to 6 p.m. (Table
NEG):

TaBLeE VIII

Oxygen consumption of hibernating garden dormice
(December/January), staying at +6°C (two measurements on
each day, regularly, on the same animal, from

9 to 12 a.m. and from 38 to 6 p.m.).

Number
of Oxygen ;
measure- consumption Standard
: ments — (mil/kg/hr) __ deviation
Morning 16 38.87 12.3
Afternoon 16 42.53 22.1

(C= 0:57)

29 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Llowever, all conditions remaining constant, the same animals
again show the normal diurnal rhythm spontaneously in Febru-
ary, at the end of hibernation: the frequency of arousals is much
ereater in the evening; the ‘‘internal cloek’’ works again.

METABOLISM

MAMMALS A. BASAL 100

—— OBSERVED
—- ~EXTRAPOLATED

TP
H AIR TEMPERATURE IN CENTIGRADL

My Basal metabolism rate =100

Metabolism

-20 -10 0 +10 +20 +30°C

Environmental tempercture

Fig. 6. Increase in heat production by tropical adapted and Arctic

adapted animals (A), and by some hibernators (B).

We have asserted already (Kayser, 1940b) that hibernation
1s realized by the double effect of internal rhythm and external
factors. But, if external factors are able to cool active hiber-
nators, it is only possible because hibernators show a deficient
physical thermal regulation and that, in autumn the chemical

1960 MAMMALIAN HIBERNATION 23

regulation also decreases (Kayser, 1939a). We insisted on this
point and reported the important increase in heat production
obtained in summer in active hibernators by lowering the en-
vironmental temperature. We compared the heat production of
hibernators in summer with the heat production of clipped rab-
bits and of rabbits with thoracic medullary section. The increase
of heat production per degree of lowering of the environmental
temperature was 6.0 per cent. This observation is in opposition
to the observations of Scholander et al. (1950), Erikson (1956)
and Irving (1958) (Fig. 6).

The values related for the Arctic ground squirrel by Scho-
lander ef al. (1950) are for the most part extrapolated, and the
values really measured by Erikson (1956) on the same animal at
—20°C are much higher. But Erikson affirms that there is no
inerease in the heat production of quiet Arctic ground squirrels
between +30° and +5°C. This seems to be real, but I think that
the quiet animals studied at 5°C by Erikson were hypothermic.
Erikson himself, like other physiologists, reports the abnormally
accentuated diurnal rhythm in oxygen consumption in the Arctic
ground squirrel. Such a lowering of oxygen consumption during
sleep cannot be understood in such a perfect homeotherm as the
animal described by Scholander.

It is difficult to understand why such a perfect homeotherm
would become hypothermic and hibernate, if its peak metabolism
is observed at temperatures near to the lowest temperatures
recorded at the coldest places on the earth.

General Conclusions

It is evident that the experimental hypothermia of the homeo-
thermic animal fundamentally differs from hibernation, as the
functioning of the nervous system of the awake and _ active
hibernator is different from that of the homeotherm; it is also
evident that a hibernating hibernator may not be confused
with a hibernator made hypothermic in summer by the sup-
pression of its thermoregulation, and that hibernation appears,
then, as a regulation to a minimum level recorded only in certain
well-defined species; it is not less obvious that between the
extremes there are intermediaries: the small-sized true hibernat-
ors — bats and garden dormice in the active state in summer —
supply us with a first example.

The states of dormaney recorded in prosimians (Bourliére,
Petter and Petter-Rousseaux, 1956), in marsupials (Coleman,

24 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

1938; Fleay, 1944; Bourliére, 1951), in the female bear (Matson,
1946; Lobatehev, 1956; Hock, 1958), and in the raccoon (Sharp
and Sharp, 1956) are other examples.

The accidental hypothermia of certain birds (hummingbirds,
swallows) is still another example (Dupond, 1987; Huxley et al.,
1939; Koskimies, 1948; Bartholomew et al., 1957); the birds
‘‘adapt’’ themselves to hypothermia.

These hypothermic states (the examples could be multiplied)
are withstood, if deep, but for rather short times: in our experi-
ments on hamsters they could not go beyond 48 hours, unless
the animal was rewarmed every other day (Kayser, 1955). If
the hypothermia stabilized itself at about 30°C (black bear), it
proceeds as the hypothermia of 5°C of a true hibernator of
the Northern [emisphere.

But in hibernation also a hypothermia of several months can-
not occur: in the ground squirrel, the longest durations we have
observed were of 85 to 40 days. If, in the garden dormouse, they
went beyond 25 days, death was the almost unavoidable conse-
quence. It is our behef that only the poikilothermic animals may
hibernate for months without any thermal ascension being neces-
sary.

Thus, studied in a single species, such as the garden dormouse
or the bat, true hibernation may show analogies with the hypo-
thermia of hibernators in summer. Studied in the whole animal
scale, hibernation places itself between the ‘‘ Winterstarre’’ or
winter rigidity of Eisentraut (1933) and the ‘‘Winterruhe’’ or
dormancy, and from the dormancy one may pass to the states of
accidental hypothermia. If the extreme eases are sharply defined,
we must, however, make allowance for the intermediaries.

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WALTHER, A.
1865. Studien im Gebiet der Thermophysiologie. Du Bois-Reymond

Arch. Physiol., 1865:22-51.

DISCUSSION FOLLOWING KAYSER’S PAPER

Two questions were raised (the first by BISHOP, the second
by SOUTH) which attempted to specify more particularly dif-
ferences observed in the uptake of oxygen by the brain slices.
In the first case, the notion was advanced that brain or nerve
has two ‘‘kinds’’ of metabolism, one seen in the resting state,
and one in activity, and that KAYSER may have been actually
measuring this difference rather than a difference truly associ-
ated with the ‘active versus the hibernating state. The second
question cast doubt on the use of competitive inhibitors such
as malonate, since malonate (as a competitive inhibitor) might
be more active at a lower temperature and hence modify the
results (rather than the effect noted being strictly physiological).

In the first instance, KAYSER was not aware of any experi-
ment which would affirm that there would be any difference in
sign of activity or of rest in brain slices from awake animals
(rats or hamsters) killed by neck section, and in the second in-
stance he stated that he had seen no difference in the critical
heat increment of the oxygen consumption of brain slices in the
presence or absence of malonate either in the hamster or rat.

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HEAT REGULATION IN PRIMITIVE
MAMMALS AND IN TROPICAL SPECIES

By Martin EISENTRAUT
The Alexander Koenig Institute
tor Zoological Research and Museum

Bonn, Germany

The state of hibernation into which some homeothermic ani-
mals fall during the period of unfavorable external conditions
is characterized, among other concomitant phenomena, by the
behavior of the body temperature. A hibernator is capable of
becoming cold just like a poikilothermic animal. In this case,
however, there is not attained a complete elimination of the heat-
regulating arrangements, but merely a change-over, an adjust-
ment of the regulation to a lower stimulus threshold. This indi-
cates to us that the problem of hibernation is essentially a prob-
lem of heat regulation and heat economy.

The varying level of development of heat economy in animals
quite generally is shown in the relation of the body temperature
to the outside temperature (Fig. 1). The poikilothermie animal
is toa large extent dependent on the warmth of the environment.
If this decreases, the body cools thoroughly and the animal in
question gradually falls into a state of torpidity. Many poikilo-
thermic animals of the temperate zones, for example, some insects,
can be sub-cooled in the winter below 0°C, without freezing of
the body fluids. When a eritical point is reached, the so-called
sudden transition of temperature occurs: the body suddenly
heats up to almost 0°C, and not until then does congealing of
the body fluids ensue, and the cooling ends in death.

The homeothermic animal, on the other hand, is able to
maintain its body temperature at a level which is optimum for it,
independent of the temperature of the environment. As the ex-
ternal temperature drops, the homeothermic animal combats the
cooling of its body by increased metabolism, by motion, and by
other precautionary measures. In this way it counterbalances the
heat loss by increased heat production. Thus, for example,
homeothermic animals of the polar region ean withstand very
considerable degrees of cold without harm. Other homeothermic
animals lack such a power of resistance against cold. With long
or intense action of cold the resistance disappears and a hypo-
thermia comes about. If in this process the temperature drops

32 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

below a certain level, which varies with the different species, the
normal nerve function is blocked; respiration and heart-action
stop. In this way the hypothermic state likewise leads to a
lethal end.

This is different in the case of the hibernator, provided that it
has the internal predisposition toward hibernation which may
be caused by hormones. If the environmental temperature drops
to a critical point, which is a characteristic one for the individual
species, the hibernator puts its regulating mechanism out of

Body Temperature

- 10° ————— —+ + = a
0

30° 25 20 15 10 5

External Temperature

Mig. 1. Schematic curve course of the body temperature versus the

external temperature.

circuit and becomes cold. Its body temperature approximates the
environmental temperature more or less, as in the case of a
poikilothermie animal. Not until the minimum temperature,
which hes around 0°C, is approached, does the regulating mech-
anism become active again; as a matter of fact, this is attributable
to the sensitivity, which is maintained even at low temperatures.
There comes about an increase of the metabolism, and with that
a production of heat, which under certain circumstances can
lead to an awakening from the lethargy of hibernation. This is
an essential property of hibernators and one which distinguishes
them from non-hibernating homeothermie and poikilothermie ani-
mals.

1960 MAMMALIAN HIBERNATION

In respect to these peculiarities of heat regulation, it is quite
venerally of interest to consider the heat economy in various
species of mammals, from a comparative standpoint. ILere we can
begin with the assumption that in the course of phylogeny homeo-
thermism has developed from poikilothermism. This process is
once more briefly repeated during the ontogeny of the individual :
the young mammal, for example, a mouse, does not aequire the
ability to maintain a temperature of its own, independent of the
environmental temperature, before the post-embryonic nestling
period.

Not all homeothermic animals have attained the highest degree
of perfection in respect to their heat regulation. Close investiga-
tions have demonstrated that, especially, phylogenetically old
mammals are often still characterized by a very primitive heat
economy. Formerly this was occasionally designated as hetero-
thermism and was separated from the homeothermie and poikilo-
thermic animals as a third group. | do not consider this tripartite
division as a very fortunate choice, but | distinguish between
higher and lower warm-blooded animals, which are contrasted
to the cold-blooded ones as a unit (Hisentraut, 1953). In this
ease, of course, it must not be expected that these two groups
of homeothermie animals can be sharply separated from each
other. Rather, it is more in keeping with our concept of phylo-
genetic development to find that there is a smooth transition
from the one into the other.

Kirst, I will single out only a few examples: man, among
others, belongs to the higher warm-blooded animals with a very
perfect heat regulation. In man, the rectal temperature varies
in the daily rhythm from about 36.7°C early in the morning to
37.5°C late in the afternoon. The Carnivora and the Ungulata
are likewise highly developed warm-blooded animals. — In
the dog and the cat, the range of variation amounts to about
92°C and extends from 37.5°C to 39.5°C. The horse and the
bovine show temperatures between 37.5°C and 38.5°C, hence a
range of variation of only one degree (Hisentraut, 1956b).
Hieher warm-blooded animals, therefore, have a relatively high
average temperature independent of the environmental tempera-
ture, and a relatively slight range of variation of their activity
temperature occurs within the daily rhythm.

In contrast to this, amone the lower warm-blooded animals the
average temperature is relatively low and the range of variation
of the activitv temperature is often, but not always, relatively
large. The Madagascan Tenrec, Centetes cecaudatus, is a typical

34 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

example of a lower warm-blooded animal. I shall return to a
consideration of it soon.

Before we discuss further examples in detail, it seems im-
portant to define briefly the individual temperature ranges. The
range of activity embraces a/l temperatures which an animal
shows during the waking state avd during dormancy. In this
euthermic state the animal is at all times capable, without re-
straint, of makine use of all its motor and sensory funetions.
If the body heat sinks below the lower limit of the range of
activity, that is, if it drops below the activity threshold, it reaches
the hypothermic range of lethargy. Correspondingly, if the
upper limit of the activity range is exceeded, we can speak of
hyvperthermic temperatures.

The data and the temperature measurements available for
mammals up to now have shown that, in general, higher warm-
blooded animals have an activity temperature above 36°C, while
in lower warm-blooded animals it lies, on the average, below 36°C,

The following compilation (Fie. 2) offers a series of further
examples of activity temperatures in mammals. Ilere, it is a
question partly of species which I was able to examine myself,
and partly of data from the literature, only a few of which, to be
sure, can be used for our purposes.

Of the higher warm-blooded animals (1) there are considered
here only the previously-mentioned representatives : man, cat and
bovine. In this group, there might be mentioned also a whole
series of lagomorphs and rodents, such as rabbits, guinea pigs
and vats. The great order of Rodentia, which has very extensive
ramifications, however, also includes representatives which pass
over into the realm of the lower warm-blooded animals on the
basis of thei very labile heat economy.

Of the lower warm-blooded animals listed in Figure 2 (IT).
primarily species are mentioned whieh we must consider old
from a phylogenetic standpoint: of the monotremes Tachyglossus
aculecatus (Sutherland, 1897; Wardlaw, 1915); of the marsupi-
als Marmosa cimerca (Hisentraut, 1955) and Metachirus nudi-
caudatus (Morrison, 1946); of the inseetivores Centetes ecauda-
fus (Hisentraut, 1955), Hemiechinus auritus, and Paraechinus
acthiopicus (Kisentraut, 1952, 1956a); from the order of the
Xenarthra the sloth Bradypus griseus (Britton and Atkinson,
1938), and the armadillos Tolypeutes conurus and Tatus novem-
cinctus (Hisentraut, 1932a,b), and of the scaly anteaters Janis
fricuspis (Kisentraut, 1956¢).

1960 MAMMALIAN HIBERNATION 39

In most of the lower warm-blooded animals considered here, in
all of which the average level of the activity temperature les
below 36°C, their ereat range of variation is striking. It is most
pronounced im the Madagascan hedgehog Centetes ecaudatus. |
had the opportunity to examine quite closely and to measure

22° 23 24 25 26 27 28 29 30 31 2 33 34 35 36 37 38 49 40 41
as
Homo
Felis IT
ee

lachyglossus
Marmosa
Metac hirus

Centetes <=
Hemiechinus
Paraechinus
Bradypus
Lolypeutes

Tatus

Citellus

Cricetus

Muscardinus
Rhinolophus “hipposideros :
Plecotus awritus

Myotis myotis

Rousettus angolensis
Rhinolophus landeri ~é=
Hipposideros caffer
Eptesicus tenuipinnis
Graphiurus” -

Galapo

Perodicticus

Fig. 2. Range of activity temperature in different mammals. Tempern

ture in degrees Centigrade,

the body temperatures of two specimens of this species, which
probably stands on the lowest stage of the placental or monodel-
phic mammals (Kutheria). The range of variation amounts to
more than 10°C, from 24.1° to 34.8°C. I should like to state
in this connection that the high values are reached only with
very intensive activity, and the low values after very long
dormancy. When the animal is aroused from its deep. sleep,
it is still able, in the lowest range of the activity temperature, to

36 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

react spontaneously to external stimuli, to move normally, to
drink and to ingest food. Only with body temperatures from
24°C on down does a lethargy, and with it an inhibition of the
bodily functions, ensue.

In IMigure 2, accordingly, the continuous lines indicate the
range of variation of the activity temperature, which occurs for
each species in the daily rhythm. We frequently find, however,
amone the lower warm-blooded animals, in addition to this, a
dependence of the body temperature on the level of the environ-
mental temperature. If the latter drops off very much, some
animals, for example, Tachyglossus, Centetes, and two species
of hedgehog, Paracchinus and Hemiechinus, and also Bradypus
and Tolypeutes, incline toward hypothermia. | have indicated
this phenomenon by dotted lnes. Conversely, with very high
environmental temperatures, the body temperature can exceed
the upper limit of the normal range of activity, as shown, among
others, by the observations made by Morrison (1946) on Meta-
chivus and Didelphis. | do not intend, however, to enter into
the details of these hyperthermic phenomena here.

We can, therefore, state quite generally that the lower warm-
blooded animals are characterized by a very labile heat economy
and a primitive regulatory mechanism.

Of the species mentioned, Tachyglossus and the three repre-
sentatives of the Insectivora belong to the hibernators or, at any
rate, to the species which are able to undergo at least a volun-
tary hibernation. With this we come to the consideration of the
hibernators themselves. In Kieure 2 (IIL), the activity ranges
of the body temperature outside of the hibernation period are
indicated for a few of the best known representatives. In their
case, too, it strikes one’s attention that the range of variation of
the normal body temperature is very broad. Thus, for example,
in the Kuropean hedgehog, Lrinaceus curopaeus, the activity
range of the body temperature extends, according to Groebbels
(1926) and according to my own observations (Hisentraut,
1956a), from 31.1° to 36.7°C. According to Wade (1930), Citel-
lus tridecemlineatus even has a range of variation from 30° to
39°C. In the hamster, Cricetus cricetus, | found a range from
32.5° to 35.5°C (EHisentraut, 1928). This imperfection in heat
regulation among hibernators has been stressed frequently in
the literature, and, on the basis of this peculiarity, we will prob-
ably have to include also all known hibernators in the group of
the lower warm-blooded animals.

1960 MAMMALIAN HIBERNATION

The order of the Chiroptera (Fig. 2, IV) assumes a special
position among the mammals in respect to heat economy. As we
know, the representatives of this order inhabiting the temperate
zones are hibernators. Concerning the flying foxes (Megachirop-
tera), which are distributed through the tropics and subtropies
of the Old World, only few data are available coneerning the
level of the heat economy. In the flying fox, Rousettus angolensis,
with habitat in tropical Africa, | was able to show an activity
temperature from 34.4° to 38.6°C (Hisentraut, 1940). With pro-
tracted action of cold it is possible to go below the lower thresh-
old of activity, and a hypothermic lethargy then ensues.

For the representatives of the Microchiroptera with habitat im
the temperate zones, a very striking lability of the body tempera-
ture was shown. The range of activity, which is very hard to
define accurately in this case, is relatively large. In Rhinolophus
hipposideros, for example, it extends from 34.4° to 37.4°C, in
Plecotus auritus from 35° to 38.2°C, in Myotis myotis from
35.6° to 39.6°C (Hisentraut, 1934), but it is possible to drop
below this range of activity even during normal day sleep. A
state of lethargy is then reached, which I designate as ‘‘day-
sleep lethargy,’’ and from which the animals awaken in the
evening. In this case the body temperature rises again up to the
activity range, starting from the inside and proceeding outward.
| should like, however, to stress expheitly that during dormaney
it is by no means inevitable that a dropping of the body tempera-
ture below the activity threshold must produce a lethargy. Every-
one who investigates the sleeping quarters of bats during the
warm season of the vear makes the observation that the animals
in question very frequently, even in a relatively cool environ-
mental temperature, are immediately ready for flight and_ fly
away, and hence are not lethargic. But it does also occur in
cool weather that some animals are in a hypothermic and
lethargic state and can be seized with one’s hand without diffi-
eulty. Animals kept in confinement very often show day-sleep
lethargy. For the bats themselves the strong inclination toward
poikilothermism is by no means a disadvantageous phenomenon,
but ean be of a certain usefulness in the lives of the animals, for
through the reduction of metabolism connected with hypothermia
the animal saves fuel in the form of insect food.

The demonstration of the primitive heat economy in bats of
the temperate zone, which, as has been mentioned, are hiber-
nators, gave rise to an investigation also of purely tropical repre-
sentatives of this order of mammals in respect to their heat regu-

38 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

lating capacity. The observations made by me in the Cameroons
(West Afriea) (Hisentraut, 1940; 1956e) on Rhinolophides and
Vespertilionides likewise indieated a very high range of varia-
tion of the activity temperatures. In this case it was shown
that the lower lmit of the range of variation hes considerably
lower than in the representatives adduced for the temperate zone.
In Rhinolophus lander: the activity range extends from 28° to
37.6°C, in Hipposideros caffer trom 314° to 38.4°C, and in Epte-
steus ftenwipiniis trom 32.2% to 39.2°C. Beyond this, however,
with sufficiently low environmental temperature — in experi-
ments condueted in a refrigerator —a drop of the body tempera-
ture into the hypothermie range occurred even in these tropical
species. A few more detailed data on this follow :

In /ipposideros calfer the body temperature can drop below
the activity threshold if the environmental temperature drops
below 24°C. [ should like to state in this connection that in the
tropical regions, especially in the West African tropical rain
forest region, the outside temperatures show only very slight
fluctuations during the day as well as during the vear; they vary
only between about 24° and 28°C. Normally, therefore, a bat
never gets into environmental temperatures which lie below 24°C,
and this explains the facet that this temperature stage represents
exactly the eritical pomt at which one can go below the activity
range of the body temperature with ensuine hypothermie day-
sleep lethargy. Beyond this a day-sleep lethargy could be ob-
served also as a normal phenomenon, hence one occurring under
natural conditions in a few species, for example, Rhinolophus
landeri: this observation was made, in fact, on the cool heights of
the Cameroon mountain range, where the external temperatures
show a greater variation and a much deeper drop than in the
purely tropical lowlands.

Just as their representatives in the temperate zones, the tropi-
cal bats also awaken of their own accord in the evening from their
day-sleep Jethareyv. They do this, in faet. under the influence of
the firmly impressed day rhythm. Likewise, they awaken when
they are taken from the refrigerator back into a warm environ-
ment. The capacity to regain heat was paralyzed, however, in
animals which had remained without food and had not stored
sufficient reserve nutrients in their bodies, so that a rise of the
hody temperature up to the activity range could no longer take
place. This strong dependence of metabolism and of heat produe-
tion on the food intake and the state of nutrition probably, im
general, plays a certain role among Chiroptera.

1960 MAMMALIAN HIBERNATION 59

In the small tropical species, Hptesicus tenurpinnis, | was
further able to show that the day rhythm disappears and no
awakening occurs in the evening if the environmental tempera-
ture drops below 20°C (Hisentraut, 1940). The day-sleep
letharey then becomes a permanent lethargy, which merely re-
sembles externally the hibernation lethargy of the bats in the
temperate zones. In respect to the lethal temperature, it could
be observed that in a few tropieal species even a protracted stay
in-an environmental temperature of 8°C or below, with a corre-
spondinely large drop in body temperature, imperils life. These
animals then lose the capacity to recover heat and perish, while
species of the temperate zones can be cooled without harm
below O°C. This behavior of tropieal bats reminds one of Weig-
mann’s observations (1929) on tropical reptiles, amone which,
likewise, even temperatures above 0°C bring about death through
cold.

In summarizing, it can be stated that among Chiroptera, es-
pecially among Microchiroptera, quite generally the devices for
heat regulation and the capacity to preserve a constant body
temperature are very imperfectly developed and have remained
at a primitive stage. Therefore, [| should like to place the
Chiroptera, in respect to their heat economy, on the lowest stage
of the lower warm-blooded animals. Kayser (1957) even goes
so far as to designate them as poikilothermic.

In connection with the investigation of tropical bats [I was
interested in the question: How do other tropical mammals be-
have, whose representatives in the temperate zones are hiber-
nators (Fig. 2, V)? During my last stay in the Cameroons, I had
the opportunity to catch a small dormouse, Graphiurus murimus,
which has its habitat there, and to bring a few lying specimens
along to Germany. The temperature measurements undertaken
on it, determined during a stay in medium environmental tem-
peratures between about 12° and 22°C, indicated a breadth of
variation of the activity range from 34.8° to 40.1°C. After
transfer into a cold environment (2.5° to 4.5°C) the lower limit
of the activity temperature still dropped a bit —in fact, to
23.5°C. But no hypothermic temperatures and no lethargy en-
sued, as | had actually expected on the basis of a very old state-
ment by Cuvier (according to Heck, 1914). Rather, the animals
were able to maintain their body temperature on the level of
the activity range. These observations, which are to be con-
tinued, have therefore shown up to now that this tropical dor-
mouse also has a great range of variation of the activity tempera-

40 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

ture like the Kuropean sleeping mouse Muscardinus avellanarius
(Hisentraut, 1929), which is a hibernator.

At the same time, | had the opportunity to make temperature
measurements on a tropical representative of the lorises, Galago
senegalensis. These measurements have shown that the activity
temperature is relatively high and the range of variability,
from 36° to 38.6°C, is only relatively small. We ean therefore
probably include Galago amone the higher warm-blooded ani-
mals, while we must classify the potto, Perodicticus potto, pre-
viously investigated by me (1956a) and belonging to the same
suborder as Galago, among the lower warm-blooded animals on
the basis of its low activity temperature. Evidently, among the
lorises, differine stages in the level of development of the heat
economy have been attained. It should be mentioned here that
a few Madagascan dwart-lemurs evidently hibernate, for ex-
ample, Jlicrocebus.

Kven the few examples given here show us the considerable
differences in the level of development of the homeothermism
of the mammals; to a certain degree they give us a picture of
the course of phylogenctie development from poikilothermism to
homeothermisim in a very general way.

On the basis of the results of investigations by Chatfield e# al.
(1948), and by Kayser (1958) on hibernators, the essential dif-
ference between the two groups of homeothermie animals seems
to me to be the different sensitivity of the central nervous system
and of the peripheral nerves to cold stimul. Lower warm-
blooded animals have a broader latitude in this respeet and are
able, even at lower body temperatures, to maintain their capacity
to function and furthermore to endure lower temperatures in
the hypothermic state. The lower warm-blooded animals are
accordingly distinguished by a physiological or constitutional
eurythermism. The same apples also to the hibernators. Physio-
logical eurythermism seems, therefore, to be the prerequisite for
hibernation. This is probably also the reason why we find no
real hibernators amone the higher warm-blooded animals, for in-
stance, among the Carnivora, which must be labeled as physio-
logically stenothermie.

I have tried to present all these phenomena concerning the
heat economy of mammals and in particular of the hibernators
only in broad outline and have touched upon many questions
only superficially. [ am aware that here a broad field of investi-
vation is still open, especially for physiologists. Tam also con-
vineed that further investigations of the heat economy in other

1960 MAMMALIAN HIBERNATION 4]

species of mammals whieh have not as yet been considered will
open up some new viewpoints or will supplement the present
ones.

Summary

Among the mammals we find a very divergent level of develop-
ment of the heat economy. We ean, therefore, distinguish be-
tween higher and lower warm-blooded animals.

In the former, the activity temperature les above 36°C and
its range of variability is small. In the lower warm-blooded
animals, the range of activity of the body temperature hes at a
lower level and the range of variation is generally greater.

Many phylogenetically old mammals still have an imperfect
heat regulation and are lower warm-blooded animals. Among
them we must also inelude the hibernators. The Chiroptera stand
on the lowest stage of the homeothermie animals in respect to
their heat economy.

Tropical Chiroptera and sleeping mice are compared, in respect
to their activity temperatures, with representatives from the
temperate zones. Within the suborder of the lorises we find
various levels of development of the heat economy.

Lower warm-blooded animals are characterized by a physiologi-
cal or constitutional eurythermism. Evidently this is the pre-
requisite for the eapacity to hibernate.

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Roy. Soe. Vietoria, (n.s.) 9:57-67.

WaAbE, O.
1930. The behavior of certain spermophils with special reference to |
aestivation and hibernation. J. Mammal., 11:160-188. |
WaARDLAW, H.S. H. |
1915. The temperature of Mehidna aculeata. Proc. Linn. Soc. New 8, |
Wales, 40:231-258.
WEIGMANN, R.
1929. Uber Unterschiede in der Kiltebestindigkeit von Froschen, |
Kidechsen und Alligatoren. Verh. phys. med. Ges. Wiirzburg,
(n.f.) 54:88-97.

1960 MAMMALIAN HIBERNATION 43

DISCUSSION FOLLOWING EISENTRAUT’S PAPER

ADOLPH asked if the measurement of body temperature was
made under controlled (laboratory) conditions or under natural
(outdoor) conditions.

KISENTRAUT replied that in most eases the body tempera-
tures were measured obviously under laboratory conditions, but
with bats it was oceasionally possible to make measurements also
in caves under natural conditions.

MORRISON made the observation that it is a wise procedure
to make body temperature measurements in the wild, when pos-
sible. He felt that hibernators may often be inadvertently placed
ina thermie group with primitive mammals, such as marsupials,
if body temperature measurements are made under laboratory
conditions.

LIL

COMPARATIVE ECOLOGY OF
HIBERNATING MAMMALS

By N. I. KauaBukHOoV
The Institute of Microbiology and Epidemiology
of the South-East of the U.S.S.R.
Saratov, U.S.S.R.

Introduction

The peculiarities of hibernating mammals have been studied
for such a long period of time that our knowledge of this re-
markable phenomenon might be considered quite ample.

Comparing the rather scanty ideas on hibernation prevalent
at the end of the nineteenth century (see Skorichenko, 1891;
Dubois, 1896) with our present diverse and profound under-
standing of this phenomenon (see Kalabukhov, 1946, 1956a,b ;
Kisentraut, 1953, 1956; Kayser, 1958, 1957; Lyman and Chat-
field, 1955; Herter, 1956) we have to admit that the various
aspects of the remarkable deep torpor im animals, which are
homeotherms in the active state, have been studied thoroughly.

But in these studies of hibernation in mammals the significance
of physiological and ecological peculiarities of mammals in the
active state has been undervalued. It is quite obvious that the ca-
pacity for going into hibernation for a long period of time should
be closely related to certain important characteristics of the mam-
mals observed in their active state, when their bodies are being
prepared for the extended period of torpor. Having studied for
three decades the phenomenon of hibernation in mammals (Kala-
bukhov, 1926, 1929a, 1933a, 1935, 1936, 1946, 1956a,b, 1959) we
also paid great attention to peculiarities of life of hibernating
mammals in the active state (Kalabukhov, 1929b, 1933b, 1938,
1939a,b, 1940, 1954, 1955; Kalabukhov and Raevsky, 1934, 1935,
1936), and we should lke to elucidate here some facts related
to the problem.

We and our collaborators have especially investigated changes
in various species of hibernating and non-hibernating rodents
at different seasons of the year. We found it possible to approach
closely the problem of ecological and physiological properties of
hibernating mammals in the active state, as these seasonal
changes were studied not only in different species of hiber-
nating rodents: ground squirrels (Kalabukhoy, 1929b, 1958,

46 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

1939a,b, 1940, 1950, 1954, 1955; Gerassimenko, 1950; Movehan,
1953; Kozakevich, 1956, 1959), and jerboas (Kalabukhov et al.,
1955; Skvortzov, 1955, 1959a,b, Mikhailov, 1956), but also in
non-hibernating species, such as gerbils (Kalabukhov and Priak-
hin, 1954; Kalabukhov, 1956, 1957a,b, 1959; Kalabukhov ef al.,
1958; Mokrievich, 1957).

Pecularities of Active Life of Hibernatine Mammals

One of the most important characteristics of hibernating mam-
mals is that the principal activities of life such as feeding, storing
fat and other reserves, moulting, breeding and growth, all take
place in a relatively short period. It is only during the few
spring and summer months of active life that hibernators
undergo all the changes which enable them to spend an extended
period in a state of deep torpor. This capability of accomplishing
during a short period of time all the functions whieh are indis-
pensable for the existence of an individual and species, is a
characteristic of hibernators, undoubtedly no less remarkable
than their prolonged state of deep torpor. The short spring-
summer period of active life causes the hibernators to intensify
and aecelerate the course of many biological and physiological
processes.

Over 30 years ago the American zoologist Shaw (1925, 1926)
and Soviet scientists Kashkarov and Lein (1927) called attention
to this peculiarity of hibernating rodents represented by ground
squirrels Citellus columbianus and C. fulvus Licht. This observa-
tion is confirmed by numerous data showing that the rate of
metabolism and the level of chemical thermoregulation in hiber-
nators while in the active state is not lower than in non-
hibernators (Gelineo, 1938; Slonim ef al., 1940; Slonim, 1945,
1952: Kalabukhov, 1946, 1956; Slonim and Scherbakova, 1949 ;
Sokolov, 1949 ; Scheglova, 1953).

In Table I we cite data on the rate of metabolism at the critical
temperature (25° to 35°C) and its fluctuations when the air
temperature is lowered to +10°C in spring (March-April-May )
for some species of hibernating and non-hibernating rodents.
lor this purpose we have taken the data on jerboas, ground
squirrels and gerbils. The species of animals are arranged in
Table | according to their weight in each group, which enables
us to compare mammals of about the same size. Thus we compare
three species of strietly night rodents, jerboas (Alactagulus,
Scirtopoda, Dipus), with three species of gerbils (Meriones),
which also are active principally at night; and two diurnal
species of ground squirrels (Citellus), with two rodents also

MAMMALIAN HIBERNATION

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1960 MAMMALIAN HIBERNATION 49

active in day hours — large gerbils (Rhombomys) and long-
toed ground squirrels (Spermophilopsis). The level of oxygen
consumption is higher in almost all hibernators, with the excep-
tion of Alactagulus acontion, than in non-hibernators of about
the same size, both at the critical temperature and at 10°C.
The same difference can be observed in small and yellow ground
squirrels (C. pygmaeus and C. fulvus, respectively), both typical
hibernators, though they are somewhat larger than the large
gerbil and longtoed ground squirrel.

The high rate of physiological processes in bibernators may
be largely accounted for by the relative stability of — their
thermoregulation which enables them to remain active even at a
considerable drop of the surrounding temperature (Table [1).

The ability to withstand cooling in the hours of their activity,
i.e. in daytime for ground squirrels and at night for jerboas, ham-
sters, hedgehogs and bats, is closely related to the regulative
effect of the central nervous system. Slonim (1945), Ponugaeva
and Slonim (1949) and Folk ef al. (1958) reported that bats and
eround squirrels in hibernation keep up the rhythm of their
activity, while in the spring-summer period a sharp rise in the
rate of all processes can be observed at night in bats (Hisentraut,
1934, 1953, 1956). This adaptation is very important, for the
activity enables the animals consuming much food to accumulate
stores of fat and other reserves indispensable for surviving the
state of continuous torpor.

A rapid increase in body weight on awakening after hiberna-
tion was observed in hedgehogs (Camus and Gley, 1901) and in
captive ground squirrels (Kalabukhoy, 1926; Kayser, 1957). A
similar rapid accumulation of fat in ground squirrels and mar-
mots under natural conditions was observed by Kalabukhov and
Raevsky (1934), Semenov ef al. (1934), Dubinin and Leshkovieh
(1945) and Bibikov and Jirnova (1956) (see Fig. 1).

The accumulation of fat at this period is accelerated by a rise
in the air temperature which in turn causes a decrease of
heat loss. In its turn, the fat stored under the skin and in body
cavities decreases the loss of heat and, being a specific factor,
inhibits the activity of the thyroid gland (Kratinoy and Shkirina,
1947: Leites, 1954), and thus lowers the rate of metabolism. The
seasonal changes of the thyroid and adrenals — whose role in
regulating metabolic rate in animals is important — have at-
tracted the attention of investigators for a long time (Adler,
1920: Suomalainen, 1938, 1940; Sokolova, 1940; Emme, 1946;

50 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Liapin, 1949; Eisentraut, 19538, 1956; Kayser, 1953, 1957;
Uuspiaé and Suomalainen, 1954; Suomalainen and Uuspaa,
1958). However, these changes were studied regardless of the
influence of environment.

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Fig. 1. Changes of body weight in woodchucks and ground squirrels
during the period of active life. Left: woodehucks, Warmota baibacina

Kastsh.; Top: body weight (———) and glucose content in blood (- --)
(after N. Trukhachev); Bottom: percent of subeutaneous (——) and
internal (---) fat (after Bibikov and Jirnova, 1956). Right: body weight

of small ground squirrels, Citellus pygmaeus Pall. (after T. Mamontoy).

Fieure 2 illustrates the changes in rate of oxygen consump-
tion in the ground squirrel and jerboa (according to Kozakevich,
1956, 1959; Mikhailov, 1956), showing these metabolic changes in
the animal preparing for hibernation. Table II] and Figure 3
illustrate gradual changes in the activity of the ground squirrels
both in captivity (Kalabukhov, 1939b) and under natural condi-
tions (Kozakevich, 1956, 1959).

That rodents spend less time on the ground surface, diurnal
species particularly, is conneeted probably with the well known
influence of shortened day-leneth on the endocrine glands and
on the hypophysis and thyroid in particular (Bissonnette, 1935,
1942; Athonskaya, 1943, 1949; Beliaev, 1950).

Males go into hibernation lone before females. The latter
store fat more slowly as they have to bear and nourish the young
(Kalabukhov and Raevsky, 1934, 1936; Kalabukhov, 1956).

1960 MAMMALIAN HIBERNATION 5)

TABLE II]

Seasonal Change of Duration of Diurnal Activity in Ground
Squirrels in Captivity
(Kalabukhov, 1939b )

Citellus suslicus Citellus pygmaeus

Guld Pall
Activity in minutes Activity in minutes
in 2+ hours in 24 hours

Species —— ————— Species San

No. Changes No. Changes
24/V- 16-31/ in 24/V- 11/VII- in
18/VI- VIII- per cent 29/VI- 8/IX- per cent

i) 120 45 37.8 90) 155 78 50.3
77 139 98 70.5 56 169 D3 31.4
61 158 94 39.8 39 176 63 35.9
64 176 90) ol. 303 181 iow 76.3
73 240 96 40.0 8) 225 230) L022
74 241 39 15.7 SS 229 110 47.7
62 245 140 57.1 SI 194 78 25.3
65 249 12 24.8 35 ‘99 218 73.0
78 252 197 78.9 S87 38o 259 67.5
70 259 158 61.u 89 406 159 9.1
60 270 G4 23.7
63 278 - 169 60.8
66 301 133 39.8
69 334 214 64.0
ol 301 123 34.9
79 afd pay ey 73.2

Mean 251 125 49.9 Mean 252 139 55.0

NOS. 35, 39, 60, 66, 69, 70, 73, 77, 81 = males, others = females.

5? BULLETIN :

TABLE IV

MUSEUM OF COMPARATIVE ZOOLOGY

Vol. 124

Susceptibility to Plague Infection in the Adult Citellus pygmaeus
Pall. Before Entering into Hibernation
(Tinker and Kalabukhov, 1984)

A. Females

The dose of

Weight microbes in Duration of life Isolation of the
in gms. thousands in days specifie culture

137 50 Over 18(X/ -—

107 150 3 4.

166 450 Over 18 —
122 1,350 7 +-
189 4,050 3 +-
101 12,150 5) +
149 36,450 Over 18 —
104 109,050 4 +

B. Males

123 50 Over 18 =
120 150 fe 18 =

72 450 f& 18 =
175 1,350 16 aL
122 4,050 Over 18 —_
182 12,150 i) f
184 36,450 Over 18 bL
175 109,050 oe ALS —

(x) “over 1 8” — The rodents were killed on the 18th day after infection.

TABLE V

Temperature At Which the Mammals Go Into Hibernation
( According to Eisentraut, 1953 )

1. Hamster

2. Marmot

3. Hedgehog
+. Haselmouse

>. Dormouse

6. Kuropean ground squirrel

Yellow ground squirrel
bat
9. European bat

‘
8. Tropical

9°10"
10°-11°
T5217 2
15°-16°
18°
20°

99°

DaPua 5°

1960 MAMMALIAN HIBERNATION 53

Still later, the young generation (born the current year) goes
into hibernation; growing rapidly, they aeeumulate fat more
slowly than the adult rodents. Dubinin and Leshkovieh (1945),
studying Siberian marmots (J/armota sibirica Radde) in the
Transbaikal region, stated that some rodents which had insuf-
ficient fat before going into hibernation due to poor feeding
conditions, either went into hibernation very late, or awakened
before the snow melted, and perished.

40+

5,07

ae)
i=)

_
Ss
T

Oxygen consumption in l per rgofh
aw
-_)
1 ia

Ly
is)
=

20r

10 — ee oo aoe! eee or =,
7c. 70° 15° 20 ied 30° F5
Aire temperature

Fig. 2. Changes in the level of oxygen consumption during the active
life of hibernating mammals in small (1) and in yellow (2) ground
squirrels, Citellus pygmaeus Pall. and C. fulvus Licht (after Kosakevich,
1956, 1959), and in jerboas (3), Scirtopoda telum Licht (after Mikhailov,

1956). Roman numerals = months.

54 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

100 F “

80 -

60+

/00 We oh eS)

%
Ss

Alcotive ty (n percent
Ss

7am lam lpm 4pm 7om

Ahours
Mig. 3. Changes in the diurnal activity eyele in small (---) and yellow
( ) ground squirrels, Citellus pygmaeus Pall. and C. fulvus Licht, in

different periods: 1) after awakening; 2) beginning of pregnancy; 3) dis-
persal of young rodents; 4+) before entering into hibernation (after Kosake-

vich, 1956, 1959).

O11

1960 MAMMALIAN HIBERNATION BE

At the time when fat is being accumulated in the spring-
summer period of the life eyele of animals, other no less im-
portant components also influence the physiology of hibernators.
Kratinov cf al. (1947) established that during this period
ascorbie acid is being stored in the organism of Citellus pygmaeus
(Fig. 4), and Isaakian and Felberbaum (1949) found the
amount of vitamin © to be in direet relation to the eondition
of nourishment of Citellus fulvus in the beginning of summer.
Suomalainen (1938, 1940) has observed similar changes in

mg %
(80 }-
/60
/40
hs bee»
/20 e9
/00
80 5 5 Thyroid
60
40 |-
a Yonads¢
; \Oo
20 | 29
Liver
0 Jd
i
8 : s 8
4 ~ 5 x
R , ee :
x Gv
$ y hes S
Bu 3 ae 8
x z XQ x

Fig. 4. Changes in ascorbic acid content in different organs of small
ground squirrels, Citellus pygmaeus Pall. (after Kratinov ef al., 1947).

hedgehogs, and has suggested that accumulation of ascorbic acid
as an active anti-oxidant inhibits the oxidation of adrenaline in
the suprarenal glands, and in this way decreases the intensity of
physiological processes.

Then, as demonstrated by M. G. Friedmann (see Kalabukhoy,
1956), fat-storing in hibernators appears to be accompanied by
storing of fat-soluble vitamin E, which plays an important role,
not only in breeding of animals (see Fig. 5), but also in regu-
lating metabolism and heat production as well (Blaxter ef al.,
1952). It is possible that while the reserves of vitamin E are
being stored in tissues, a disconnection of cellular respiration

56 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

and phosphorylation takes place and is responsible for inhibition
of thermoregulation in mammals (Nason ef al., 1957).

Hibernating mammals breed, as a rule, only once a year. Fig-
ure 6 illustrates the relationship of the time of breeding for
Citellus pygmaeus Pall. in different years to the time of their
awakenine (Kalabukhov, 1929b, 1936, 1956). In Figure 7 is
eiven the periodicity of breeding of certain species of jerboas
(Fenjuk and Kazantzeva, 1937; Kondrachkin and Edikina, 1957 ;
Mokroussoyv, 1957).

It is emphasized that, in spite of the idea that all vital processes
are inhibited in hibernation, the very short period of active life
sugeests a paradoxical fact — namely, that in the period of deep

= gonods with speemarozolds
LJ gonads without speematozoids

booy weight at the day
400 of investigation

300 -

ogy welghl ingm
%
S
S

Fig. 5. Influence of the degree of fatness in males of Citellus pygmaeus
before hibernation (in October) on the preparation for breeding (in
Mareh) (after Friedmann; ef. Kalabukhov, 1956).

torpor the body of the hibernator is preparing itself for breeding.
This was established for marmots by Rasmussen (1917), and for
ground squirrels by Shaw (1926), Sokolova (1940), and Kayser
(1953). Asdell (1946), in his book, cites other examples for
hedgehogs, bats and rodents.

The peculiarity of the breeding process in hibernating mam-
mals can be explained by the phenomenon of storing vitamin E
together with fat. This peculiarity seems to be of great import-
ance as another regulator of the eyelie alterations in the gonads,
for changes in the duration of daylight have no effect on hiber-
nators (Allanson and Deansley, 1984; Wells, 1935).

The exclusion of light as a stimulus for the chain reaction from
eye to central nervous system to hypophysis to gonads may be

1960 MAMMALIAN IIIBERNATION 5

explained by the fact that hibernators stay for months, while in
torpor, in deep, dark holes and other covers.

Besides Friedmann’s experiments which demonstrated the
relationship of the degree of fatness in small ground squirrels
before hibernation and the store of vitamin E to their eapability
of breeding in spring (see Fig. 5), we can refer to the observa-
tions of these rodents under natural conditions by Orlova
(1955a,b). She reports that when ground squirrels live in sum-
mer near wheat fields, they breed next spring more intensively

CTT TT TTT

O O |
Las = Hal Au

Ob Awarening

NOY
30 Ha —— finding of /-st

nt female
Sibyl iis erearent reme

Re oi

§ 20 |— aH : He, finding of last
sissececray ON | pregnant female

_flast appearing

of young rodents

ry .
/ —+—e— Beginning

otdispeesal
w | “Dispersal 4

— Pe or Young s

/0 == a

& :

5 20 =

S «End of dispersal
30

1926 27 28 29 ul 3/ 32 years

Fig. 6. Influence of period of awakening in small ground squirrels,
Citellus pygmaeus Pall., on their breeding (after Kalabukhov, 1929b, 1936;
Kalabukhov and Raevsky, 1934).

than populations of this species inhabiting other localities. The
endosperm of wheat is especially rich in vitamin E. Thus it seems
quite possible that going into aestivation in some regions and a
retardation of hibernation in others is caused, not only by the
drying up of vegetation, but also by different rates of accum-
ulation of fat and other reserves, which in turn depend on
different kinds of vegetables and other foodstuffs.

Last of all, the peculiar character of molting in the active
period of hibernating mammals distinguishes them from the non-
hibernating species which are closely related to them (IKuznet-
zov, 1940; Hansen, 1954; Kalabukhov et al/., 1958). This pecul-
arity is obviously also influenced by the lack of external stimulus

8 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

at the time of torpor, for hght is the primeipal regulator of the
process of molt in mammals and in birds active throughout the
vear (Bissonnette, 1935, 1942; Athonskayva, 1943, 1949; Beliaev,
1950; Kalabukhoy, 1951).

---- /932
1935

so0- Sciztopoda

10+ oe
—) ah. = = 4 aes f =e 4 at J

90+ Mlactagulus
80} acontion

Number of peegnant females in peecents
S
S

LLM@WVWWWmiKxnxd
months
Fig. 7. Cycle of the breeding in different species of jerboas in Pre-
caspian steppes: Dipus sagitta Pall. (after Fenjuk and Kazantzeva, 1937),
Scirtopoda telum Licht (after Mokroussov, 1957) and Alactagulus acontion
Pall. (after Kondrachkin and Edikina, 1957).

It is evident also that the beginning of torpor in hibernating
mammals must be influenced not only by the direct action of
external factors on physiological mechanisms but also by the
effect of the environment on the behavior of animals. It is an
excellent example of the conditioned-reflex relationship in the
central nervous system of mammals, or the action on the organism
of some external ‘‘signals’’ connected with the approach of
unfavorable conditions (Pavlov, 1937). For instance, Ryabov
(1948) observed that woodchueks (Marmota sibirica Radde)

1960 MAMMALIAN HIBERNATION 59

were active in late autumn, when snow had not yet covered the
eround surface, although the air temperature was below 0°C, but
went into hibernation on the day after the first night snowfall.

Although desert jerboas (Dipus sagitta Pall.) do not usually
hibernate in Turkmenia (Vinogradov and Argiropulo, 1938;
Kalabukhov et al., 1958; Skvortzov, 1959a), the last author
found that they did hibernate after an occasional winter rainfall
which caused a dense crust of frozen sand in the region of their
dens. Similar lowering of activity of animals under the influence
of some external factors is connected evidently with the falling
of the level of metabolism and body temperature in hibernators,
under conditions of forced, prolonged rest and fasting (Rall,
1932; Isaakian and Felberbaum, 1949; Strumwasser, 1959).

About a decade ago the Soviet investigators discussed the
significance of such ‘‘signal’’ changes in the environment, which
influence the nervous system of hibernating mammals (Kala-
bukhov, 1948; Byikov and Slonim, 1949). Now, in the light of
modern data about ‘‘stress reactions,’’ other scientists have
developed ideas concerning the inhibitory effeet of external fac-
tors on the nervous and endocrine system when hibernators @o
into torpor (Suomalainen and Herlevi, 1951; Kisentraut, 1956;
Kayser, 1957; Strumwasser, 1959). These data confirm our state-
ment that the principal features which characterize the hibernat-
ing mammals are closely related to the peculiarity of the active
phase of their annual cyele of life.

There are other numerous data illustrating the profound and
eradual changes occurring in the organism of hibernating mam-
mals in their active period. Their sensitivity to infection from
plague, for instance, is greatly changed. This change in sensi-
tivity is not uniform in males, females and the young, as the
rate of physiological modification in their respective bodies is
different (Gaisky, 1926; Tinker and Kalabukhov, 1934; Kozake-
vitech, 1956).

In Table LV these data are given for the small ground squirrel,
according to Tinker and Kalabukhov (1934).

According to Kozakevitch (1956) the LD;,» of plague bacilli
increases 37 times for the males of small ground squirrels from
the moment of their awakening (March) to the period of prep-
aration for hibernation (June) and only twice for the females.
Some fluctuations in the sensitivity of hibernating mammals to
various poisons have been recorded at the time of their active
life (Borodina, 1956: Kalabukhov, 1956).

60 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The influence of carcinogenic substances, too, seems not to be
uniform at different periods of their active life, though this
question has been studied as yet only on the mammals in state of
torpor (Finkelstein and Rukhov, 1950; Lyman and Faweett,
1954). Some data on this question is found in a paper by
Finkelstein and Belogrudova (1957), who observed the influ-
ence of 9,10 dimethyl-1,2 benzantracen on ground squirrels (Cit-
cllus erythrogenus Br.) in a state of aestivation produced by
feeding on dry oats at a room temperature of 14-15°C. Fluctua-
tions in body temperature of 28 torpid rodents ranged between
13.5° and 33.5°C, and between 31.7° and 38°C in 37 active ones.
By the end of hibernation neoplasms at the site of injection
were considerably fewer in the shg¢htly torpid ground squirrels,
than in controls.

All the above-mentioned data and conclusions are of great
assistance in another respect, as they help to elucidate a dift-
ference having a practical importance, i.e. comparison of the
state of hypothermia in non-hibernating animals and in man,
to the state of natural hibernation. These phenomena are doubt-
less not the same. There is little in common between the torpor
of hibernators produced by profound and gradual changes in
the physiology of the animals in the period of active life and the
sudden inhibition of the heat-regulating mechanism produced
by drugs or by a rapid cooling of non-hibernating mammals.

The data on hibernation clarifies the rather simplified ideas of
artificial hibernation prevalent amone some biologists and medi-
cal men (Giaja, 1953; Laborit and Huguenard, 1954; Kayser,
1955; Saakov, 1957; Starkov, 1957; Smith, 1958).

All the physiological changes which occur in the active period
of the hfe of mammals that hibernate take a reverse course
during hibernation. By the end of hibernation the animals
restore their ability to maintain body temperature through a
high metabolic rate and other physiological processes, although
the surrounding temperature is even lower than at the time when
they entered into hibernation (Shaw, 1925, 1926; Kashkarov
and Lein, 1927; Kalabukhov, 1929a). This appears to be due
to the gradual freeing of the organism from the factors which
inhibit the rate of metabolism, e.g. from fat and vitamin E
accumulated before going into hibernation which results in
restoration of the function of the endocrine glands, especially the
thyroid and suprarenal.

1960 MAMMALIAN HIBERNATION 6]

The experiments by Suomalainen and Uuspiaé (1958) which
showed that during the awakening of hedgehogs the lowering
of adrenaline content and the rise of the noradrenaline level in
the adrenal glands took place, also illustrate this regularity, as
well as the valuable experiments by Popovie (1955) on the
action of desiccated thyroid and methylthiouracil on ground
squirrels.

Pecuharities of the Influence of Cooling in Different Species
of Hibernating Mammals

That the ability to hibernate appeared in mammals as a
result of a definite combination of effects of various external
conditions just at the time of their active life can be most clearly
confirmed by the fact that the influence of the surrounding tem-
perature on them is not uniform. Hisentraut (1953, 1956) not
only divided the hibernating animals into two groups according
to the length of their respective periods of hibernation, but he
recorded the different degree of their dependence on cooling as
well (see Table V).

These data alone are sufficient to disprove the widespread
opinion that entering into hibernation is always a result of a
drop in the surrounding temperature. However, within one
systematic group of mammals such as ground squirrels, jerboas,
or hamsters one can find species which become torpid in the
high temperatures of early summer, others which become torpid
in the low temperatures of late fall, while others do not
hibernate at all. Thus, two species, the yellow ground squirrel
(Citellus fulvus Licht) and the small ground squirrel (C. pyg-
maeus Pall.) go into aestivation in the arid regions of the South-
Kast of the USSR at the end of May and the beginning of June,
when the temperature in their burrows is above 15°-18°C (Kash-
karov and Lein, 1927; Kalabukhov, 1929a), while in other
localities these rodents and a third species of ground squirrel,
Citellus suslica Guld., go into hibernation only in September
When the effeet of the low temperature is quite obvious (Tikh-
vinsky and Sosnina, 1939; Kalabukhov, 1950, 1956).

Still more striking is the difference in the time when two
species of jerboas inhabiting the same place — Scirtopoda telun
Licht and Alactagulus acontion Pall. go into hibernation in the
Precaspian steppes (Kalabukhovy et al., 1955). While the former
species become fat as early as July and den up by the end of

62 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

October, the latter remain active in November, and sometimes
even in December. Thus the double breeding season that was
recorded by Kondrachkin and Edikina (1957) in Alactagulus
acontion Pall. (which distinguished them from the ‘‘long-
sleeper’? Scirtopoda telum, studied in the same region by Mok-
roussov In 1957) does not appear accidental (see Fig. 7). Skvort-
Zov (1955, 1959a,b) established in his experiments that Alacta-
gulus @oes into torpor only when the surrounding temperature
drops below +5° to 6°C, while according to Mikhailov (1956)
hibernation in Scirtopoda occurs at a higher temperature.

The third species of jerboa, Dipus sagitta Pall., is reported to
enter into hibernation in the Volga-Ural sands at the same time
as Alactagulus (Fenjuk and Kazantzeva, 1937; Skvortzov,
1959b), while in Turkmenia it usually remains active throughout
most of the winter, although Alactagulus becomes torpid there
(Vinogradov and Argiropulo, 19388; Skvortzov, 1955, 1959a). It
is characteristic of the latter that in the Caspian steppes as well
as in the Turkmenian deserts it builds special ‘‘hibernating
chambers’’ before becoming torpid (Skvortzov, 1955; Kondrach-
kin and Edikina, 1957). These chambers have no nest and are
situated near the ground surface where the small animal would
be easily cooled.

The very pecuhar picture of the close correlation between the
level of metabolism and thermoreeulation and the surrounding
temperature has been found in the smallest of hibernating
rodents, such as the birehmouse, Siersta, or poeket-mouse, Perog-
nathus (Suomalainen, 1947; Bartholomew and Cade, 1957).

The greatest variety of reactions to cooling can be observed
in the different species of hamsters, among which only the com-
mon hamster (Cricetus ericetus L.) is a typical ‘‘long sleeping”’
species (Eisentraut, 1928, 1953, 1956) which easily becomes
torpid when cooled. Hamsters of the genus Jesocricetus such
as the golden (JM. auratus Waterhouse) and the Caueasian (M.
raddci Nehr.) are less sensitive to cooling (Farrand cet al., 1956:
Panuska and Wade, 1958). It was in the golden hamster that
South (1958) discovered that the rate of oxygen consumption
in the cardiae muscle was lower than in the bat, and thus the
‘heat of activation’? is higher in the eolden hamster than in true
hibernators such as the bat.

inally, many species of hamsters (genus Cricetelus) which
‘nhabit the steppes and desert regions of Europe and Asia
‘Cricetelus migratorius Pall., C. eversmanni Brandt, C. bara-

1960 MAMMALIAN HIBERNATION 63

bensis Pall., Phodopus sungorus Pall.) fail to hibernate, and
stay active throughout the year (Kalabukhov, 1956).

In a state of torpor the sensitiveness to cooling in different
species of hibernating mammals is also not uniform. Some of
them awaken when the surrounding temperature drops to 0°C
or below which suggests quite a paradoxical ability of restoring
heat regulation on cooling as well as on warming. This was
recorded in the ground squirrel by Horvath (1881), and in
the hedgehog by Suomalainen and Suvanto (1953), and Chao
(1955). On the contrary, other species can stand cooling to a
temperature below 0° for a lone time, the liquids in their body
being in the state of superecooling at —1.0° to -1.5°C in small
and long-tailed ground squirrels, or even at —5° to —9°C in bats
(Bakhmetiev, 1912; Kalabukhov, 1933a, 1935, 1958; Murigin,
1937, 1948; Nekipelov and Peshkov, 1958).

Conelusion

All the above data permit us now (Hisentraut, 1953, 1956;
Kalabukhov, 1936, 1946, 1956; Lyman and Chatfield, 1955) to
reject the doubtful idea that the ability of some species. of
mammals to hibernate is but a manifestation of the primitive
characters of poikilothermal animals.

We believe that the above data show that the phenomenon of
hibernation is not only a result of adaptation in animals to the
unfavorable conditions of life in autumn and winter but also to
consequent extreme shortening of the duration of the phase of
their active life.

Without considering many other facts mentioned in our book
(Kkalabukhov, 1956b), we wish to complete our paper calling
to mind the statement by Charles Darwin (1839), in the fifth
chapter of the ‘‘ Journal of the Vovage of the H. M. S. Beagle,’’
on the significance of the temperature factor in hibernation :
“This shows how nicely the stimulus required to arouse hiber-
nating animals is governed by the usual climate of the district,
and not by absolute heat.’” If his idea ineluded also entering
into hibernation, we find an excellent example of how profoundly
the great scientist understood the degree of relativity in all
adaptations to the conditions of existence.

I am sure that recalline these wise words is particularly ap-
propriate in the current vear, since the 150th anniversary of
Darwin’s birthday occurred in February, and in a few months
the centennial of his ‘‘Origin of Species’’ will be celebrated.

64 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

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SOME INTERRELATIONS BETWEEN
WEIGHT AND HIBERNATION FUNCTION'

By Prerer Morrison
Departments of Zoology and of Physiology
University of Wisconsin
Madison, Wisconsin

The phenomenon of hibernation in mammals which has at-
tracted the interest of zoologists for more than 200 years, pro-
vides the first demonstration in eomparative physiology of a
striking adaptation to special environmental requirements, and
at the same time emphasizes the contrasting homeostatic condi-
tions in other mammals. The general conditions of hibernation
were laid out in several studies early in the last century, and
these showed the depressed level of metabolic aetivity as mani-
fested by a low body temperature and reduced heart and _ res-
piratory rates and suspension of all general activities. Since that
time a considerable body of data has been collected and with
the improvement of physiological technique and instrumentation
we now have detailed deseriptions of many aspects of this condi-
tion. These data have shown common features in many animals
but also substantial quantitative differences between species in
such factors as the duration of hibernation, the accumulation of
metabolic reserves, the reduction in body temperature (Tp), and
its relation to the ambient temperatures (T,), the degree of
depression of metabolie activities, and the specialization of indi-
vidual tissues for functioning at low temperatures. Thus, the
jumping mouse, the hamster, the hedgehog, the ground squirrel,
the bat and the bear may each be distinguished from the others
in respect to at least one feature of its hibernating behavior. As
a consequence, there is some difference of opinion as to the precise
definition and limits of the condition of hibernation, and as to
the animals which should be classed as hibernators. Thus, how
greatly must the metabolic level be reduced for an animal to
qualify as a hibernator? Must the body temperature be lowered
by a specified amount, or must it approach the ambient tempera-
ture within a specified limit?

1 Studies on hibernation at the University of Wisconsin have received continu-
ing support from the Wisconsin Alumni Research Foundation.

76 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The examination, which follows, of some underlying principles
related to energy balance and hibernation will not directly
answer these questions, but 1tf may provide some basis for a eom-
mon treatment of various animals, and clarify the relation of
questionable species both to acknowledged hibernators and to
the bulk of non-hibernatine mammals.

All animals may encounter periods during which food is
unavailable or in short supply so that a deficit must be met from
body reserves. During hibernation the ordinary metabole de-
mands, which might otherwise exhaust the metabolic reserves,
are reduced to such a level that the reserves last through a whole
inclement season. Although protein is depleted during prolonged
starvation, this represents a loss of structural material, and the
metabolic reserve may be considered as effectively represented by
the body fat. The fat content? may vary widely, but its maximum
level (Fy;) may be taken as about equal to the fat-free* body
weight (W)

Fy = W(g) (1)

Although wild animals ordinarily earry much smaller amounts
of fat (G. C. Pitts, personal communication), such values, i.e.,
50 per cent of total body weight as fat, are observed in species
spanning almost the entire weight range from the jumping mouse
(101e@) to the blue whale (108e@), and there are no obvious in-
trinsic Lmitations which would prevent such an accumulation
in any animal.’ Thus the potential energy reserve will be pro-
portional to body weight in animals of all sizes and may be
expressed as

Fy); = 7W (keal) (W in g) (2)

By contrast, the basal metabohe rate (My) is weight depend-
ent, following the familar relation,

M = 0.443/4 (keal day-1) (W in g) (3)
(exponent rounded; Brody, 1945). Therefore, he fasting po-
tential will vary direetly with weight and represent the quotient
of these two heterogenie relations (Adolph, 1949) in animals with

a ‘‘maximum’’ fat supply and a ‘‘standard’’ metabolic level.
F,,/Mzg = 16 W1/4 (days) (4)

yr the fasting duration can be more generally deseribed as
F/M = f/m 16 W1/4 (days) (5)

where f is the fat content (fraction of W) and m is the meta-
bohe level (fraction or multiple of Mp).

2 Por the purposes of this discussion “fat will represent adipose tissue, the
triglyeeride content of which will be taken as 75-80% (Pitts, 1956).
3 Bats may represent an exception in that the requirements of fHght could

limit the load of fat. In Eptesicus, a maximum fat content of 15 (f = %) has
been observed prior to hibernation (Beer and Richards, 1956).

—~!

1960 MAMMALIAN HIBERNATION

This relation is shown in Figure 1, which relates log days of fast
to log weight. The heavy curve shows the relation when f = m
1.0, and the light curves show the effect of lowered fat content
with f ranging down to a value of 1/8. An adequate duration
for survival might be taken as 200 days (7 months), the time
between first and last frosts in our northern states, although
dormant periods as long as 300 days have been deseribed (Vol-
canezky and Furssajeu, 1984), and shorter periods, of about 100
days, may be adequate under other circumstances. At the Mz, a
20 ke animal (beaver) might just survive 200 days with the maxi-

ie) 2 4 6 8
LOG W in G
Fig. 1. Influence of body fat on fasting time in mammals of different

size (m= 1).

mum fat content, but a 300 ke animal (bear) would need only
half this amount. A 5-ton animal (elephant) would meet the
200 day requirement with only 1/4 its weight in fat (20 per cent
total weight), and our largest mammal (blue whale) would last
with f= 1/8. Animals smaller than 20 ke could not survive with-
out modifying their metabolic level. It may be noted that our
largest unquestioned hibernator, the marmot, has a weight just
below this value. A 200 @ animal (squirrel) could survive about
2 months, and our smallest mammal (3 @ shrew) only 20 days
even with 50 per cent of the total body weight as fat.*

t No hibernating shrews are known, and how small shrews meet the substantial
requirement of several times their body weight in meat each day during the
winter is a question. However, it is amusing to consider that the long-tail shrew
might have a vicarious connection to hibernation. In Alaska these shrews may
be found in areas with Arctic ground squirrels, and should the latter be avail-
able, one large hibernating carcass could support half a dozen 8 g shrews over

the entire winter. Reports of shrews seavenging animals as large as a moose
suggest that this possibility is not as fanciful as it may seem.

78 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Figure 2 shows the effect of a reduced metabolic level on sur-
vival (f=1). It may be seen that with a factor, m= 1/2, a 2 kg
animal (marmot) will survive for 200 days; at m= 1/4, a 100 g
animal (ground squirrel) ; and at m=1/8, a 10 @ animal (small
bat). However, in interpreting these curves it will be judicious
to allow some margin, perhaps 2-fold, for increases in metabolism
either during the periodic awakenings which appear necessary
or at the end of the fasting period in the spring. For example,
the metabohe rate in naturally hibernating Myotis, estimated

) 2 4 6
LOG W in G.

Fig. 2. Influence of metabolic level on fasting time in mammals of

different size (f= 1).

from fat depletion, is 0.1 ce 0o/g/hr (Mann, 1936) while the
measured metabolism at 5°C is 0.05 ce/g¢/hr (Hock, 1951). Simi-
larly, oxygen consumed during periodic awakenings in ground
squirrels and hedgehogs is of the same magnitude as that used
in the intervening dormant periods.

It may also be more appropriate to estimate fat on the basis of
{ =0.5, rather than f=1.0. Although the latter value is a rea-
sonable maximum, the former (33 per cent of total weight) may
better represent ordinary values.” So, for these average values
(f/m =1/4).

F,/M, =4 W!/4 (days) (6)

With this conservative estimate, a weight of more than a ton is
necessary for a 200 day fast.

1960 MAMMALIAN HIBERNATION 79

As the temperature of a hibernating mammal falls, so does its
metabolism, and this reduction may be calculated if a tempera-
ture coefficient is assumed. Values near 2.0 are characteristic of
many biological systems and have been described for isolated
tissues in hibernating and other mammals (South, 1958; Meyer
and Morrison, 1960).

Figure 3 shows the effect of lowering the temperature in such
a system. With a 30° drop, a small bat would last the 200 days,
but with no margin for a lower average fat level or a higher
average metabolism (cf. above). For f/m = 1/4, a kg or more of
body weight would be required. A further depression of the

0) 2 4 6
LOG Win G.

Fig. 3. Influence of body temperature on fasting time in mammals
of different size (Qio = 2.0 and //m=1).

metabolism is required in smaller mammals and such is mdeed
seen (Kayser, 1940), but this necessitates a higher Q, 9. The
influence of variation of Q, 9 on the fast duration at a representa-
tive hibernating temperature of 10°C (T= —25*°) is shown in
igure 4 for various Q; 9 values. At f/m —=—1/4, a Qy9 of 4.0 is
necessary for survival of a small bat. Interestingly, a Qo close
to this, 3.8, may be calculated from the data of Hock (1951) on
Myotis, between 2 and 40°C,

Another quantity relating to hibernation that may depend on
body weight is the difference between the ambient and body
temperatures, Figure 5 shows this in an experiment on a thirteen-
lined ground squirrel, in which the Ty, passively followed the T),

8O BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

but always at a slightly higher level by virtue of its heat produc-
tion. In popular terms such hibernation represents a turning-ott
of the physiological **‘thermostat.”’ It may be left off indefinitely,
in which case the temperature will always ‘‘float’’ just above the
ambient temperature, as in the bat; or, as in the hedgehog or
dormouse it may be subsequently ‘‘set’’? at a new level in
response to a low Ty, so that the animal again regulates but at a
Tn far below that of the active animal (Eisentraut, 1929). Ani-
mals just entering hibernation may also show intermediate states
(Strumwasser, 1959) or an alteration of ‘‘on’’ and ‘‘off’’

0 2 4 6
LOG W in G.

Fig. 4. Influence of Qio (Tr = 25°) on fasting time in mammals of
different size (f/m = 1/2).

cycles, but the passive condition with the Ty, close to the Ty
should probably be considered the ordinary situation.

These small temperature differences are difficult to measure
unless the Ty is carefully controlled. But they may be directly
estimated from the metabolic rate (M) under the given condi-
tions and the thermal conductance (C). These quantities are
related through Newton’s law of cooling as applied to a thermo-
regulating animal (Scholander et al., 1949). Thus,

M=€C (T,-Ta.) = CAT (7)
or AT=M/C (8)

Metabolic estimates of C (Morrison and Ryser, 1951), supple-
mented by in vitro measurements on pelts (Scholander ef al.,
1949), give the relation:

1960 MAMMALIAN HIBERNATION 8]

C=K W1/2 (9)
That is, because of the decreasing surface to weight ratio and
the increasing pelt thickness, larger animals cool less rapidly than
smaller ones. If we now combine equations (3), (8) and (9),
M/C =aT=mk W?1/4 (10)
and the temperature difference will vary directly with weight.
Hlowever, the use of this equation for animals in hibernation at
any given Ty presumes a common temperature coefficient. If, as
Kayser (1960) suggests, the metabolic-weight function during
hibernation has an exponent near 1.0, then the temperature
difference may be even more weight dependent :

AT =k W!1/2 GI)

30 CITELLUS T NO 5
) RUN 30
@® RUN 29
2
Te
IN 20

1200 2400
TIME IN HOURS

Fig. 5. Body temperatures in hibernating thirteen-lined ground squir-
rels (after Ryser, 1952).

Equation (10) may also be applied to non-hibernating animals
to define the critical temperature differences (Tce) and the
associated critical temperatures (Tae) which mark the lower
limit of thermal neutrality and the beginning of chemical reeu-
lation.”

M;/C = Ts-Tac= ATe = 4 W!/4 (12)
It has, moreover, a possible relation to hibernation, since an ani-
mal in the ordinary hibernating posture which allows minimum
heat dissipation cannot reduce its body temperature in the zone of

5 By coincidence, this equation (12) is identical to equation (6). That is to
say, the critical temperature differential in °C is equal to the fasting period
(f/m = %) in days.

82 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

thermal neutrality without lowering the metabolism below the
basal level. While some animals possess this ability to abruptly
lower their metabolic level (V. Popovie, personal communication )
this capacity may be limited to species which become dormant in
warmer latitudes or seasons." In our experience temperate or
northern hibernators do not show abrupt metabolic depressions.
In one species, the Jumping mouse (Zapus hudsonius), indi-
viduals ready for hibernation were observed to starve to death at
25°, neither eating available food nor lowering their body
temperature or metabolism. In Myotis (Hock, 1951) or in the
Arctic ground squirrel (Fig. 6), the metabolism follows along a
single temperature function during the course of cooling. This
Function (Q, 9 =—4-6) is considerably steeper than can be ac-
counted for by the intrinsic behavior of the isolated tissues
(South, 1958; Mever and Morrison, 1960) and so must involve
regulatory components. Ilowever, the observed regularity in
behavior appears more compatible with a passive response to
temperature than to an active depression of the metabole level
at any point.

If these extrinsic influences, whatever their nature, are
thought of as acting to multiply the tissue metabolism then the
overall Q,o could represent the sum of those for the tissue
respiration and its ‘‘control.’’ Thus, in an analogue :

| ka | ky

A + }} ——>_ CU
k

If the concentrations of A and B depend on their rates of
formation, k, and k,, which are in turn governed by tem-
perature coefficients, Qioa and Qo» then k=k, ky and
Qio = Qroa + Qiov. By such a concept we might account for
the high observed Q,, values as the resultant of two Qo values
of ordinary magnitude, which act together to maintain a single
overall temperature coefficient over the whole range.

6 Such a capacity to depress the metabolic level with little reduction in Ts
might provide a physiological criterion for differentiating aestivators from hiber-
nators. Since both classes appear to behave in a similar manner during dormaney,
this would represent an added specialization in the aestivator.

1960

LOG CC Op PER G HR

MAMMALIAN HIBERNATION

|
ae |

: It 1 =
30 40

=
10 20
Tg IN °C

Fig. 6. Body temperature changes in Spermophilus undulatus no. 4 duc-

ing a single cycle of arousal from/return to hibernation. (Run H-19, 2/27
Ryser and P. Morrison). Warm-

Heavy dotted curve shows main-
The difference between this

9
o/

ing time, 3 hrs; cooling half-time, 3 hrs.
tenance metabolism during the cooling eyele.
curve and the ‘fenter H’’ curve at any point defines the calorie deficit, and

of cooling, eg., 2.7°/hr. @

(2/58, unpublished observations of KF. A

corresponds closely to the observed rate
“M7? maximum observed metabolism in this individual in summer:

202.

‘6°’? maintenance metabolism at Ta = 6° and Ty = 38°: ‘*B,’’ basal

metabolic rate in summer; ‘f©,’’ maintained metabolism during 4-hour
Note the identity in

period. Values refer to Qio for each curve.
Qio between ‘‘leave H’? and Nght dashed curves which differ only in rate

(factor of 60). The fine dotted curve was drawn with the Qo
of 2.2 seen in isolated tissues. The difference between its value at 38°
(SHE???) and My (factor of 6) may define the ‘‘extrinsic’’? depression of

The light solid line curve (Q1o = 3.8) cor

constant

the metabolism in hibernation.
responds to the values for the transition into hibernation reported for other

e
hibernators. designates early and later readings from other animals in

hibernation.

84 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Accordingly, the critical ambient temperature may represent
a limiting condition, and only below this value could our (north-
ern) hibernators lower their Ty, by failing to supply heat for
thermoregulation. Model curves for two animals of different
size are given in Figure 7. The 40 @ animals (e.g., a jumping
mouse) could cool below 25°, but the 4 ke animal (e.g., a large
marmot) would need a temperature of less than 5° to cool. Of
course, these relations are based on the standard curve of metab-
olism and weight, in which MJarmota is a conspicuous exception
with a metabolic rate about one-half the ‘‘predicted’’ value
(Benedict and Lee, 1938). Thus, the critical temperature for

a a ae aca i

M/Mpg

Ty IN °C

Fig. 7. Model curves for two animals showing the influence of size and
basal metabolic rate on the critical temperature, below which the body
temperature can be reduced while metabolism remains at the basal level.

the marmot would be 20°C, and not 5° which might be difficult
to attain. One wonders if this correlation with hibernation
function and considerable size could provide an explanation for
the unusually low metabolic rate in this genus.

The relation between conductance and weight is only valid up
to a size of perhaps 50 ke since the fur length does not increase
in larger animals. At this weight, however, the critical differen-
tial would be 65° and the critical temperature —30°C,

1960 MAMMALIAN HIBERNATION 85

It will be of interest to evaluate the bear in terms of the previ-
ous discussions. This animal oceupies an anomalous position
among hibernating mammals. Although it is popularly the ani-
mal first thought of in reference to this phenomenon, some natu
ralists and physiologists suggest that the bear is not a hibernator
at all. This view stems from observations of reactivity and body
temperature during the dormant phase. These show that while
some bears are certainly inactive for a considerable season, they
do not become torpid and helpless as do most hibernators, but
may react with considerable vigor at any time and ean carry
out activities, e.@., parturition, which seem imeompatible with
hibernation. Further, the body temperature clearly does not fall
near the ambient temperature, although depression of the Ty
below the normal range (to 31°+) is reported from measure-
ments on a denned bear (Hoek, 1957). Other values on a captive
bear (Svihla and Bowman, 1954) and a recent measurement,
from our laboratory, on a denned bear in Wisconsin indicate Ty
values within the normal range.

We have just noted that beeause of its low critical tempera-
ture (equation 12) the bear should have difficulty in cooling,
and this would be so even with a considerable increase in con-
duetanee. Further, it may also be calculated that because of its
low thermal conductance (equation 9) it could not maimtain a
Ty, near the Ty. In any event, it is clear (equation 5) that the
bears lie in the interfacial zone between animals which might last
through the winter at or near their basal metabolie rates and
those which cannot. A small bear with little fat would fall in
the latter group, while a large bear with abundant fat would
definitely fall in the former eategory. Our Wisconsin bear (ef.
above) had more than enough fat to last through the winter
without metabohe alteration,

Even in the bears which need some extension of metabolic
reserves, the requirements will be much more modest than in
smaller animals. Thus, the limited observed depression of T,
and metabolism (Hoek, 1957) would be sufficient to extend the
fasting period by a eritical factor of perhaps 2-fold, and thus
allow survival in nature. This may qualify the bear as an
‘ecological’? hibernator. On the other hand, the limited extent
of these changes and the lack of adaptations for functioning at
low temperatures’ may disqualify the bear as a ‘‘ physiological”?
hibernator.

7VThe properties of peripheral nerve in the bear are similar to the non-

hibernating white rat rather than to the hibernating ground squirrel (Wehl and
Morrison, 1960, and unpublished observations).

86 BULLETIN ; MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Given the ability to hibernate at all, 1.e., to turn off or readjust
the ‘‘thermostat,’’ one may ask: Why did these species not
evolve toward an even more economical hibernation with even
lower Ty and metabolism in the winter. This eould be very
important in some situations, e.g., a poor season with limited
fattening. Perhaps, the size relations (equation 10) prevent
substantial lowering of the body temperature. However, even
were it achieved, this lower TR would present a distinet eon-
comitant disadvantage to the bear which, because of its size, 1s
often only poorly protected from disturbance. No one who has
encountered a denned bear will question the survival value of
this intermediate condition which, while permitting a substantial
saving of energy by complete inactivity and perhaps some lower-
ine of Ty, at the same time maintains the animal in condition to
take immediate defensive action. This is clearly in eontrast to
smaller hibernators, which, though helpless when disturbed at
a low temperature, are in nature effectively protected from such
disturbance by niches or burrows. It may be significant that the
Tr, near 30° in the denned bear is just low enough to provide a
reasonable reduetion in metabolic rate (faetor of 2), and is
also just high enough to provide effective activity for defense. At
any rate, a temperature near 30° appears critical for effective
action by other hibernators such as the bats (Reeder and Cowles,
1951), which on occasion actively maintain their T;, at this level
(Morrison, 1959).

The behavior of the bear is, then, indisputably distinet from
most hibernators, but the question of whether it hibernates or
merely enters a deep sleep will depend on our definitions
of these respeetive states. However, some of the controversy
regarding this animal may be resolved in terms of a flexible
response to meet different needs and circumstances.

Other medium-sized carnivorous aninals ineluding the skunk
and badger have been suggested as possible hibernators because
of their disappearance in the winter over considerable periods
when their normal food supply may be unavailable. But their
dens or burrows do not permit direct observation or removal
without disturbances, and they have not been induced to hiber-
nate in the laboratory. However, negative evidence may merely
indicate Imadequate conditions, since captive bears are seldom
dormant. Slonim (1952) has deseribed instability of body tem-
perature and metabolism in the badger during the winter and

1960 MAMMALIAN HIBERNATION 87

although deep hibernation was not observed, the general behavior
appeared not unlike that of the bear.

The energetic considerations advaneed above for the bear
are even more cogent as applied to these smaller species. No
conceivable amount of fat would tide these animals over a winter
season without a very substantial reduetion in metabolism. How-
ever, their reserves would be sufficient to last them over periods
of more than a month or perhaps two months with a modest
metabolie reduction of 2-fold as seen in the bear. Disappearances
of such duration have been noted during periods of inclement
weather.

Animals with adequate food either in stores or aecessible im
the environment need not fast. Fasting (and hibernation) is only
found in annals with inadequate external food reserves, what-
ever the reason. Animals without external reserves must use
internal reserves and often hibernate. However, there are inter-
erades such as the hamster which may either hibernate or use a
storage food. These might be referred to as permissive hiber-
nators, which may regularly have an option between hibernating
or not during the winter. These contrast strikingly to sueh a
form as the jumping mouse which does not store food, but is
so obligated to become dormant in winter that, when eonditions
are not suitable, it starves to death rather than modify its habits
or its metabolism.

Although we have confined our attention to mammals, It
should be noted that the problem of the extension of food reserves
over an inclement season is a common one to most cold-blooded
animals as well, and equations (1)-(6) may be modified for
their use. But the problem is more critical in homeotherms for
several reasons. The first is their higher metabolic rate, whieh
in part reflects their higher temperature. Thus, at 20° the
rattlesnake requires only 0.1 ce 0o/g¢/hr and is therefore com-
parable to the whale in maintenance efficiency. The second dis-
advantage in homeotherms is that their winter requirements may
increase due to the demands of thermoregulation, while those of
poikilotherms are substantially reduced. Finally, the organiza-
tion in homeotherms may be considered as of a more critical
nature with fixed maintenance requirements while in the lower
animals a considerable lability is tolerated. Table T summarizes
these concepts.

88 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

TABLE [

Equivalent Influences to Extend Fasting Duration by Two-fold.

FACTOR CHANGE
W x 16
m x72
f x2
Tr —10° (Qyo = 2.0)
—7 (Qip = 3.0)
Qio x 1.42 (Tp —20°)

x 1.26 (Ty. — 30° )

In conclusion, we have brought together several simple equa-
tions which interrelate body weight, fat content, metabolic rate,
ambient and body temperature and thermal conductance. These
equations which relate common aspects of the energetic balance
in hibernating and non-hibernating animals can be used as a
framework on which to present various hibernating species.
These equations conveniently show the quantitative substitution
of the various factors which both necessitate and permit hiber-
nation. They also show the gradient in the summed requirements
for hibernation in animals of inereasing size, and, with the
decrease of the sum to zero in larger animals, allow some predic-
tion as to the size limit of hibernators. The qualitative influence
of the various factors described here are well known, but it is
hoped that these relations may be useful in bringing them to-
gether and allowing the synthesis of a variety of observations
into a common pattern.

REFERENCES
ADOLPH, E. F.
1949. Quantitative relations in the physiological constitutions of mam-
mals. Science, 109:579-585,

Brer, J. Ro AND A. G. RICHARDS
1956. Hibernation of the big brown bat. J. Mammal., 37:31-41.
BENEDICT, F. G. AND R. C. LEE
1938. Hibernation and marmot physiology. Carnegie Inst. Washington
Publ., 497:1-239.
Bropy, 8.
1945. Bioenergetics and growth. Baltimore, 1023 pp.
MIseNTRAUT, M.
1929. Beobachtungen itiber den Winterschlat der Uaselmaus (Mis
cardinus avellanarius). Zschr. Siugetierk., 4:213-239.

1960 MAMMALIAN HIBERNATION 89

Hock, R. J.
1951. The metabolie rates and body temperatures of bats. Biol. Bull.,
101 :289-299,
1957. Metabolie rates and rectal temperatures of aetive and hibernat
ing black bears. Fed. Proe., 16:440.

IXAYSER, C.
1940. Les échanges respiratoires des hibernants. Théses, Univ. Stras-
bourg. 3864 pp.
1960. Hibernation versus hypothermia, (This volume, Pp. 9-30.)

KHL, T. H. AND P. MORRISON
1960, Peripheral nerve function and hibernation in the 13-lined ground
squirrel, Spermophilus tridecemlineatus, (This volume, Pp. 387-
403.)

,
1936. Veranderung im Fett Winterschlafender Fledermiiuse. Fette
und Seifen, 43:155-156.

Meyer, M. P. AND P. Morrison
1960. Tissue respiration and hibernation in the 13-lined ground
squirrel, Spermophilus tridecemlineatus. (This volume, Pp. 405-
420.)

MORRISON, DP.
1959. Body temperatures in some Australian mammals. I. Chiroptera.
Biol. Bull., 116:484-497.

MORRISON, P. AND F. A. RYSER
1951. Temperature and metabolism in some Wisconsin mammals. Fed.

Proe., 10:93-94.

Pirrs, :G: C;
1956. Body fat accumulation in the guinea pig. Am. J. Physiol., 185:
41-48.

REEDER, W. G. AND R. B. CowLES
1951. Aspects of thermoregulation in bats. J. Mammal., 32:389-403.

Ryser, F. A.
1952. The body temperature and its variation in small mammals.
Thesis, Univ. Wisconsin.

SCHOLANDER, P. F., V. WALTERS, R. Hock AnD L. IRVING
1949. Body insulation of some arctic and tropical mammals and birds.
Biol. Bull., 99:225-271.

SLONIM, A. D.
1952. Animal heat and its regulation in the mammalian organism.
Acad. Sei., U.S.S.R., Leningrad and Moscow.

Q() BULLETIN ;: MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Soutu, F. E.
1958. Rates of oxygen consumption and glycolysis of ventriele and
brain shees, obtained from hibernating and non-hibernating
mammals, as a function of temperature. Physiol. Zool., 31:6-15.

STRUMWASSER, F.
1959, Factors in the pattern, timing and predictability of hibernation
in the squirrel, Citellus beecheyi. Am. J. Physiol., 196:8-14.

SvinLa, A. AND H. S. BowMAN
1954. Hibernation in the American black bear. Amer. Midl. Nuat.,
§2 : 248-252.

VOLCANEZKY, J. AND A. FURSSAJEU
1934. Uber die Okologie von Citellus pygmacus Pall. im pestendemis
chen gebiete des westlchen Kasahstan. Zschr. Siugetierk.,
9:404-423.

DISCUSSION FOLLOWING MORRISON’S PAPER

STRUMWASSER suggested that it becomes a semantie prob-
lem to decide on criteria of hibernation when the quantitative
aspects become the all important considerations. Instead, one
might define hibernation qualitatively, yet operationally. Broadly
speaking, hibernation might be considered the regulation of a
set of physiological parameters at preferred levels leading to
minimal energy expenditure; obviously there are different de-
grees of hibernation. The regulation of a set of physiological
parameters at some new preferred level is hardly an unknown
phenomenon in biology ; the principle is evident in animals eseap-
ing from predators and might be expected in preparations for
migratory flight in birds. STRUMWASSER then asked MOR-
RISON what he meant by ‘‘turning off the thermostat.’” MOR-
RISON rephed that to his mind ‘‘turning off the thermostat”’
was the simplest way of envisioning hibernation taking place.
He pointed out that adaptations are generally conservative, that
if possible the animal modifies what is available rather than
evolve a new mechanism. Among bats, for example, there is no
question but that the ‘‘thermostat’’ is turned off rather than
reset at some lower level and some marsupials seem to have the
same ability.

A second question by STRUMWASSER was, ‘‘ What is the
Qo of metabolism during the actual entrance into hibernation ?”’
The reply was that to his (MORRISON’s) interpretation, an
animal could simply ‘‘slide down’? a temperature-metabolism

1960 MAMMALIAN HIBERNATION Q]

function. In general, he saw no difficulty with this as a response
pattern, and coneluded that onee the ‘‘thermostat was turned
off’? the system could respond passively.

PENGELLEY asked whether a marsupial which ‘‘turned
down its thermostat’? was able to lower its body temperature
to levels comparable to hibernation. MORRISON replied that
he had recorded marsupial body temperatures of below 30° at
ordinary ambient temperatures, but observations at lower am-
hbient temperatures have not been made.

DENYES stated the well-known biochemical doctrine that
fat is burned in the flame of carbohydrate.’’ Thus, if an animal
is using fat, it must use carbohydrate to make this possible.
In fat loss the limiting factor may be carbohydrate supply and
the animal then arouses. If an animal ends the hibernating
season with a residue of fat, this strengthens the contention that
carbohydrate is the true limiting faetor. MORRISON stated
that the problem of intermediary metabolic rate and fat utiliza-
tion in these animals was extremely difficult of solution.

V
TORPIDITY IN BIRDS

By Oniver P. PEARSON
Museum of Vertebrate Zoology
University of California
Berkeley, California

Birds and mammals are called homeotherms in spite of the
fact that their body temperature changes under varying ¢ir-
cumstances. <All of them operate near the upper temperature
tolerance of their bodies and so all resist rising body temperature
with whatever behavioral and physiological tricks they can per-
form. The response to lowered body temperature, however, is
quite different in different species. When considering this re-
sponse in mammals | have found it convenient to set up three
categories: (1) obligate homeotherms; (2) stubborn homeo-
therms; and (3) indifferent homeotherms.

Obligate homeotherms. The species in this category, when in
a cold environment, work with all their resources to retain a
high body temperature. When they have exhausted themselves
in this effort their temperature drops to a level at which they
cannot survive and from which they cannot recover. Man and
probably most other mammals fall in this category. These are
the chaste mammals, defending their thermal virginity to the
death against the onslaughts of the environment. Not all mem-
bers of this category are as precise regulators as fully clothed
man; drops of several degrees centigrade when in cold surround-
ines are not unusual, as well as shghtly lowered temperatures
during sleep.

Stubborn homeotherms. These animals maintain a warn
hody temperature over a wide range of environmental tempera-
tures, but under the influence of excessive cold or of hunger the
body temperature drops drastically. The animal can, however,
recover from the accompanying torpor and return to normal
active life. Peromyscus (deer mice) indulge in this emergency
torpor in the wild (Howard, 1951), and several species of Perog-
nathus (pocket mice) drop into torpor in captivity when food is
withheld (Bartholomew and Cade, 1957). These are the sub-
missive creatures whose thermal virginity will yield) to ap-
propriate environmental pressures.

Q4 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Indifferent homcotherms. In this category fall the least precise
temperature regulators, such as most species of Temperate Zone
hats, in which the body temperature drops almost to that of
the surroundings whenever they fall asleep in cool or even in
moderately warm surroundings. From this torpor they arouse
themselves spontaneously just as other animals awaken from
sleep. These are the animals of easy virtue, the promiscuous ones.

True hibernators (those that remain in deep torpor for many
days or weeks) can belone either to the second or to the third
category. Syrian hamsters in captivity seem to belong to the
second category (stubborn homeotherms) because they usually
require a little push (exposure to cold) to make them torpid.
In addition to bats, various hibernators such as jumping mice
(Zapus) belong in the third category, because a Zapus kept at
room temperature with an excess of food and water will spon-
taneously enter hibernation. Admittedly, the boundaries between
the three categories are not sharp. A species or an individual
may shift back and forth between the second and third category
depending upon the time of year, endocrine balance, etc. Never-
theless, I find the classification useful. Whenever | meet an un-
familar species, I like to know into which category it falls, for
this helps to define its metabolic personality.

Among birds the same three categories are appropriate. Most
birds fall into the first category, obligate homeotherms, and
maintain a warm, fairly stable body temperature throughout
a wide range of environmental temperatures. Most birds are
strictly diurnal and probably more lax than mammals about
body temperature while they are asleep, but, although many
birds permit their body temperature to drop 2°C or more when
they sleep (accompanied by a drop of 10 per cent or more in
metabolic rate), unlike sleeping bats they continue to regulate
their temperature nicely at the lower sleeping temperature.
Therefore, the birds in this category, the obligate homeotherms,
are of no further interest in this discussion. It is in the other
two categories that we find torpor.

Much of the literature on torpidity in birds has been discussed
in-a valuable review by MecAtee (1947). He pointed out that
scientists have been slow to acknowledge the oeeurrence of
torpidity in birds, in spite of dozens of seeminely reliable reports.
Within the past 15 vears, however, torpidity has been demon-
strated conclusively by physiological criteria in several species,
and it is reasonable to assume that other species will be added
to the list in the future. It is certain that we still have much

1960 MAMMALIAN HIBERNATION 95

to learn about the nature of torpor in those species already known
to become torpid. I shall review what has been learned so far
about torpor in the few species that have received more than
minimum attention,

Swifts. Swifts make their living by sweeping through the air
in pursuit of flying msects. One of the risks accompanying: this
way of life is the danger of starvation when bad weather keeps
the poikilothermous prey grounded while the warm-blooded
predator must keep its machinery running at almost full speed.
Swifts and many other birds migrate to warmer lands and thus
are not concerned with the over-winter scareity of flying insects,
hut this does not solve the problem of reduced food supply
during spells of stormy weather in the summer nesting season.
Most species of small birds will die in a few days without food,
vet swifts live in many regions where several consecutive days
of bad weather are not only a possibility but a probability. Ap-
parently in response to this environmental challenge swifts have
developed the ability to drop into temporary torpor, thereby
slowing until the return of good weather the rate at which they
use up the food resources stored in their tissues.

Koskimies (1948, 1950) has done the most pertinent research
with swifts. He reports, for example, that nestling European
swifts (Apus apus), which become so fat that they frequently
weigh more than their parents, can survive at 24°C without food
or water for more than 10 days (and this despite frequent dis-
turbances). For the first few days of this starvation they main-
tain a warm body temperature and lose weight rapidly, but
thereafter they drop each night into torpor, then warm them-
selves up again each morning. During the nightly torpidity their
body temperature drops to within a few degrees of the sur-
roundings (ambient temperatures were 19° and 24°C), and
oxygen consumption and carbon dioxide production are reduced
accordinely. The birds can be handled and probed without
arousing them. The adults also become torpid, but not as
readily.

Bartholomew, Howell, and Cade (1957) investigated white-
throated swifts (Aéronautes saratalis) and found that adults
of this species also become torpid after food deprivation. When
the birds were kept at 5°C there was some evidence that they
would not let their body temperature fall below about 18°C.
Furthermore, under the experimental conditions, birds whose
body temperature dropped below 18°C died. This suggests that
these swifts have only a limited eapacity for hypothermia. It is

96 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

interesting that their point-of-no-return is close to the tempera-
ture from which laboratory rats are unable to warm themselves
by their own efforts (Andjus and Smith, 1955).

It is clear that both species of swifts that have been tested
are able, under laboratory conditions, to enter torpor and_ to
emerge from it spontaneously. It also appears that they must
pushed’’ by food shortage before they will become torpid.
This places them in the second category of animals outlined
above (stubborn homeotherms). Knough reliable reports have
been published about swifts found torpid in the wild so that it
seems fairly safe to assume that torpidity does play a role in the
energy economy and survival of swifts in nature.

Poor-wills and other caprimulgids. Poor-wills and nighthawks
are nocturnal, insect-catchine birds with some of the same meta-
bohe problems as swifts: what to do when bad weather cuts off
the supply of flying insects? Poor-wills (Phalaenoptilus nuttali) ,
the species about which most 1s known, respond under some cir-
cumstances by becoming torpid. In fact, they hibernate. This
habit seems to have been known to the American Indians of
the Southwest, but not until the reports of Culbertson (1946)
and Jaeger (1948, 1949) was the hibernation of poor-wills
acknowledged to be firmly established. Jaeger discovered a
torpid poor-will in a crypt in a granite cliff, banded it, and
found that the same bird returned to the same erypt for three
or more winters. Week after week the bird was found torpid at
the same spot with a body temperature of 18° to 20°C. Air
temperatures during the mornines when the bird’s temperature
was taken varied from 17° to 24°C. Maximum-minimum temper-
atures during the same interval at a weather station nearby
fluctuated between —6° and 25°C. It is not known how fre-
quently the bird roused itself, if at all, but in view of the fact
that it was found to be torpid whenever visited, no matter
whether it was day or night, there scems to be little doubt that the
torpor lasted for many days consecutively and may have lasted
for weeks. Since poor-wills are known to accumulate enormous
amounts of fat in the autumn (Marshall, 1955), we must con-
sider the bird to have fulfilled all the requirements of true
hibernation.

The poor-will’s potential for prolonged hibernation can be
estimated. Their metabolic rate in deep torpor at air tempera-
tures lower than 10°C is one-tenth to one-twentieth that of resting
poor-wills, and cloacal temperatures are frequently within
O.1°C of the surroundings (Bartholomew, Howell, and Cade,

1960 MAMMALIAN HIBERNATION 97
1957; Howell and Bartholomew, 1959). These authors estimate
that ten grams of fat could sustain a torpid poor-will for more
than three months if the bird’s temperature remained below
10°C.

Marshall (1955) kept poor-wills over the winter in an outdoor
cage at Tueson, Arizona, and found that they frequently became
torpid. They never remained torpid for more than four days,
but this may have been because of frequent disturbances and
because of the sunny exposure of the shed in which the birds
were kept. There was no series of more than three cloudy days in
a row. He found that cold weather alone would not make the
birds torpid. It was necessary to withhold food for one or more
days before any of the birds would drop into torpor. There
is some evidence that poor-wills cannot arouse themselves while
the surrounding temperature remains below 15°C (Howell and
Bartholomew, 1959).

Brauner (1952) raised a young poor-will and kept it in cap-
tivity for many months. Below-freezing cold, starvation, and
reduced day-length all failed to push it into hibernation. Its
body temperature remained above 86°C during these manipula-
tions. Presumably its hibernation machinery was not vet im
readiness.

During an average day, poor-wills have brief periods of activ-
ity at dusk and before dawn. Their total period of activity lasts
scarcely more than one hour out of 24 (Brauner, 1952). Wild
poor-wills collected in the evening during one of their active
periods (ambient temperatures between 8° and 30°C) have body
temperatures between 40.5° and 43.1°C (Miller, 1950; Brauner,
1952; Marshall, 1955). In between their two periods of activity,
however, they sit quietly with body temperature lowered 1°
to 38°C. Their temperature is high during exercise, no matter
what the time of day or night. This spread between the tempera-
tures of active and inactive or sleeping poor-wills is similar to
the spread found in non-hibernating small birds. On some
occasions, however, the temperature of resting poor-wills drops
considerably lower. Miller (1950) measured the temperature
of a wild poor-will flushed from its daytime roost in August at
10:30 a.m. (air temperature 20°C, light rain) and found it to
be only 34°C. Although able to fly, this bird obviously was in-
dulging in a bit of the poikilothermia available to this species and
no doubt was benefiting from the resultant saving of energy.

In summary, poor-wills are capable of reversible deep torpor
and actually engage in true hibernation in the wild. The evidence

98 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

accumulated so far suggests that they will not become torpid
while food is abundant, and accordinely they are classified as
stubborn homeotherms. While they are at rest in the daytime,
their body temperature sometimes drops to a level lower than
‘hat usually found in other birds.

Marshall (1955) has reported on two trilline nighthawks
(Chordeies acutipenns) kept in captivity in an unheated room
in Tucson, Arizona, durine’ one summer and autumn. They
hecame quite fat and at the end of November both fell into
torpor (body temperatures 18.6° and 19.2°, room 18.7°C) and
both emerged from their torpor when warmed. Since nighthawks
face the same danger of weather-induced shortage of flying in-
sects as poor-wills, it is not unhkely that nighthawks make use
in the wild of the capacity for torpor shown by these captives,
but evidence is still lacking.

Hummingbirds. Most birds have no sense of smell and poor
night vision — deficiencies that force them to be diurnal. When
these deficiencies are combined in the same animal with an
extremely high rate of metabolism, as in hummingbirds, the night
becomes a dangerously long period of fasting. Presumably in
response to the threat of overnight starvation hummingbirds
have developed an ability to become torpid. In addition, it is
possible that poor weather reduces the supply of nectar and
insects on which hummingbirds feed, and that this weather-
induced food shortage also exerted a strong selective pressure
for torpor on humminebirds, as it no doubt did on swifts and
poor-wills.

Ruschi (1949) noted that many of the hummingbirds in his
aviary in Brazil became torpid overnight, some for as long as
14 hours. For nine genera of tropical hummingbirds, average
body temperatures at 3 p.m. varied between 39.5° and 44.6° (air
temperature approximately 26°C). Body temperatures of sleep-
ine birds at 10 p.m. varied between 36.6° and 40.5° (air approxi-
mately 23°C), and the body temperature at which mdividuals
of these species entered torpor varied between 32.0° and 36.3°C,
ITe states that the body temperature must drop about 7°C below
the normal daily temperature before the bird becomes torpid.

Anna and Allen hummingbirds (Calypte anna and Selasphorus
sasin) have been studied more than other species. Figure 1
shows the rate of oxygen consumption during a 24-hour period
of a captive bird kept at 24°C. The rate of oxygen consumption
of the bird when awake but resting was about 14 c¢e/e¢/hr (Pear-
son, 1950). At dusk, however, the rate dropped briefly to a

1960 MAMMALIAN HIBERNATION 99

‘‘sleeping’” level and then plunged to a torpid level at about
0.8 ce/g hr. Before daybreak the bird spontaneously roused
itself. During the nightly torpor of hummingbirds their body
temperature drops to within a few degrees of the surroundings
(Pearson, 1950; Bartholomew, Howell, and Cade, 1957). Pearson
(1950, 1953) found torpid hummingbirds at night in the wild,
so it is clear that this is not just a laboratory-induced torpor.

Since hummingbirds are able to maintain a high body tempera-
ture for many hours without food, and yet drop quickly into
torpor after dark, even at mild temperatures, they belong in
the third category, indifferent homeotherms, members of which
can enter torpidity without thermal or hunger stress. In many
respects such as this one, the metabohe personality of hummine-
birds is like that of the small insectivorous bats.

HUMMINGBIRD

CC/G/HR
i)

NOON | 2 3 4 5 6 7 8 9 10 tI NIGHT | 2s ¢ 8 ¢ * 6 9-0

Fig. 1. Rate of oxygen consumption of an Anna hummingbird for 24
hours at air temperature of 24°C compared with the rate for a shrew o4
comparable size (Sorex cinereus). From Pearson (1950).

Some idea of the amount of energy saved by overnight tor-
pidity may be estimated by comparing (Fie. 1) the rate of
oxygen consumption of the humminebird with that of a shrew of
about the same size. Both species have about the same rate of
metabolism when resting, but the shrew keeps its body warm at
all times, an expense that it can afford because its senses enable
it to forage for food at all hours. The energy that the hummine-
bird saves per day by becoming torpid is indicated by the area
between the two curves. At cold temperatures the saving would
be even greater.

High in the Andes, where a few species of humminebirds are
able to survive, the air temperature at might frequently drops
far below freezing, even in the summer. This would endanger
torpid birds, but the hummingbirds avoid this danger by going
into relatively thermostatic caves at meght, and by building their
hests in eaves (Pearson, 1953).

100 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

It is difficult to imagine how nightly torpor could be combined
successfully with incubation and brooding of young. Howell
and Dawson (1954), by mserting a thermocouple into an Anna
hummingbird nest, have shown that the incubating female main-
tains a high body temperature overnight and that the advanced
nestlings also do not become torpid. Obviously, hummingbirds
are able under certain favorable circumstances to survive over-
night without torpor. The thick insulation of the nest undoubt-
edly aids them in maintaining a warm temperature. It will be
interesting to learn more about the physiological control of tem-
perature in these nesting hummingbirds. Among mammals, body
temperatures are elevated during the luteal phase of the estrous
evele and after injection of the luteal hormone progesterone
(Landau, Bergenstal, Lugibihl, and Kascht, 1955), and pregnant
sloths maintain a tighter control of their body temperature
durine exposure to cold than do control animals (Morrison,
1945). Perhaps an analagous or even homologous endocrine
control operates in birds.

Size X torpidity. A humminebird, living in the wild, uses
about 10.3 Calories per 24 hours if it sleeps at might without
becoming torpid, but only 7.6 Calories if it becomes torpid during
the night (Pearson, 1954). Why don’t more species take ad-
vantage of similar metabolic savings? An animal the size of
asmall bat or a hummingbird can warm itself up from torpidity
at a rate of about 1°C per minute (Bartholomew, Howell, and
Cade, 1957) so that the entire process takes less than half an
hour, sometimes as little as ten minutes. Swifts, which are
larger, Warm at a rate of about 46°C per minute, and poor-wills,
Which are larger still, take more than one hour to emerge from
torpor. Entrance ito torpor takes even longer. Consequently,
large birds or large mammals could not afford the time necessary
to enter and emerge from torpidity each day in the manner of
humminebirds and bats.

Furthermore, the energy expense of warming up a large mass
of tissue is too great. Assuming a specific heat for flesh of
0.95 Cal/ke/°C (calculated from White, 1892), it costs a 4-¢gram
hummingbird 0.114 Calories to warm its body from 10° to 40°C.
This is a mere 1¢5th of the total 24-hour energy expenditure of
an active hummingbird in the wild. In contrast, a 200-ke¢ bear
would need 5100 Calories to warm up from 10° to 37°C — and this
is a full 24-hour energy budget for a bear. So even if there
were time enough in 24 hours for a large animal to enter and
emerge from torpidity, it would be metabolically unprofitable.

1960 MAMMALIAN HIBERNATION 10]

Granted that short periods of torpor would not be profitable
for large birds and mammals, why do they not take advantage
of the metabolic savings inherent in prolonged periods of hiber-
nation or deep torpor? One reason seems to be that because of
their low rate of metabolism they can survive without food for
long periods without resorting to torpor. By being relatively
inactive, a bear without food can draw for many months on its
supply of fat, and a bull fur seal is able to make his supply of
fat last during months of vigorous activity while he is ashore
during the breeding season. Such large animals clearly do not
need to resort to hibernation to survive lone periods of starva-
tion.

In conclusion, the occurrence of torpidity among birds. is
best documented for certain ecaprimulgids (poor-wills and night-
hawks), mieropodids (swifts), and troehilids (hummingbirds) —
groups usually considered to be fairly closely related taxo-
nomieally and evolutionally. The species exhibiting hypothermia
are all relatively small birds, and all feed, at least in part, on
insects in a manner that exposes the birds to starvation during
poor weather. A fourth unrelated group, the swallows, also feeds
on flying insects, and numerous field reports make it seem likely
that torpidity plays a role in their biology, but physiological
measurements are still lacking for them as well as for a few
other kinds of birds suspected of torpidity.

REFERENCES

ANpDgus, R. K. anp A. U. SMITH
1955. Reanimation of adult rats from body temperatures between 0°
and +2°C. J. Physiol., 128:446-472.

BARTHOLOMEW, G. A. AND T. J. CADE
1957. Temperature regulation, hibernation, and aestivation in the little

pocket mouse, Perognathus longimembris. J. Mammal., 38:60-72.

BARTHOLOMEW, G. A., T. R. HOWBLL AND T. J. CADE
1957. Torpidity in the white-throated swift, Anna hummingbird, and
poor-will. Condor, 59:145-155.

BRAUNER, J.
1952. Reactions of poor-wills to light and temperature. Condor, 54:
152-159.

CULBERTSON, A. E.
1946. Occurrences of poor-wills in the Sierran foothills in winter.
Condor, 48:158-159.

102 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

llowarp, W. E.
1951. Relation between low temperature and available food to sur-
vival of small rodents. J. Mammal., 32:300-312.

Ilowruh, T. R. AND G. A. BARTHOLOMEW
1959. Further experiments on torpidity in the poor-will. Condor,
61:180-186.

Hloweun, T. R. AND W. R. DAwson
1954. Nest temperatures and attentiveness in the Anna hummingbird,
Condor, 56:93-97,

JAEGER, EK. C,
1948. Does the poor-will ‘‘hibernate’’? Condor, 50:45-46,
1949. Further observations on the hibernation of the poor-will. Con-
dor, 51:105-109.

IXOSKIMIES, J.
1948. On temperature regulation and metabolism in the swift,
Micropus a. apus ., during fasting. Experientia, 4:274-276.
1950. The life of the swift, Micropus apus (L.), in relation to the
weather. Helsinki, 151 pp.

LANDAU, R. L., D. M. BERGENSTAL, K. LUGIBIHL AND M. E. KAsScutr
1955. The metabolic effects of progesterone in man, J. Clin. Endo-
erinol. and Metab., 15:1194-1215.

MARSHALL, J. T.
1955. Hibernation in captive goatsuckers. Condor, 57:129-154.

McAtTer, W. L.
1947. Torpidity in birds. Amer. Midl. Nat., 38:191-206,

MILLER, A. H.
1950. Temperatures of poor-wills in the summer season. Condor,

52:41-42.

MorRISON, P. R.
1945. Acquired homiothermism in the pregnant sloth. J. Mammal.,

26 : 272-275.

PEARSON, O. P.
1950. The metabolism of hummingbirds. Condor, §2:145-152.
1953. Use of caves by hummingbirds and other species at high
altitudes in Peru. Condor, 55:17-20.
1954. The daily energy requirements of a wild Anna hummingbird.
Condor, 56:317-322.
Ruscul, A.
1949. Observations on the ‘Trochilidae. Bull. Mus. Biol. Prof.
Mello-Leitao, Santa Teresa, Brazil, No. 7. (Seen only in trans-
lation prepared by C. H. Greenewalt).

1960 MAMMALIAN HIBERNATION 103

Whitt, W. I.
1892. A method of obtaining the speeifie heat of certain living warm
blooded animals. J. Physiol., 13:789-797.

DISCUSSION FOLLOWING PEARSON’S PAPER

STEEN mentioned several other species of birds as possessing
the faculty for going into torpor at night. These were the tit-
mouse, the red poll, and the tree sparrow. He also pointed out
that these animals arouse (warm up) from this torpor within
five minutes after beine disturbed, and arouse also when left
alone, undisturbed, and in the dark.

PEARSON replied that there are field reports on other birds,
but he had mentioned only those on which body temperature
changes had been measured. He also indicated that reports on
more species surely will appear in the future. There have been
many reports of torpid swallows in the wild.

WIMSATT asked if torpidity was correlated with the insecti-
yorous or with the seed-eating habit. PEARSON noted that
the seed-eater’s food goes through its body in 144 hours. Thus,
dietary factors may be of importance in distinguishing a
tendency to hibernate.

STEEN noted the similarity in behavior in aboriginal human
heings, for they also are found to be torpid in the morning with
hody temperatures of 34.5°C. They also cool during the night
although they appear to eat well.

DAWE asked if nocturnal torpidity in birds was correlated
with season. PEARSON indicated that not enough examples
from the wild have been observed. The poor-will seems to be
seasonal, the hummingbird shows torpidity throughout the vear.

FOLK asked the speaker to comment on the correlation be-
tween the cyche torpidity behavior and oxygen consumption.
PEARSON indicated that cyclic behavior in birds was tied in
with feeding, and that a burst of activity appeared before the
animal went into the torpid state for the night.

VI
ENDOCRINES IN HIBERNATION’

By VoJin Popovic
Division of Applied Biology
National Research Council

Ottawa, Canada

In the last few years a number of data have shown hiberna-
tion in a new light. Hibernation is an active process which
consists essentially of 4 phases: 1) preparation for hibernation:
2) changing the ‘‘lfe speed’’ from the high to the low one;
3) regulating at the new low level and 4) returning to euthermie
life.

If the endocrines have a role in hibernation, and a number
of data have suggested it, then the effect of the endocrines is
probably to be observed in all stages. However, up to now the
most striking manifestation, and therefore the easiest to detect,
was the change in the endoerine glands during the preparation
period.

Gemelli (1906) was the first to deseribe the winter involution
of the anterior hypophysis in hibernators. Since then, many
investigators found involution and hypofunetion of the anterior
hypophysis, as well as of other endocrine glands, in hibernators.
These observations were made during the fall and winter months.
Today, it is generally agreed that most endocrines involute
before, not after, the hibernator enters wintersleep. The anterior
hypophysis (Kayser and Aron, 1938), thyroid (Adler, 1920;
Coninx-Girardet, 1927), adrenal cortex (Kayser and Aron, 1950)
and gonads (Kayser and Aron, 1950; Kayser, 1955a) change
considerably before winter lethargy appears.

Involution of the endocrines before entering hibernation is
accompanied by a decrease in the basal metabolie rate (Gelineo,
1938; Popovie, 1951), a decrease in resistance to low oxygen
tension of the inspired air (Popovic, 1952a), decreased mobil-
ity, and diminished food intake. It is probable that a number
of physiological processes are changing during this period. At
the end of that period the animal enters hibernation. The en-
vironmental temperature is not a decisive factor in this process.
Ground squirrels, for instance, will begin to hibernate in autumn

1 Issued as N.R.C. No. 3709.

106 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

and will come out of hibernation in spring even if kept in a room
at a constant high thermoneutral temperature of 30°C (Popovie
and Popovic, 1956).

The appearance of regressive changes of the endocrines before
entering hibernation is highly regular. This is why Kayser
(1955b) states that there is no hibernation without involution
of the endoerine elands.

Kayser (1957a) as well as Eisentraut (1956) consider hiber-
nation essentially as a problem of thermoregulation. However,
even if hibernation is considered as a complex problem, a simi-
lar approach could be methodologically useful — especially to-
day when data on that problem are not too numerous and often
contradictory. This is the reason why attention should be paid
also to the possible role of the endocrines in the thermoregu-
latory mechanism of hibernators as well as non-hibernators. H1-
bernation is also a kind of adaptation to the cold and other
trying conditions associated with cold weather. Therefore, hor-
monal control of adaptation to cold in hibernators and non-
hibernators should also be discussed.

Anterior Hypophysis

A number of experiments, performed on different homeo-
therms, show that the anterior hypophysis has a considerable
role in temperature regulation. It was found that the homeo-
therm’s resistance to cold was greatly reduced after hypophys-
ectomy (Tyslowitz and Astwood, 1942). The inability of
hypophysectomized mammals to keep their body temperature at
the normal level when exposed to moderate cold is so marked
that some investigators consider that such animals are no longer
homeotherms (Schaeffer and Thibault, 1946). These findings
fitted quite nicely with the picture of hibernators as ‘‘lower
homeotherms,’’ i.e., hibernators overcome by external cold and
passively entering hibernation when the temperature of the
environment decreases. Such opinion was widespread even 10-15
years ago and was also one of the reasons why the anterior
hypophysis was studied in hibernators. During the summer
months, hibernators do not enter hibernation even if the en-
vironmental temperature suddenly decreases, because their an-
terior hypophysis is active and their heat production (measured
chiefly by the basal metabolic rate) is as high as in non-hiberna-
tors. But when the fall approaches the hypophysis involutes and
heat production (BMR) decreases. Soon afterwards, when heat

1960 MAMMALIAN HIBERNATION 107

loss exceeds the decreased heat production, the animal becomes
more and more hypothermic and hibernation begins. However,
some of the postulates of that theory are not acceptable today
and therefore the theory loses some of its validity. Thus, for
example, in rats and other non-hibernators both BMR and cold
thermogenesis (chemical thermoregulation) are decreased after
hypophysectomy (Sahovie, Popovie and Anaf, 1953). On the
other hand, in ground squirrels and hamsters, before entering
hibernation, the BMR is decreased but cold response is increased
when compared with summer animals (Popovic, 1951, 1953; Fie.
1). This fact is quite unexpected: in spite of the involution of
the anterior hypophysis and hypofunection of other endocrine
elands prior to hibernation, the biggest part of the total heat
production (cold thermogenesis) is increased during the eold
seasons.

The anterior hypophysis of a hibernator, as well as some other
endocrine glands, show a pronounced biological rhythm in the
course of a year. The anterior hypophysis involutes in the be-
ginning of the fall (Kayser and Aron, 1950) before any sub-
stantial decrease in environmental temperature, and returns to
the high spring and summer activity in the middle of the winter.
considerably before hibernation ceases (Petrovic and Kayser,
1957). After extirpation and transplantation of the hypophysis,
the graft does not show the same rhythm, as judged by its effect
on gonads (Petrovic and Kayser, 1957).

The effect of extirpation of the hypophysis was studied in
ground squirrels (Foster ef al., 1939), and hamsters (Kayser,
1950). Foster et al. found that hypophysectomized ground squir-
rels ean hibernate only for 3 days and, unless they were re-
moved from the cold, death occurred.

In our opimon, the hibernation of ground squirrels in the
aforementioned experiments is only hypothermia which occurs
after hypophyseectomy in hibernators as well as in non-hiberna-
tors, and which differs in every respect from hibernation (Popo-
vic, 1952b, 1960a) except that the body temperature is low in
both cases. Thus, for instance, a non-hibernator, the rat, can
stand lethargic hypothermia at 15°C body temperature for 9
hours only, while an artificially cooled ground squirrel can live
119 hours at 10°C body temperature. This long survival of
artificially cooled hibernators could be easily misinterpreted
as hibernation, as Foster ef al. (1939) and some other investi-
gators studying endocrines or different physiological processes

108 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

did. It is difficult to distinguish hibernation from the hypo-
thermie state in a hibernator. But if, after a few days, hiber-
nation ‘‘ends in death’’ it suggests strongly that only a hypo-
thermie state was involved. The most eonvineing proof would
be obtained by measuring the oxygen consumption which is sev-
eral times lower in hibernation at the same low body temperature
than in hypothermia (Popovie, 1952e, 1959). Even a few hours
before death, the oxygen constunption is considerably higher in
hypothermia than in hibernation.

Hypophysectomized hibernators rarely survive long enough
(Kayser, 1950) to determine whether or not they eould hiber-
nate. When exposed to low external temperature, hypophysec-
tomized hibernators soon die, as do the non-hibernators, before
it is possible to see if hibernation will oceur or not. Our opinion
is that similar experiments could be performed now under better
conditions. It has been shown recently (Popovie and Popovie,
1956) that normal ground squirrels will hibernate during the
winter months even if kept at 30°C room temperature. Simi-
larly, Bartholomew and Hudson (1959) observed torpor in
desert ground squirrels during the summer. Thus, it seems that
the temperature of thermal neutrality, or slightly lower, is suit-
able for keeping hypophyseetomized hibernators alive for long
periods of time and allows the investigator to observe hiberna-
tion if it exists.

We conclude that the anterior hypophysis has a temperature
regulation role in homeotherms. In hibernators this endoerine
gland has a seasonal rhythm but the data purporting to show its
possible role in hibernation should not be accepted until per-
formed under better conditions.

Parathyroid

This gland does not seem to have any effect on thermoregula-
tion. At least the parathyroidectomized rats stand the cold as
well as the controls (Leblond and Gross, 1948).

There are only a few observations upon the role of the para-
thyroid gland in hibernation. Adler (1926) thought the para-
thyroid of hibernating animals was hypoactive, contrary to
Skowron and Zajaezek (1947) who saw a hyperactive parathy-
roid during hibernation, and to Kayser (1957a) who eonsidered
it as normally active.

In conclusion, the very few collected data about the parathy-
roid gland in hibernators do not indicate any decisive role of
this endocrine gland in hibernation.

1960 MAMMALIAN HIBERNATION 109

Thyroid

A number of data show that the thyroid has a definite role in
the thermoregulation of non-hibernators.

In anesthetized rats (Gergely, 1943) as well as in unanesthe-
tized rats (Schaeffer, 1946), the heat production is decreased
after thyroidectomy. The decreased heat production is prob-
ably the cause of somewhat diminished cold resistanee found

SUMMER WHITE RATS
GROUND (DEPOCAS ET AL)
SQUIRRELS
(GELINEO)

WINTER GROUND SQUIRRELS

GROUND SQUIRRELS

SUMMER
15°—20°G

=s0°C O 30°C —30°C O 30°C
ROOM TEMPERATURE

Fig. 1. Oxygen consumption of active ground squirrels. Upper left, active
summer ground squirrels adapted to 18-23° C and 29-32° C. Upper right,
white rats adapted to 6° and 30° C. Lower left, active winter ground squir-
rels adapted to external temperature of 2-5° C or 30° C. Lower right, active
summer and winter ground squirrels adapted to similar external tempera-
tures.

after thyroidectomy (Leblond and Gross, 1943) or after admin-
istration of thiouracil (Ershoff, 1948). After thyroidectomy the
basal metabolic rate is diminished but the cold response per se
(cold thermogenesis, i.e., maximal metabolic rate of a homeo-
therm exposed to cold minus basal metabolie rate) is not altered
(Sahovie and Popovie, 1953). The drop of BMR in thyroidee-
tomized animals is considerable, up to 30-40 per cent (Popovie

110 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

and Popovie, 1951). A similar difference in BMR is found be-
tween cold- and warm-adapted rats (Depocas et al., 1957) (Fig.
Ee

Boatman (1959) coneluded that the thyroid has a role in
maintaining body fluids in an efficient equilibrium for rapid
adjustments in a cold environment.

Thyroid hormones increase the 0. consumption of tissue slices
too (Barker, 1951, 1955) ; after thyroidectomy the Oo consump-
tion of tissue slices is 30 per cent smaller (Spach eft al., 1955).
However, it is not certain how thyroid hormones affect metabol-
ism, whether centrally or not. The vegetative nervous system as
well as adrenaline could play a role.

Thyroxin or thyroidectomy affect the level and activity of
some enzymes (Tipton ef al., 1946; Tipton and Nixon, 1946;
Maley, 1957).

The thyroid aetivity of homeotherms is increased during cold
exposure as shown by histological investigations (Uotila, 1939)
or by incorporation of iodide into the thyroid gland (Leblond
et al., 1944; Cottle and Carlson, 1956). In cold adapted non-
hibernators the increased activity of the thvroid (over twice
that of warm adapted as caleulated by Hart, 1958) is sustained
for long periods of time (Cottle and Carlson, 1956; Woods and
Carlson, 1956). The thyroid is also the seat of seasonal changes
independent of environmental temperature (Bernstein, 1941).

The activity of the thyroid gland is greatly reduced when
deep hypothermia is induced in non-hibernators, as measured
by the histological picture (Ariel and Warren, 1948) or by in-
corporation of [151 (Verzar and Vidovie, 1951; Andjus et al.,
1954).

In hibernators the thyroid gland changes considerably from
season to season. The chanees are even greater than in non-
hibernators and in contrast with non-hibernators the cold sea-
sons induce regressive changes, in hibernators, as ilustrated by
the histological picture (Adler, 1920; Coninx-Girardet, 1927 ;
Kayser and Aron, 1938; Zalesky and Wells, 1940), blood studies
(Kratinov and Skirina, 1947), or by the incorporation of [1#1
(Lachiver, 1952a,b; Vidovie and Popovic, 1954). The incorpo-
ration of [1°1 into the thyroid of active ground squirrels is very
low in autumn, hardly higher than in hibernating animals at a
body temperature of 5-10°C (Vidovie and Popovic, 1954). In
active hibernators prolonged cold exposure does not change the
histological picture of the hypoactive thyroid gland (Kayser,
1939; Deane and Lyman, 1954) except in the midwinter cold

1960 MAMMALIAN HIBERNATION 11]

(Suomalainen, 1956) but increases the basal metabolic rate
(Popovie, 1955a; Adolph and Richmond, 1956). Sinee the in-
crease or decrease of BMR, a consequence of acclimatization to
cold or warm environments, is present in thyroidectomized guinea
pigs (Popovic and Popovic, 1951), it seems that the adaptive
changes of BMR are not to be associated with thyroid gland.
In the middle of the winter the incorporation of [1°1 returns
to the higher level (Vidovie and Popovic, 1954). Thus in active
ground squirrels the thyroid becomes active again, considerably
before the period of hibernation ceases. These findings were eon-
firmed on other hibernators (Lachiver et al.. 1957). However
the reactivation of thyroid is accompanied by more frequent
awakenings and shorter hibernating periods.

Total extirpation of the thyroid does not prevent ground
squirrels from hibernating (Foster et al., 1939). The frequeney
and the length of periods spent in hibernation are similar to
that of the controls (Popovic, 1955a). The same results were
observed after prolonged administration of thiouracil (Popovie,
1955a). It was mentioned that the basal metabolic level of active
hibernators is low during autumn and winter. A further lower-
ing of BMR was observed when hibernators (ground squirrels)
were exposed during the cold seasons to the temperatures of
their thermal neutrality (30-32°C) or treated with thiouracil
(Popovic, 1952b). These tests show that although the involuted
thyroid is not essential for entering hibernation it has a certain
role in keeping the BMR at the winter level.

Injected thyroid extracts wake up the hibernating animal
(Adler, 1926). Hibernation of hedgehogs stops when thyroid
extracts of either winter or summer hibernators are used (Uiber-
all, 1934). Injected thyrotropic hormones decrease the number
of days spent in hibernation (Foster ef al., 1939) and injected
homogenates of the anterior hypophysis stimulate the thyroid
during the cold seasons but not in summer (Petrovic and Kayser,
1958).

Seasonal changes of many endocrine glands, their involution
during hibernation, and particularly his own results (which
indicated that injected extracts of a number of endocrine glands
wake up hibernators) gave Adler one of the basic postulates for
developing his endocrine theory of hibernation. However, the
experimental basis of Adler’s work often has been submitted to
strong criticism. It has been said that Adler was not careful
about the right temperature of injected solutions, that thyroid
and other endocrine gland extracts were not properly prepared,

112 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

that they had very small or no hormonal content, ete. The most
important objection is that the glandular extracts had to be in-
jected by needle, and this technique evidently is not appropriate.
It is known that the nerves of hibernators conduct impulses even
when the body temperature is very low (Chatfield et al., 1948).
The injection by needle will wake most hibernating animals
even if no substance or only a physiological solution is injected.
Even smaller stimuli interrupt hibernation, such as measure-
ments of colonic temperature by a small thermometer or han-
dling of the animal, ete. For this reason Lyman and Chatfield
(1956) stated that ‘‘more delicate and better controlled methods
must be developed before the cause of spontaneous arousal is
clarified.’’

However, the new technique of permanent cannulation of the
aorta and vena cava in small laboratory animals (Popovie and
Popovic, 1960) now permits clarification of this question. The
hormones of any gland can be injected into the blood stream
of the hibernating animal without disturbing normal winter-
sleep; thus all these questions about hormones and their effect
on hibernation could be investigated now under better experi-
mental conditions.

Feeding the hibernators with dry thyroid gland results in
cessation of hibernation (Adler, 1926). Moreover, a very small
amount of that gland given with the food every third day gives
the same effect. Elevation of the BMR is very small in these
experiments. Values of the BMR do not exceed those in summer
in the same animals (Popovic, 1955a) so that the toxie effect of
thyroxin probably should not be considered at all as the cause
of cessation of hibernation.

In summary, the thyroid gland in hibernators shows a strongly
accentuated biological rhythm. In contrast with non-hibernators,
cold seasons induce hypofunction of thyroid in’ hibernators.
Without the thyroid, hibernators can continue to hibernate the
same as with a slightly activated gland. But hibernation will
stop if the thyroid is very active — for instance, when the BMR
is at high spring-summer level.

Pancreas

It is not known if insulin has any appreciable role in the tem-
perature regulation of non-hibernators. However, when injected
in massive doses, insulin induces a marked lowering of the level
of blood sugar and of the body temperature. The drop in body

1960 MAMMALIAN HIBERNATION 113

temperature, at least in man, is a consequence of increased heat
loss while heat production remains the same (Lundback and
Magnussen, 1941).

Heat production in alloxan diabetic rats was found to be nor-
mal (Sahovie et al., 1952). Injected insulin increases the amount
of glycogen in intrascapular fat tissue of hypophyseectomized
rats (Engel and Scott, 1950) and it could be that it has a similar
effect in winter hibernators, with an involuted anterior hypo-
physis.

In artificially cooled rats, non-hibernators, the level of blood
sugar was very low when lethargic hypothermia was sustained
for several hours (Popovic, 1955b). However, the hypothermic
rat cannot be revived by rewarming. In artificially cooled
ground squirrels with stabilized body temperatures at 10°C
the level of the blood sugar tended to decrease all the time. After
100 hours, blood sugar values were 40 mg %, similar to that in
hibernation where hypoglycemia is usually observed (Bierry and
Kolmann, 1928; Ferdmann and Feinsehmidt, 19382; Agid and
Popovic, 1957), but in hibernators as well as non-hibernators
death ensued when the animals were rewarmed.

Because insulin injected in large doses decreases the body tem-
perature of a homeotherm, non-hibernator or hibernator, investi-
gators used this hormone in trying to produce artificial hiberna-
tion. Dworkin and Finney (1927) reported that insulin and
moderate cold induce artificial hibernation in woodechueks. Suo-
malainen (1939b) found the same to be true in hedgehogs using
Mg and insulin, and Suomalainen and Petri (1952) produced
the same results in hamsters using insulin only. A similar tech-
nique was used to induce deep hypothermia in non-hibernators
(Giaja et al., 1955; Popovie, 1960b) and in hibernators (Kayser,
1955b).

As for most endocrines, cyclic changes have been observed in
the pancreas of hibernators (Skowron and Zajaezek, 1947). It
was also shown that the relationship between B and A cells
differs from season to season (Skowron and Zajaezek, 1947:
Aron and Kayser, 1956).

We conclude that there is not enough evidence to assume that
the pancreas has a role in hibernation. What was called artificial
hibernation in hibernators is, in all likelihood, only induced
hypothermia. Since hibernators withstand induced hypothermia
longer than non-hibernators, this state was misinterpreted as
hibernation.

3ULLETIN : MUSEUM OF CON > ZOOLOG Tol. 12
114 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Gonads

Some investigators (Hemingway, 1945) consider the gonads as
one of the endocrine glands which controls body temperature.
But even if gonads have a part in the temperature regulation of
homeotherms, it is probably small. Thus, for instance, the body
temperature of homeotherms is not changed after extirpation of
the gonads.

Involution of the gonads is very pronounced before and during
hibernation (Kayser and Aron, 1950; Kayser, 1955a; Lyman and
Chatfield, 1956). Foster et al. (19389) were convineed that,
among other endocrine glands which have a definite role in
hibernation, gonads play a part too. These investigators found
an inverse correlation between hibernation and activity of gon-
ads, as well as a ‘‘resistant state’’ to hibernation when gonads
are active. In the second part of the winter, castrated animals
will continue to hibernate more often than controls. At that
time of the year a small activation of the gonads of hibernators
may be seen. However, hibernation is observed after administra-
tion of anterior hypophysis hormones and a consequent activa-
tion of the gonads. Similarly, Lyman and Dempsey (1951)
showed that after testosterone was injected golden hamsters
could continue to hibernate.

Some hibernators, for instance ground squirrels, breed only
once a year. The breeding period is usually immediately after
arousal from hibernation. Bats are in a state of prolonged
oestrus during the winter (Guthrie and Jeffers, 1988). Ovula-
tion of follicle and implantation of the egg occur in April (Wim-
satt, 1944). The response of reproductive organs of winter bats
to injected gonadotrophie hormones is similar to reactions found
in immature or hypophyseectomized non-hibernators (Sluiter
et al., 1952).

In conclusion, the gonads of hibernators are sites of profound
seasonal changes. However, hibernation occurs not only in au-
tumn and the first part of the winter when the gonads are in-
voluted but also in the last part of the winter when the gonads
show some activity. Also, activation of the gonads after admin-
istration of anterior hypophysis hormones or injection of testo-
sterone is compatible with hibernation. Therefore, it seems that
the gonads do not exert any major effect on the process of hiber-
nation.

1960 MAMMALIAN HIBERNATION 115

Adrenal Medulla

The cold resistance of homeotherms is greatly diminished
after extirpation of the adrenals (Schaeffer and Thibault,
1945b). After demedullation, the cold resistance is diminished
in white rats (Ring, 1942) but not in dogs (Morin, 1942).
Greatly decreased cold thermogenesis after adrenalectomy in
white rats is chiefly restored after administration of desoxycor-
ticosterone, DOC (Sahovie et al., 1951b). Cold thermogenesis is
not appreciably changed after adrenalectomy if the rats are car-
riers of cortical grafts (Sahovie et al., 1951a).

A considerable number of investigators failed to find a calori-
genic effect of adrenaline (see Griffith’s review, 1951), but the
recent data on high calorigenic action of adrenaline are becom-
ing more and more convineing (Griffith et al., 1940; Morin,
1943). Adrenaline and noradrenaline are hormones which cause
an immediate metabolic reaction. The calorigenic effect of
adrenaline is higher in rats with an active thyroid, after lone
cold exposure (Ring, 1942; Hsieh and Carlson, 1957). Calori-
genic action of adrenaline is small in animals which have a low
BMR as a consequence of adaptation to the temperature of
thermal neutrahty (Schaeffer and Thibault, 1945a). After thy-
roidectomy the adrenaline effect is small; after thvroxin treat-
ments it is high (Issekutz and Harangos6-Groszy, 1942). The
calorigenic effect of noradrenaline is also increased in cold-
adapted animals but not in warm-adapted ones, either anesthe-
tized (Hsieh and Carlson, 1957) or unanesthetized (Depoeas,
1960).

In rats (Fisher et al., 1955) and dogs (Malmejae et al., 1956),
when cooled below 20°C body temperature, the adrenal medulla
is no longer activated by cold.

During hibernation the adrenal glands of some hibernators
seem to be involuted. The involution is very pronounced, up to
50 per cent of the volume of the glands (Valentin, 1857), but
in the hibernating hamster the weight of the adrenals is not
changed (Holmes, 1955). Some investigators found that the
adrenal medulla is involuted (Suomalainen, 1938b, 1940), while
others did not find medullar involution (Kayser and Aron, 1950).

The adrenaline content of the adrenals is increased during
hibernation, while noradrenaline content is decreased (Suomal-
ainen and Uuspai, 1958). It was suggested (Kayser, 1940) that
the highly variable content of adrenaline in the adrenal medulla
of winter hibernators could be associated with the frequeney of
arousals from hibernation.

116 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The medullar hormones probably play an important role in
arousal from hibernation. Chatfield and Lyman (1950) found
an increased effectiveness of sympathetico-adrenal activity during
that process. Even if eviscerated the hibernating animal shows
some ability to rewarm.

Adrenalectomized ground squirrels with cortical grafts are
able to hibernate and to wake up as do control animals (Popovie,
1952b). This suggests that if calorigenic hormones, adrenaline
and noradrenaline, have a role in hibernation, especially during
arousal, then the medulla is not their only source.

In conclusion, it seems that the adrenal medulla in hibernators
shows certain biological rhythms. It seems also that without this
gland, hibernators may enter hibernation and wake up normally.

Adrenal Cortex

Like some other endocrines the adrenal cortex has a role in
temperature regulation. Cold resistance is highly reduced in
cortico-deficient rats. Treatment with ecortin or with DOC par-
tially restores cold resistance (Tyslowitz and Astwood, 1942;
Sahovie ef al., 1951b). The cold resistance of a normal man is
increased after DOC treatment (Zarrow, 1942).

After exposure to a cold environment the cortical funetion
seemed to be increased only during the first days of exposure
(Levin, 1945). Adrenal cortical functions return to the pre-
vious level of activity after a few days as judged by ineorpora-
tion of P®? (Nicholls and Rossiter, 1955, 1956). Similarly, it
was found that adaptation to cold does not inerease the need
for cortical hormones (Sellers ef al., 1951; Heroux and Hart,
1954). The homeotherms are able to adapt to cold environment
without the adrenals (Sellers e¢ al., 1951; Heroux, 1955) as well
as without thyroid (Popovie and Popovic, 1951). However, in
hibernators, some investigators could not find the manifestation
of adaptation to cold even after partial extirpation of these
vlands (Kayser, 1939).

In induced hypothermia of non-hibernators the output of keto-
steroids from the adrenals is decreased (Ganong et al., 1955).
After rewarming from hypothermia, dogs showed stress mani-
festations (Sarajas ef al., 1958).

In hibernators, the cortex shows similar biological cycles to
the thyroid, gonads or the anterior hypophysis. The adrenal
cortex is involuted prior to hibernation (Kayser and Aron,
1950). Similar involution of the adrenal cortex has been ob-

1960 MAMMALIAN HIBERNATION 117

served in non-hibernators after hypophyseetomy. Suomalainen
(1938b, 1940), too, saw pre-hibernal involution of the cortex in
hibernators. Later, however, Suomalainen (1954) eoneluded on
the basis of his new observations that the cortex of hedgehogs is
hyperactive during cold seasons. The same author was able to
confirm these findines in hamsters (Suomalainen and Granstrom,
1955). He coneluded that the ‘‘alarm reaction’’ of Selye is
present in hibernators during cold months. Zalesky and Wells
(1940) also observed a well developed cortex in ground squir-
rels exposed to the low environmental temperature.

ADRENALECTOMIZED
GROUND SQUIRRELS

SURVIVAL TIME IN DAYS

OCT. NOV. DEC. JAN. FEB. MAR.

Fig. 2. Survival time of adrenalectomized ground squirrels during autumn-
spring period. From Popovie and Vidovie (1951).

Survival of summer-active hibernators after adrenalectomy
is Similar to that of non-hibernators. But during the winter,
survival time of adrenalectomized marmots (Britton, 1931; Brit-
ton and Silvette, 1937) and of adrenalectomized ground squir-
rels (Popovie and Vidovie, 1951; Vidovie and Popovic, 1954)
was much longer. This contrasts with the non-hibernators in
which cold decreases the survival of adrenalectomized aninals.
Figure 2 shows the survival of adrenalectomized ground squir-
rels during the cold months. This is similar to Kayser’s graph
(1952) showing the time spent in hibernation during the same

118 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

period of time. The adrenalectomized ground squirrels live
longer during the cold seasons but none of them enter hiberna-
tion (Popovie and Vidovie, 1951; Vidovie and Popovie, 1954).
However, the adrenalectomized ground squirrels hibernate
either when a small cortical fragment of one of the adrenals is
left in situ (Vidovie and Popovie, 1954) or with a cortical graft
in the anterior chamber of the eve (Popovic, 1952b). Treatment
with cortical hormones was also able to restore the ability to
hibernate in adrenalectomized ground squirrels, sometimes after
2-3 days of DOC or cortisone injection (Popovie et al., 1957).
Some of these results were confirmed on another hibernator, the
hamster. Adrenaleetomized hamsters hibernate ten times less
than the controls (Kayser, 1957b). In a repetition of the same
experiments on the same species, Kayser found that none of the
adrenalectomized hamsters would hibernate (Kayser and Petro-
vie, 1958) unless treated with cortisone or, even more readily,
with DOC,

Hibernating Gland

Up to now the hibernating gland did not justify the name
viven it by Vienes in 1913. At present it is not known if the
hibernating gland has any role in hibernation. It is not known
if it is an endocrine gland, and all research has failed, up to now,
to extract from that tissue any active principle. However, it is
known that hibernating gland tissue is also found in non-hiber-
nators (Wertheimer and Shapiro, 1948).

The hibernating gland is a fatty, reserve tissue, brown in
color and situated between the scapulae. The respiration of
this tissue is much higher than that of other adipose tissues, and
the decrease of respiration is smaller during cooling (Hook and
Barron, 1941). Some investigators found that injected extracts
of this gland into active hibernators decrease the metabolie rate
and the body temperature (Wendt, 1943) ; but the same effect is
found for the extracts of other tissues even in non-hibernators.
Moreover, Klar (1941) was not able to confirm the findings of
Wendt.

The hibernating gland shows a rhythm similar to certain endo-
erine glands. During the winter it is involuted (Valentin,
1857), but Suomalainen and Herlevi (1951) coneluded on the
basis of their observations that the hibernating gland has a role
in the *‘alarm reaction.’” Immediately after arousal: the hiber-
nator is in a state of stress.

1960 MAMMALIAN HIBERNATION 119

In conclusion, the hibernating gland is a reserve tissue of
high activity showing strong seasonal changes. For the moment
we do not have sufficient information to understand its role in
hibernation.

Summary

After half a century, the endocrine theory of hibernation
offers little more than it did when first postulated. The data are
often contradictory and at times obtained by usine teehniques
not particularly appropriate. In spite of this, there are positive
indications that some of the endoerines play a role in hiberna-
tion.

The data presented here suggest that the endocrine glands
have the same function in hibernators and non-hibernators as
far as temperature regulation is concerned. But there are ex-
ceptions. For example, in active winter hibernators exposed to
cold there is no increased activity of the thyroid and other
endocrines. Adrenalectomized ground squirrels and marmots
survive winter temperatures longer than summer temperatures.

Most of the endocrines involute prior to hibernation and _ re-
sume nominal functioning in active winter hibernators even
before winter sleep finally ends. Hibernators in which the ac-
tivity of the endocrines, especially the thyroid, has been artifi-
cially increased either do not hibernate at all or hibernate less
than the controls. However, reports in recent years indicate that
involution and hypofunetion of some endocrines, particularly
the adrenals, do not occur before or during the hibernation
period.

Adler’s experiments on injections of endocrine extracts and
hormones are, for the most part, unacceptable today because of
inadequate experimental techniques. A new technique is sug-
vested here.

Using the extirpation and hormone replacement method it was
shown that without the adrenal cortex or its hormones, ground
squirrels and hamsters do not hibernate. Both desoxycorticoster-
one and cortisone restore the ability to hibernate in adrenoin-
sufficient animals, but to different degrees. Adrenalectomized
ground squirrels with cortical grafts in the eve hibernate nor-
mally.

120 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

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1960 MAMMALIAN HIBERNATION 127

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1960 MAMMALIAN HIBERNATION 129

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DISCUSSION FOLLOWING POPOVIC’S PAPER

ZIMNY opened the discussion by pointing out three endo-
crinological observations she has made on the tissue of hibernat-
ing animals :

(a) Using the Sudan-Black B reaction for lipid and the Schultz
reaction for cholesterol, she found that these materials increased
in the adrenal cortex during hibernation. In this connection she
helieves the material is stored there to be mobilized for use in
arousal, and not for use as a slowly releasing material during
hibernation.

(b) Using the chromaffin technique she found an increase in
adrenalin in the adrenal gland during hibernation.

(ec) Upon staming the pancreas, she found an increase in eranu-
lation of the beta cells during hibernation.

SUOMALAINEN indicated that he, too, had observed an
increase of adrenalin in the adrenals and observed that beta cells
of the pancreas were proportionately more numerous in hiber-
nating animals than in non-hibernating animals.

POPOVIC asked SUOMALAINEN if injection of insulin
could produce hibernation in the summer animal. SUOMAL-
AINEN rephed that injection of insulin without magnesium
salts puts the animal into an ‘‘artificial hypothermic state.”’

130 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

ZIMNY said she had heard of an interesting case of a diabetic
man in the Aretie who found he did not require insulin while
doing field work. She speculated as to whether, therefore, cold
alone might not produce an increased beta cell population.

WIMSATT asked what results were obtained using radioac-
tive analysis of thyroid function. POPOVIC said his tests by
this means show some thyroid activity. KAYSER said that his
experiments with hamsters using [!%! showed him that neither
the uptake nor the release of [1°! measured by counts with the
CGeiger-Muller apparatus could provide a correct evaluation of
thyroid activity. WIMSATT remarked that he also had tried
such a study with bats, and could get no sense out of the results.
Ile (WIMSATT) concluded that conditions were not standard
enough, and that the bat is more easily prodded to arousal than
other hibernating animals, hence acquisition of data on hiberna-
tion in the bat is far more difficult than in other species. JO-
ILANSSON stated that the protein-bound iodine in the hedgehog
is decreased during hibernation as compared with the situation
during the summer. JOHANSSON asked if infection occurred
in the preparation in which a cannula was permanently im-
bedded in a vessel. POPOVIC rephed that they had both arte-
rial and venous cannulae in vessels of ground squirrels longer
than 40 days without infection. LYMAN volunteered the infor-
mation that he had used arterial cannulae, and animals had
carried these devices as long as four months without infection.

Vil

HISTOLOGICAL CHANGES DURING THE
HIBERNATING CYCLE IN THE
ARCTIC GROUND SQUIRREL”’

By WiuuiamM V. Mayer
Department of Biology
Wayne State University

Detroit 2, Michigan

On the Arctic slope of Alaska, where the field work was done
for this particular research problem, the Aretic ground squirrel
is active from May until October and hibernates during the
rest of the year. The animal completes its life cycle during these
brief months. It awakens from hibernation early in May and
mates almost as soon as it emerges from its burrow. The young
are born in mid-June after a 25-day gestation period and are self-
sufficient by mid-July. They attain approximately adult weight
and are ready to enter hibernation by the latter part of Septem-
ber or the early part of October.

On the flat, wet, treeless Arctic slope, one would hardly expect
to find a burrowing animal. However, on the few high spots
of ground which stand above the normally wet tundra, the
permafrost table is low enough to allow these animals to burrow
beneath the surface and be protected from the full fury of winter
Plate 1, fie; 1).

Ilibernation is not a continuous process involving entering
the torpid state in September followed by a single emergence
only in May. Periodically, during the winter, the animal awakens
at intervals varying from two to three weeks. The typical hiber-
nation pattern consists of a relatively slow drop of temperature
as heat radiates from the body, a rather consistently low body
temperature during hibernation, and an almost explosive awak-
ening which the animal undergoes during a relatively short
period of time. The temperature at which hibernation takes place
is correlated with that of the environment, and it is possible
to have animals hibernating at body temperatures varying from
OF tomo:

1'This research was supported, in part, by the United States Air Force under
Contraet Number AF 18(600)-848, monitored by the Arctic Aeromedical Labora-
tory, Alaskan Air Command, Ladd Air Foree Base, Alaska.

2 Contribution Number 39 from the Biology Department of Wayne State Uni-
versity, Detroit 2, Michigan.

132 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Due to the profound etfect of temperature on metabolic proe-
esses it is essential to know the body temperature at which an
animal hibernates. After taking rectal temperatures on 55 dif-
ferent individuals over a period of a year, the 425 records thus
obtained on animals considered to be warm and active showed
the average body temperature to be 36.4°C. The environmental
temperature for this period averaged about 19°C. These captive
animals in the laboratory were used to being handled and, while
never becoming completely resigned to the taking of rectal
temperatures, did not struggle or fight as much as animals caught
in the field. Seventy-five rectal temperatures taken on squirrels
captured in the field during July and August showed an average
temperature of 59.1°C, the heightened temperature in all proba-
bility due to the fear which the newly caught animals evidenced
on being trapped and handled.

In addition to animals kept at an average environmental tem-
perature of 19°C, six animals were kept at a more or less constant
temperature of 11.4°C. Thirty-nine rectal temperatures taken
throughout the year on these six warm and active squirrels
averaged 34.8°C or only 1.6°C less than the body temperatures
of those kept at 19°C. In five warm and active animals kept at an
average temperature of 1.6°C, a total of 48 rectal temperatures
taken throughout the vear averaged 36.1°C, or only three-tenths
of a degree centigrade less than that of the animals kept at
19°C. Thus, the average body temperature of the warm and
awake squirrel varies shehtly with environmental temperature,
and also between captive and wild animals, although the latter
difference is probably due to the difference in reaction to bemg
handled. The minor rise in body temperature as the environ-
mental temperature approaches 0°C is indicative of an increase
in metabolism necessary to remain warm and _ active.

Amone animals in hibernation, a similar series of measure-
ments was made. Thirty rectal temperatures on 25 different
animals, hibernating in an environmental temperature of 19.7°C,
averaged 18.5°C. In an environmental temperature of 18.4°C,
28 rectal temperatures on 20 different animals averaged 16.6°C.
Thirty-three rectal temperatures on six different animals, hiber-
nating in an environmental temperature of 11.4°C. averaged
13.7°C. Twenty-five rectal temperatures of five different ani-
mals, hibernating in an environmental temperature of 1.6°C,
averaged 4.2°C. The body temperatures during hibernation
helow those of the environment ocevr only at relatively hieh
environmental temperatures and probably are due to heat loss

1960 MAMMALIAN HIBERNATION 133

through respiration and evaporative cooling from the surface
of the squirrel.

The typical picture of an animal in hibernation at tempera-
tures above 0°C was determined by the use of a rectal thermis-
tor inserted to a depth of 12 em. The thermistor was connected
to a continuous recording milliammeter. The pattern thus ob-
tained, as typical, was of an initial rise at the insertion of the
thermistor and then a slow drop of the body temperature (Fig.
1). As the decrease begins, it is more rapid than after the body

TYPICAL HIBERNATION PATTERN - SPERMOPHILUS UNDULATUS

OLGREES CENTISRADE

2400 2400 2400 2400

TIME - MIDNIGHT TO MIDNIGHT (2400)

Fig. 1. Aretie ground squirrel hibernation pattern. Slow loss of heat
from body, relatively consistent low temperature during hibernation, and
explosive awakening from hibernation are typical.

temperature has been lowered to about 11°C. In the typical
picture, the time taken for the temperature to drop from 22°
to 11°C was 10 hours. The time necessary for the temperature
to be lowered from 11° to 6°C was 24 hours. The animal then
remained at about this temperature for a period of up to three
weeks before awakening. The temperature pattern at the awaken-
ing is explosive.

Upon awakening from hibernation, the temperature first rises
from 4° to 17.5°C in one and one-half hours, and then takes the

MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

154 BULLETIN :

next one and one-half hours to rise from 17.5° to 32°C (Fig. 2).

Aecompanying this rapid rise in body temperature, there is also
At the hibernating

a typical awakening pattern of behavior.
body temperature, there are about three irregular respirations
per minute. The heartbeat is not noticeable at 5°C even with a
diaphragm stethoscope. An investigation of the heartbeat pat-
tern reveals that the animal in hibernation has so slow a heart
action that the heart operates by slowly wringing; too slow to
produce the characteristic sounds heard through the chest wall

of the normally awake and active animal.

AWAKENING FROM HIBERNATION SPERMOPHILUS UNDULATUS

'
'
'
'
'
Uy
U
'
'
'
'
i
Uy
U
'
i
!
'
'
'

DEGREES CENTIGRADE

SQUIRREL

ENVIRONMENT

1400

'200 1300

TIME IN HOURS

Fig. 2. Temperatures during awakening from hibernation. The steep rise
between 12°C and 17.5°C is occasioned by the shivering of the animal. The

second steep rise, at 24°C, marks the time the animal opens its eyes and

sits up.

1960 MAMMALIAN HIBERNATION 135

The behavior pattern is also characteristic, and certain actions
can be correlated with temperature. By the time the tempera-
ture has reached 11°C, the increasing respiratory rate as indi
cated by chest movements is obscured by other movements of
the body. At 12°C, the first shivering reaction sets in, with a
corresponding rise in body temperature. About this time, the
hind limb motions begin. When a temperature of 16°C has been
reached, the animal tries to right itself. At a temperature of
17.5°C, the shivering stops and the animal begins to move its
tail. At 24°C, the squirrel opens its eyes and suddenly sits up.
At this time, a second, more rapid temperature rise is noted.
By the time the body temperature has reached 25.4°C, the animal
is sitting up, flicking its tail and is considered essentially warm
and active.

The problem of the temperature patterns of animals exposed
to temperatures of less than 0°C and whose body temperatures
drop below 0°C was explored. Twelve animals awakened when
the body temperature reached 0°C. Seven animals, exposed to
—15°C, either awakened or froze. One survived in this tempera-
ture for 50 hours without the protective insulation of a nest.
Wiule several animals recorded body temperatures in hiberna-
tion below O°C, only two dropped below —3°C. These two
animals had been repeatedly stressed by prolonged exposure to
cold and by-at least three awakenings from hibernation prior
to the below zero body temperature experiments. They attained
body temperatures of —5.5°C and —4.6°C, respectively, before
the formation of ice in the body was indicated by a rapid rise
of the rectal temperature to O0°C (Fig. 3). This work confirms
the experiments of Kalabukhoy (1985) who supercooled a mouse
to —2.2°C and a bat to —7.5°C before ice formation occurred.

In a natural environment, the animal protects itself by the
manufacture of a large nest of dried leaves, grass, lichens, moss,
and hair in a burrow system constructed in such a fashion as
to face away from prevailing winds. The snow cover in winter
varies from negligible to a depth of about 92 em. In order to
determine the temperatures to which the animal would be ex-
posed durine hibernation, thermistors were introduced into
burrows on 25 foot cables fastened to the ground squirrels by
a harness made of cloth bandage. The squirrels were chased into
the burrows and the cable payed out behind them. The cable
would be taken into burrows for a distance of from 10 to 22 feet.
Any cable taken in less than 10 feet would be withdrawn and
the experiment repeated. The ends of the cable protruding from

136 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

the burrow were attached, under inverted cans, to poles mounted
in the ground by the burrow entrance. They were tagged to
identify them and were read on the average of every two days
during the winter so that the weekly averages are those of
three temperatures in most cases. This experiment was conducted
from September 12, 1954 to May 7, 1955, the period approximat-
ine the time from entrance into hibernation in the fall to the
time of emergence in the spring.

3 RECTAL TEMPERATURE - SPERMOPHILUS URXDULATUS

CEGREES CENTIGRADE
o

love 2400 2400 2400

TIME IN HOURS

Fig. 3. Temperature of an animal supercooled to —5.5°C, The rapid rise
in temperature is occasioned by ice formation within the tissues,

The thermistors were recovered by digging out the burrows
the following spring as soon as the ground had thawed enough
for this purpose. The deepest penetration beneath the surface
was found to be 3.8 feet; the shallowest, 1 foot. For all ther-
mistors, the depth averaged 1.72 feet. During the winter, the
variable snow cover averaged 59 em.

During this same period, the maximum and minimum environ-
mental temperatures were recorded. There was a 100° Fahren-
heit range in the temperature during this period from a high of
o8°F to a low of —42°F, the average environmental temperature

1960 MAMMALIAN HIBERNATION 137

being 22.8°F. The burrow temperatures varied during this
period from 36.5°F to 16°F with a range of 20.5 IF degrees to
average 25.2°F for the season, which is 2.4° higher than the
environmental average. The average temperature for the warm-
est burrow was 26.1°F and that for the coldest 23.7°F (ig. 4).

53 SPERMOPHILUS BURROW TEMPERATURES PAXSON LAKE 1954-1955

DEGREES FAHRENHEIT

MAX. EMVIRON, TaMP
-22 Wim. EHVIRON. TaRP.
- AVE BURROW TEMP

TCMP. OF WARMEST BUD

-27: TEMP. OF COLDEST BURROW

10 17241 TIME IN WEEKS
APR MAY

26 3 1017 2437 424 285 219262 916 23906 20276
SEPT OCT NOV DEC JAN FEB MAR

Vig. 4. Comparison of burrow temperatures with those of the environ
ment. Paxson Lake, Alaska.

In correlating these temperatures with snow cover, it was
found that the burrow covered with the greatest depth of snow
had a temperature range of from 32 to 21°F or 11 F degrees and
an average for the winter of 25.8°F. The burrow with least
show covering, in which the bare ground could be noted during
the winter, was occupied all winter. The thermistor was 50 em
beneath the surface of the ground, and the temperature here
ranged from a high of 31.7°F to a low of 16.5°F or through

138 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124
15.2 I degrees, for a season average of 23.7°F. In contrast, in
another burrow in which the thermistor was likewise 30 em
beneath the surface of the ground the temperature ranged
through 18 I degrees from a high of 34° to a low of 16° for an
average of 23.7°1l*, or the same as for the above, even though
this burrow was covered by S87 em of snow. It is significant
that the temperature on any one day in all the burrows never
varied one from another by more than 5 °F. The seasonal
average varied only 2.31°F from the highest to the lowest burrow
temperatures despite the difference in depth of the thermistors
beneath the surface and the difference in snow cover over the
burrows. It is thus concluded that the depth of snow cover and
the distance of the burrow beneath the surface, in excess of 30
em, does not give appreciably greater protection to the hiber-
nating squirrel than a shallow, less well covered burrow.

Wherever the environment makes it possible, the burrows tend
to be oriented with their openings away from the prevailing wind
and in areas of favorable drainage. Burrow temperatures during
the winter are well above the environmental minima and show
relatively little fluctuation. Im the wild, burrow temperatures
do not drop below a minimum of 16°F which is considerably
ligher than the temperatures at which the animals have been
kept in the laboratory for a long period of time. From this, it is
apparent that a simple hole in the ground would give a great
deal of protection from the cold. When this is coupled with the
large and well insulated nest, the combination makes it possible
for the Aretic ground squirrel to maintain itself through the
severe Aretie winter.

Ilistological Changes

The etfect of hibernation upon the various tissues of the body
is a subject of considerable interest. In general, it might be
said that all of the tissues of the body apparently involute dur-
ing the period of hibernation. If we begin with the digestive
tract, there have been references in the literature to the fact
that the digestive tract and the exocrine pancreas are involuted
during hibernation. Valentin (1857-58) and, recently, Lyman
and Ledue (1953), and Mayer and Bernick (1957) indicated the
changes that take place in the digestive tract of various animals
during hibernation. The lining of the stomach of the warm and
active Arctic ground squirrel, stained by the periodic acid
Schiff (PAS) method, shows mucoid material limited to the

1960 MAMMALIAN HIBERNATION 139

surface epithelial cells and a few of the superficial mucous
neck cells. However, after the squirrels are in hibernation for a
period of three weeks, the stomach lining shows an increase in
the amount of mucus stored in the superficial cells. It has been
shown by others that the golden hamster, still containing food
in the esophageal portion of its tract, likewise will show the
presence of neutral or shgehtly acid mucus. Concurrently, with
this increase in mucus in the superficial cells, the chief cells also
contain mucoid material. After three months of hibernation,
there is an even more marked increase in mucus with the super-
ficial cells, the mucous neck cells, and the chief cells filled with
PAS-positive material. However, within twenty-four hours after
awakening from hibernation, the stomach lining displays a
marked loss of mucoid material and mucus is restricted to the
cells of the surface epithelium and the underlying mucous neck
cells.

The parietal and chief cells, which can be seen plainly in the
stomach wall of normally warm and active squirrels, become
less obvious after one week in hibernation; and, after three
weeks, there is an inactivation of both the parietal and the chief
cells and an absence of zymogen granules within the chief cells.
The large, rounded parietal cells do not give a typical response
when stained with hematoxylin and triosin as they appear vacuo-
lated and contain very fine cytoplasmic granules. The smaller
chief cells also have a vacuolated appearance. After three
months of hibernation, neither the parietal nor the chief cells
respond to staining with hematoxylin and triosin.

In this instance, however, twenty-four hours after awakening
the animal from hibernation, the parietal and chief cells are
again apparent with the reduction of the contained mucus noted
previously.

While these changes are taking place within the stomach, the
mitotic figures in the erypts of Lieberkthn, which are a common
feature of the sections of the jejunum of the warm and awake
squirrels, are reduced in number after as little as one week of
hibernation. By the end of three weeks a very few mitotic
figures are demonstrable and, after three months in hibernation,
no mitotie activity can be noted, and little further change occurs
in the erypts of Lieberkihn.

Within twenty-four hours after awakening the squirrel from
hibernation, mitoses are again taking place in the crypts of
Lieberkiihn. In warm and active squirrels, Paneth cells, which

140 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

are pyramidal in shape, contain fine purple granules when stained
with Gomori’s aldehyde fuchsin. They do not stain as darkly
in sections taken from animals in hibernation.

No major changes are noted in the large intestine during
hibernation except that the amount of mucus in the goblet cells
of the epithelium is reduced during the period of hibernation
from that normally noticed in warm and active animals. Twenty-
four hours after the squirrel’s awakening from hibernation,
however, the colon shows a normal amount of mucus in the goblet
cells.

We can see here that, in general, the changes in the digestive
tract during hibernation are those that one would normally
expect to occur in an animal which is not taking in food. The
empty stomach collapses until it is possible to note its opposite
walls in one single histological section. There is a progressive in-
crease in both the secretion and storage of mucus as long as the
animal remains in hibernation, and this covering of the epi-
thelium with mucus may have the effect of preventing adhesions
between the closely opposed walls of the stomach and may also
serve to prevent autodigestion of the mucous membrane by resid-
ual acids or enzymes. Immediately after death, autodigestion of
the mucous membrane begins and a similar pattern can be ex-
pected to develop during hibernation. Failure of such a process
to take place durine hibernation can most lkely be charged to
the mucus-containinge cells of the stomach. The presence of
mucus in the chief or zymogenic cells may serve to delay the
diffusion of pepsin precursors or to inhibit the action of pepsin
itself and thus prevent damage to the cells of the inactive
mucosa. The decrease in activity and, apparently, in numbers
of both parietal and chief cells would be expected with the
inaction of the digestive tract and the absence of the necessity
of producing the antecedents of pepsin and hydroehlorie acid.

The mitoses in the erypts of Liberkiihn serve to replace
cells subject to normal attrition in the digestive tract. With
the cessation of digestion during hibernation this is no longer
a necessary activity. The Paneth cells do not respond in hiber-
nation as in starvation, perhaps due to the lowered temperatures
accompanying the condition of hibernation.

The submaxillary gland of the Arctic ground squirrel is simi-
lar to that of the rat, with a separate lobe for its mucous and
serous portions. When sections of the submaxillary are treated
by the periodic acid Schiff method, a heavy eoncentration of

1960 MAMMALIAN HIBERNATION 14]

PAS-positive granules appears in the distal parts of the cells
adjacent to the lumen of the well defined duets (Pl. 1, fig. 2).
In sections stained with hematoxylin and eosin, the intralobar
ducts from a warm and active squirrel show columnar, striated
cells with centrally placed nuclei surrounding the definite lumina
of the duets.

The intralobar ducts of animals in hibernation demonstrate
inactivity. There is a progressive loss of PAS-positive granules
in the cytoplasm of the ductule cells in the process of hiberna-
tion. This loss is observable as early as one week after entering
hibernation and continuing diminution of the granules is demon-
strable after about three weeks of hibernation, when only
isolated granules are observed in the cells treated by the periodic
acid Schiff reagent (Pl. 1, fig. 3). After three months of hiber-
nation, there is no evidence of granules in these cells.

The general configuration of the cells likewise changes with
time during hibernation. A hematoxylin and eosin stained sec-
tion of the submaxillary from an animal in hibernation for three
months demonstrates a decrease in the size of the cells with an
apparent ‘‘piling up’’ of the nuclei. The lumina of the duets
appear narrowed with many being collapsed.

Within twenty-four hours after awakening from hibernation,
the PAS-positive granules can be noted. They are first seen at
the basal part of the cell and migrate toward the distal part.
In sections stained with hematoxylin and eosin, the cells again
appear columnar in shape and have centrally placed nuclei. The
lumina are also again well defined.

The acinar part of the submaxillary of the warm and active
squirrel contains pyramidal cells with basally located nuclei
When the serous part of the gland is stained with hematoxylin and
eosin. The cytoplasm shows a finely granular appearance (PI.
1, fig. 4). With the onset of hibernation, there is a gradual
collapsing of the acini, and, in a section stained with hema-
toxvlin and eosin and taken from a squirrel in hibernation for
three months, the acini appear smaller and give a disorganized
appearance. The nuclei are centrally placed with only a small
amount of cytoplasm surrounding them. The cytoplasm has lost
much of its granular appearance (PI. 1, fig. 5). However,
twenty-four hours after awakening from hibernation, there is
a reactivation of the acini which again appear pyramidal and
contain basally placed nuclei. The cytoplasm resumes its nor-
mally granular appearance.

142 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The atrophy of the exocrine pancreas during hibernation was
observed first by Carlier (1893) who noticed the characteristics
of the islands of Langerhans. Bierry and Kollmann (1928)
as Well as Vendrely and Kayser (1951) utilized Brachet’s tech-
nique which reveals the ribonucleic acid in these cells. In our
studies, the appearance of the exocrine pancreas of a warm and
awake squirrel, when stained with Gomori’s aldehyde fuchsin,
shows pyramidal cells resting on a delicate reticular membrane
and converging toward a central lumen. These cells possess
centrally located nuclei and a granular cytoplasm (PI. 1, fig. 6).
The exocrine pancreas from an animal in hibernation for three
months shows acini so densely packed with coarse positively-
staining granules as to mask the appearance of the nuclei (PI. 2,
fig. 1). Kayser (1957), in citing the work of Genest, points out
that, after twenty-four hours of hypothermia, the cells of the
exocrine pancreas of the white rat are filled with secretory
concretions and there is apparently some resemblance between
the appearance of the exocrine pancreas of the hypothermic
rat and that of the hibernating ground squirrel, although the
time clements involved are greatly different. As early as twenty-
four hours after awakening from hibernation the granular ae-
cumulation has disappeared from the acini.

This behavior is essentially what one would expect in the
elands of the digestive system during hibernation when no food
is either swallowed or digested. The rapid recovery of these
lands might be taken as an adaptation to the necessity of the
recently awakened animals’ taking food and being able to
swallow and digest it. It has been suggested that the collapse
of the glands during hibernation might be indicative of the fact
that protein material is being withdrawn from these inactive
cells for use in essential body metabolism.

The work on the effects of hibernation on teeth has been frag-
mentary indeed. Sarnat and Hook in 1942 utilized the thirteen-
lined ground squirrel and concluded that all stages of tooth
development were retarded by hibernation. A closer investiga-
tion of this phenomenon in the Arctie ground squirrel indicates
the following changes.

In the normal situation within the warm and active animal,
the ineisal dentin is a wide, homogeneously calcified, outer layer
which extends from the surface of the tooth to the unealeified
predentin (Pl. 2, fig. 2).

In contrast, the process of hibernation is accompanied by a
deterioration of bone and teeth. There is an indication of a

1960 MAMMALIAN HIBERNATION 143

disturbed calcification process in the region of the incisal dentin
after an animal has been in hibernation for a period of three
weeks. Here, contrasted to the more recently formed, disturbed
dentin, there is still present a thin outer zone of homogeneously
calcified dentin (PI. 2, fig. 3).

In addition to the effects on dentinogenesis, the alveolar bone
also undergoes similar degenerative changes during hibernation.
The interradicular and interseptal bone of the molariform
teeth show a high degree of calcification in the warm and active
animals (PI. 2, fig. 4). This can be contrasted to the condition
in an aninal in hibernation for three months for, in the hiber-
nating animal, the progressive loss of both imterradicular and
interseptal bone is obvious (PI. 2, fig. 5).

Iuxamination of skulls of squirrels kept in captivity for ex-
tended periods of time shows a marked development of caries
rather consistently in the molar and premolar teeth. Field-caught
animals do not show such a predilection for caries, and it is
believed that caries are not related to hibernation but more
likely to conditions of captivity, including: diet.

Investigation of the pattern of caries shows all stages of
involvement from early lesions to the fracturing of the coronal
part of the tooth. More advanced carious lesions are actually
characterized by the invasion of the dentinal tubules.

Pulp involvement is not an uncommon occurrence. Invasion
of pulpal areas to produce degenerative changes often oceurs
and the disintegration of the pulp may extend into the root to
produce an apical abscess. The pulpal inflammation likewise
may result in a hollowing out of the tooth canal and, in addi-
tion, the epithelial attachment may proliferate deeply alone
the sides of the root.

In addition to the problem of caries and the effeets on bone
and dentinogenesis, animals in hibernation for from three weeks
to three months exhibit various degrees of periodontal involve-
ment. Osteoporosis of the alveolar bone has been demonstrated
in animals which have been in hibernation for three months.

In an animal in hibernation for three weeks, the interproximal
region of the two premolars shows a loss of trabeeulation, al-
though much less than that observed in some animals which
have been in hibernation for a longer period of time. However,
there may be pocket formation on both the mesial and distal
surfaces of two adjacent teeth. The breakdown of surface epi-
thelium with the presence of caleulus on the cementum and a
loss of interseptal bone has also been noted. The downgrowth

144 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

of the epithelial attachment may proliferate into the radicular
area and may be accompanied by a disorganization of the attach-
ment fibers and a lowering of the alveolar bone. A further
progression of this effect involves periodontal pockets in the
mesial and distal surfaces with such extensive bifureation in-
volvement that no bone remains in the interradicular region
(PI. 2, fig. 6). In addition, apical abscesses may be observed with
inflammatory invasion of the surrounding connective tissues.
It is obvious that hibernation produces changes in the bone,
periodontum, and the process of dentinogenesis, and that these
changes become more marked the longer the animal remains in
hibernation.

Other participants in this symposium are charged with dealing
with endocrines during hibernation and with the brown fat prob-
lem, neither of which I will attempt to mention here.

The relationship of the lipids and glycogen in selected ground
squirrel tissues has been the subject of much research. IKayser
(in press) has distinguished two nutritional behaviors during
hibernation as exemplified by (1) hamsters which often awaken
in the course of their hibernation and feed, but do not become
excessively fat prior to entering hibernation, and (2) those
hibernators, such as the ground squirrel, which store a great deal
of fat in the autumn and have less frequent periods of awakening.
While this is not a hard and fast rule because squirrels have
been noticed by myself, and also by Wade as early as 1930, to
take food during their periods of awakening, it is sufficient for
a simple division. In the hibernators of this second group, the
energy is provided apparently by lipids. As early as 1857,
Valentin showed that 99.3 per cent of the stored lipids were
consumed during a five month hibernation. Valentin divided
the organs into two types: those whose weight loss during hiber-
nation was proportionally greater, and those whose weight loss
was proportionally smaller than the weight loss of the whole
animal. Thus, he indicated that the main function of certain
organs was to constitute energetic storages whereas other organs
wore out to a certain extent during fasting.

Investigations of the liver, tongue muscle and heart of hiber-
nating and non-hibernating squirrels indicate a reduction of
liver and muscle glycogen in hibernators below that of the warm
and awake animal. The reverse situation, however, is true for
heart muscle. Lipids were noted to be in greater concentration
in the livers of hibernating squirrels than in the livers of non-
hibernators. The problem of glycogen synthesis during hiberna-

1960 MAMMALIAN HIBERNATION 145

tion has not yet been solved. Forssberg and Sarajas (1955),
in experiments with carbon-14 labeled glucose, could not resolve
this question which was raised as early as 1849 by Regnault and
Reiset.

Coupled with the unsolved problem of glycogen synthesis is
the fact that the cold environment itself may qualitatively alter
lipid metabolism in hibernators as Faweett and Lyman (1954)
proved. The iodine number of the stored fat rose in a hamster
kept at cold temperatures, but that of a rat left in a cold
environment did not particularly change.

In addition to glycogen synthesis, modern histochemical tech-
niques have made it possible to detect concentrations of lipids
and alpha amino acids in tissues as well. Utilizing the ninhydrin-
Schiff reaction as deseribed by Lillie in 1954, it was possible to
demonstrate the content of alpha amino acids in liver and tongue
muscle of hibernating Arctie ground squirrels. In hibernating
squirrels, there is a marked reduction in the amount of demon-
strable alpha amino acid. In the livers of warm and _ active
squirrels, however, there is, as one would expect, an abundance
of alpha amino acids from normal protein hydrolysis. A similar
picture is presented by tongue muscle of hibernators and warm
and active animals in which the protein degradation is quite
readily demonstrated during the active stages.

The absence of appreciable alpha amino acids in the livers of
hibernating squirrels indicates a low rate of protein metabolism
during hibernation. We know from previous work that digestion
has ceased and no alpha amino acids are coming from the digestive
tract. This, however, does not mean that there is no hydrolysis
of protein occurring. It is possible that protein degradation
is taking place at such a low rate as to have the resultant products
used almost as rapidly as they are produced and thus leaving
none to accumulate in the liver of the hibernating squirrel.
There is also the possibility that the reduetion and perhaps
cessation of protein metabolism may simply be due to the lessened
body temperature of the hibernator. At lowered body tempera-
tures it is possible to postulate that the reactions incident to
protein catabolism may not be able to proceed. Haurowitz (1950)
has mentioned a reduction in the rate of denaturation of protein
solutions by storage at low temperatures, perhaps indicative of
reduction of other types of protein metabolism as well at lower
temperatures.

146 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The muscle alpha amino acid content likewise follows the
pattern of that of the liver in showing neghgible protein degrada-
tion in the muscle cells of hibernators while the presence of the
alpha amino acids in the muscle cells of the warm and awake
squirrels is Indicative of a greater protein catabolism in those
animals.

Tissue lipids were investigated using frozen sectioned tissues
stained with Sudan black B (Lilhe, 1954). The lipid content of
the livers of those animals which were warm and awake was
negheible. The livers of the animals in hibernation, however,
showed definite fat droplets in the cytoplasm of the parenchymal
cells. One aninal, supercooled to —4.6°C in hibernation, showed
an even more dramatic concentration of fat in the liver with the
fatty degeneration of the organ quite pronounced. On staiming
with hematoxylin and triosin, large intra- and extracellular
fatlike vacuoles were observed. In the sections stained with
Sudan black B, large fat globules were present in and around
the imdividual liver cells.

While the liver is not normally mentioned as a fat depot im
warm and active animals, this condition apparently changes with
the onset of hibernation. The livers of the active animals showed
a negative sudanophilia, while the livers of squirrels in hiberna-
tion showed the presence of fat droplets. With the exception of
the supercooled animal mentioned previously, the liver lipids
and elycogen are present in inverse proportion one to another.
The lessened liver glycogen and the presence of pids strongly
suggest the metabolism of lipids to account for the energy of
awakening from hibernation.

It is apparent that the process of hibernation places great
demands upon the tissues of the body. All the tissue resources
are directed toward the problem of maintaining the animal's
metabolism at the minimal level necessary for life. This involves
disruption of glands and a cessation of all activity not mmedi-
ately germane to the process of living at the lowest possible
metabolic level. While such structures as the digestive tract
und its related glands appear not to suffer from this process
and recover relatively rapidly, the effeets on certain other struc-
tures such as teeth may be deleterious. The mobilization of ma-
terials from individual tissue cells to be utilized elsewhere wun-
doubtedly stresses the tissues concerned, as indicated by the eosi-
nopenia and leucopenia observed by authors investigating the
circulatory changes during hibernation (Lyman ef al., 1957;
Villalobos et al., 1958).

1960 MAMMALIAN HIBERNATION 147

The hibernator apparently balances on a very narrow line
between maintenance of life at such a level that recovery is
possible and maintenance at such a level as will eventually lead
to death. From the evidence of the tissues, the process of hiberna-
tion seems to be a precarious method of survival at best and one
from which many animals do not awaken.

REFERENCES

Bierry, H. and M. KOLLMANN
1928. Activité exocrine du pancréas et ilots de Langerhans. Cas de
Vhibernation. C. R. Soc. Biol., 99:456-459.

CARLIER, E. W.
1893. Contribution to the histology of the hedgehog. J. Anat. Physiol.,
27:85-111.

Fawcett, D. W. AnD C. P. LYMAN
1954. The effect of low environmental temperature on the composition
of depot fat in relation to hibernation. J. Physiol., 126:285-247.

Forsssere, A. AND H.S. 8S. SARAJAS
1955. Studies on the metabolism of !4C-labelled glucose in awake and
hibernating hedgehogs. Ann. Acad. Sci. Fenn., (A)IV (28) :1-8.

HavrowitTz, F.
1950. Chemistry and biology of proteins. New York, 374 pp.

KALABUKHOV, N. I.
1935. Anabiose bei Wirbeltieren und Insekten bei Temperaturen unter
0°. Zool. Jahrb., Abt. Allg. Zool. u. Physiol., 55:47-64.

KAYSER, C.
1957. Physiological aspects of hypothermia. Ann, Rey. Physiol., 19:
83-120.

KAYSER, C.
Hibernation. Jn: Physiological Mammalogy. New York (in

press ).

LILLIE, R. D.
1954. Histopathologic technic and practical histochemistry. Phila-
delphia, 501 pp.

Lyman, C. P. anp E. H. Lepuc
1953. Changes in blood sugar and tissue glycogen in the hamster during
arousal from hibernation. J. Cell. Comp. Physiol., 41:471-492.

Lym an, C. P., L. P. Weiss, R. C. O’BRIEN AND A. A. BARBEAU
1957. The effect of hibernation on the replacement of blood in the
golden hamster. J. Exp. Zool., 136:471-486.

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Mayer, W. V. AND S. BERNICK
1957. Comparative histochemistry of selected tissues from active and
hibernating Arctic ground squirrels, Spermophilus undulatus.
J. Cell. Comp. Physiol., 50:277-292.

Reagnauur, V. AND J. REISET
1849. Recherches chimiques sur la respiration des animaux des diverses

classes. Ann. Chim. Phys., (3) 26:299-519.

SaRNAt?T, B. G. AND W. E. Hook
1942. Effects of hibernation on tooth development. Anat. Rec., 83:
471-498.

VALENTIN, G.
IS57-58. Beitriige zur Kenntniss des Winterschlafes der Murmelthiere.
Moleschott’s Unters. Naturl., 1:206-258; 2:1-55; 22-246; 3:
195-299; 4:58-83.

‘

VENDRELY, C. AND C. IXAYSER

1951. Reeherches sur le fonctionnement du systeme nerveux des hiber-
nants. Differences entre le comportement du hamster ordinaire
(Cricetus frumentarius) et le spermophile (Citellus citellus). C.
RK. Soc. Biol., 145:1125-1126.

VILLALOBOS, T. J., HE. AbELSON, P. A. Ritey, Jr., AND W. H. Crossy
1958. A cause of the thrombocytopenia and leukopenia that occur in
dogs during deep hypothermia, J. Clin. Invest., 37:1-7.

Wapbk, O.
1930 ‘Phe behavior of certain spermophiles with special reference to
sestivation and hibernation. J. Mammiatl., 11:160-188.

DISCUSSION FOLLOWING MAYER’S PAPER

LYMAN asked if, in the case of an animal hibernating for
three weeks at a time, MAYER knew whether or not this was
continuous hibernation for three weeks. MAYER replied that it
was continuous hibernation, since animals were attached to a per-
manently recording device. LYMAN then addressed KAYSER
and asked if his record for continuous hibernation was 130 days,
as he (LYMAN) understocd. KAYSER replied, ‘‘ Lately I have
again become interested in the problem of the durations of the
phases of uninterrupted hibernation, particularly in contrasting
hibernation of mammals to hibernation of poikilotherms. — |
think — but I have no evidence — that hibernation of poikilo-
therms is characterized by the absence of any discontinuity. This
seems to be the general rule. In hibernating mammals, hiberna-
tion must be interrupted. Thus, they occupy an intermediate

1960 MAMMALIAN HIBERNATION 149

position between poikilotherms and artificially cooled homoio-
therms. In the latter the duration of hypothermia may not
exceed 24 hours; in poikilotherms it lasts for several months; in
hibernators there are interruptions — every fourth day, on the
average, in the European hamster (Criectus ericetus), a ‘“bad’’
hibernator, and about every 21st day in the European ground
squirrel (Citellus citellus), a ‘‘good’’ hibernator.

“With this thought in mind, | made precise measurements
during the winters of 1957-58 and 1958-59. The above data are
the results; they have not been published separately, but appear
in my manuseript on hibernation sent in February, 1959, to
Pergamon, Ine., publishers.

“Tn 1952 (Arch. Sei. Physiol., 6:193) I presented curves of
the whole hibernation of ground squirrels (Citellus citellus).
In these curves the longest uninterrupted sleeps lasted 18 and
21 days, respectively. My experiments of 1957-58 and 1958-59
showed phases of 21 days to be ‘‘normal’’ in ground squirrels
in. November-December.

“‘In 1940, I gave an example of periodic hibernation pro-
longed for nearly a year in the common dormouse (Glis glis).
I said, ‘We have kept a dormouse without food or drink, shel-
tered from noises and tactile stimulations, from Mareh till the
end of July. The animal slept for weeks and months, without
interruption .

“At that time, I did not want to determine the duration of
uninterrupted sleep, but to show that by keeping the external
conditions constant (temperature +5.0° £0.5°C), it is possible
to have a hibernator hibernating for nearly a year, and that the
endocrines of the hibernator remain in winter condition. I saw
no arousal from April Ist, 1988 (a day on which the thermo-
stat failed to work correctly) till the end. It is thus possible
that the animal did not awake for more than 90 days. I cannot
affirm it with certainty, having no longer any formal document
on this point. My attention was called to the facet that I had
made an ‘‘abnormal’’ observation by Lyman and Chatfield
(Physiol. Rev., 35 :403, 1955, p. 413). This is why I tried later
to determine the durations of uninterrupted hibernation. I saw
then that in refrigerators regulated at 5 = 2.0°C this duration
was 21 days for the ground squirrel. No dormice were available
(100-150 gm), so I could not repeat the same experiments in
this species. In the garden dormouse (Eliomys quercinus),
weighing 30-60 em, I found that the arousals may be incomplete :

150 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

the animal increases its temperature, but does not awake en-
tirely. The actograph (statograph with a tambour and a record-
ing tambour) showed no activity in spite of the increase of the
superficial body temperature. For this reason I think that eon-
tinuous recording of the microclimate by thermistors in contact
with the animal would be the best method of monitorine the
phases of uninterrupted hibernation. Actography, as I used it
with small-sized animals, may lead to inexact conclusions. The
longest continuous hibernation [ have seen in the hamster is
8-9 days.’’

FISHER remarked that it would be good to know not only
the extremes, but the average of such a function. LYMAN said
the average varied with the species. KAYSER said the average
for the hamster was about 4 days, for ground squirrels in No-
vember, 20 days, and in December, 25-27 days. FISHER indi-
eated that when such figures are given one must be careful to
specify the temperature of the environment.

LYMAN then asked MAYER what ambient temperature was
used in his animal experiments. MAYER replied that they had
animals hibernating at different temperatures, but their standard
for low temperature was 4.2°C.

FOLK then referred again to KAYSER’S remarks, indicating
that he was about to give figures for continuous hibernation
similar to KAYSER’S, with a maximum of 26 days in one
animal. STRUMWASSER asked FOLK for his criteria of con-
tinuous hibernation. FOLK replied that they had observed
animals three times a day (first winter) and twice a day (2nd
winter) in considerable detail, using a marking technique and
counting respirations, and that he was positive that animals had
not come out of hibernation. STRUMWASSER then remarked
that convictions, based on visual observation, that animals con-
tinuously hibernate for lone periods of time are quite subjective ;
actographie recording of the animal’s activities is at least objec-
tive, but still subject to a great deal of criticism when involved
in determining the length of maintained natural hypothermia.
FOLK said he was convinced actograph recordings were not
necessarily more sensitive than counting respirations.

SMITH asked MAYER if there had been any histological
studies made during natural arousal, and whether one would
expect partial changes to occur. MAYER said he would expect

1960 MAMMALIAN HIBERNATION 15]

changes to occur in proportion to the time involved, but no one
yet has sacrificed animals at short intervals during the process
of arousal from hibernation to show this in detail.

WIMSATT asked if the animals in MAYER’S experiments
were fed prior to sacrifice. MAYER replied that they were not
fed between time of arousal and sacrifice, although they were
allowed water.

DAWE asked if MAYER holds to a point of view that the
animal hibernates ‘‘all over,’’ that all tissues change. MAYER
replied that in his opinion every tissue participates in hiberna-
tion, that changes in hibernation are not simply changes in an
endocrine or two, but a change implicating all tissue. LYMAN
inquired specifically as to the changes in the heart during hiber-
nation. MAYER said that glycogen was the only thine he had
studied in the heart. He did notice that myocardial elycogen
increases during hibernation. LYMAN agreed that this is what
he had found also.

KAYSER asked if MAYER knew of any specific cause for
the deterioration of teeth in hibernation. MAYER said the sub-
maxillary gland is not operative, hence no protection is afforded
from that area. However, a majority of the caries seen seem not
to be directly attributable to hibernation. The development of
caries Is more a dietary problem. People in dental schools have
trouble getting animals to develop caries, but these animals do
it automatically,

MENAKER asked where the thermistor used in the experi-
ments was located. MAYER said it was not next to the animal,
but that most of the thermistors ended up lying on the floor of
the burrow. The temperature in the nest where the animal is
would be higher beeause of the insulating value of the nest.
He said that when he indicated that none of the burrow tempera-
tures went below 16°F, the temperature at the animal wasn’t
anywhere near this low.

SOUTH mentioned a recent paper on caries which showed
that carious conditions correlate directly with involution of
the thyroid (hypothyroidism) since such involution results in a
change in the buffering capacity of the salivary gland secretions.
In this ease, caries do not correlate with dietary factors directly,
but with thyroid activity which, in turn, might be correlated

152 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

with pre-hibernation involution of the gland. SOUTH also re-
marked that, with respect to MAYER’S figures indicating the
lowering of body temperature, the difference between that tem-
perature and the environmental temperature would require
(according to a rapid calculation he had just done) the evapora-
tion of too much total body water over a 24-hour period to make
it a probable explanation; in other words, such an explanation
would require a heat pump, which is highly unlikely. Granted
the accuracy of the recordings, such results may be caused by
the lag which may exist in the methods of measurement. MAYER
rephed that he did not postulate a heat pump but felt that the
evaporative losses at the higher environmental temperatures at
which the animal hibernated, together with the low relative
humidity, would give the effect described.

Plate ]

Fig. 1. Typical ground squirrel burrow site on Arctic slope of Alaska.
Near Meade River, Alaska, September, 1952. Fig. 2. Section of the intra-
lobar duet of the submayillary gland from a warm and active Arctic ground
squirrel. Heavy coneentrations of PAS-positive granules surround the
lumina. Fig. 3. The intralobar ducts of the submaxillary gland of an Arctic
ground squirrel in hibernation for three weeks. Only isolated PAS-positive
granules can be seen. Fig. 4. The acinar part of the submaxillary gland of the
Aretie ground squirrel stained with hematoxylin and eosin, Finely granular
cytoplasm, pyramidal cells and basally located nuclei are typical of the warm
and awake squirrel. Fig. 5. The acinar part of the submaxillary gland of
the Arctic ground squirrel stained with hematoxylin and eosin. The small,
disorganized acini with centrally placed nuclei and non-granular cytoplasm
are characteristic of the ground squirrel in hibernation for three months.
Fig. 6. Pancreatic acini of the Arctic ground squirrel stained with Gomori’s
aldehyde fuchsin. Finely granular eytoplasm is characteristic of the non-

hibernating squirrel.

Plate 2

Fig. 1. Panereatic acini of the Aretie ground squirrel stained with
Gomori’s aldehyde fuchsin. Coarse granules mask the cellular nuclei and
ave characteristic of the animal in hibernation, Fig. 2. Homogeneous
calcified dentin of the incisor of a warm and active Arctic ground squirrel.
Fig. 3. Incisal dentin from a ground squirrel in hibernation for three weeks.
Deficient dentin is characterized by an increased number of interglobular
spaces. Fig. 4. Upper molar from a warm and active Arctic ground squirrel.
The interseptal and interradicular bone is compact. Fig. 5. Upper molar
from a squirrel in hibernation for three months. Osteoporosis of the spongi-
osa of both interseptal and interradicular bone is obvious, Calculus is present
in the interproximal gingivae. Fig. 6. Extensive carious involvement of the
upper first molar of a squirrel in hibernation for three months. Obvious
bifurcation involvement with the loss of attachment fibers and bone,
Periapical abscess present.

VII

SEASONAL VARIATIONS IN PHYSIOLOGIC
FUNCTIONS OF ARCTIC
GROUND SQUIRRELS AND BLACK BEARS**

By Raymonp J. Hock
Arctic Aeromedical Laboratory
Ladd Air Force Base
Alaska

Hibernation is a evelic phenomenon, exhibited annually except
in the ease of the bats (EHisentraut, 1933; Hoek, 1951). It ap-
pears that, due to the shortened season into which hibernators
must compress their annual cyele of activity, nearly all facets
of their life history and consequently their physiologic functions
reflect the fact that they are constantly preparing for hiberna-
tion, hibernating, or reeoverine from that condition. Thus, the
whole vear is involved in the eyelie phenomenon that is hiberna-
tion.

Let me define hibernation as ‘‘a periodic phenomenon in
whieh body temperature falls to a low level, approximating am-
bient, and heart rate, metabolie rate, and other physiologic
funetions fall to correspondingly minimal levels.’’ The <Aretie
ground squirrel fits this definition as a hibernator well, and is
in fact as deep a hibernator as any of the more intensively
studied European or temperate American species.

On the other hand, the black bear is not a hibernator by this
definition, and elsewhere I have proposed the term carnivorean
lethargy to deseribe the condition found in bears and presumably
some other carnivores (Hoek, 1958).

Annual Cyeles

The Aretic ground squirrel, Citellus widulatus, is the hiber-
nator found farthest north in North Ameriea, as it extends nearly
to Point Barrow in Alaska. Due to the short season in which it
can remain active in Alaska, this species has greatly compressed
its annual sequence of activities. In my study area in the

1The contents of this manuscript refleet the personal views of the author
and are not to be construed as a statement of official Air Force policy.

2 The animal experimentation was conducted according to the “Rules Regarding
Animal Care” as established by the American Medical Association.

156 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Talkeetna Mountains near Anchorage, the male squirrels emerge
rather precisely beginning April 21, despite variation in the
seasons. Figure 1 shows a summary of five years of observation.
This figure also shows that the time of entrance into hibernation
is nearly as precise, for the later dates shown here reflect the fact
that I was present in the area a little later each year.

GROUND SQUIRREL ENTRANCE & EMERGENCE DATES
SPRING FALL

LAST DATE APPR. 2 OCTOBER
NO OBSERVATIONS COLD,LIGHT SNOW | OCTOBER
"NORMAL SEASON"

FIRST DATE, APPR. 22 APRIL LAST DATE 5 OCTOBER
3' SNOW, BARE SPOTS COLD, HEAVY SNOW 1&5 OCTOBER
"NORMAL"SEASON EARLY SEASON

FIRST DATE 21 APRIL LAST DATE 7 OCTOBER
4'4- 6' SNOW, NO BARE GROUND WARM (+10°C ), RAINY
3 WEEKS LATE SEASON LATE SEASON.

FIRST DATE 21 APRIL LAST DATE 9 OCTOBER
2-4' SNOW, '/e BARE GROUND WARM, CLEAR. I* SNOW 18 OCTOBER
2 WEEKS EARLY SEASON LATE SEASON

FIRST DATE 21! APRIL LAST DATE 12 OCTOBER
2% - 4' SNOW, PART BARE COOL , LIGHT SNOW
3-4 WEEKS EARLY SEASON "NORMAL" SEASON

NONE SEEN UNTIL 27. APRIL
HEAVY BLIZZARD ON 16 APRIL NO OBSERVATIONS
5/2 —- 8' OF SNOW. LATE SEASON

Fig. 1. Dates of first and last seasonal appearance of Arectie ground
squirrels near Anchorage, Alaska, in 1950-55, with notes on weather, ground
cover and seasonal comparisons. (Reprinted from Cold Injury. Trans. 5th
Cont. Josiah Macy Found., 1958.)

Figure 2 shows the events that comprise the seasonal cycle of
the Aretic ground squirrel. On May 1, the females begin to
emerge and breeding begins almost at once. By about May 10-15,
all females are pregnant, and the gestation period is 25 days
(Mayer and Roche, 1954), so that the young are born in late May
to early June. The voune stay in the nest about six weeks, and
begin to emerge about July 5-10. The mother eares for the young
for another 10 days to two weeks, after which they are left to
eare for themselves. At the end of this period of eare (early
August), all the population rapidly acquires sufficient fat re-
serves for hibernation. The males have already started increas-
ing in weight in early July. By about September 10-15, the

1960 MAMMALIAN HIBERNATION 157

number of squirrels is noticeably reduced, and it appears that the
females have started to hibernate. The adult males are active
until about the end of September to October 5, and after that
date I have found only young of the year active. Normal last
date of activity is about October 12, although a few aberrantly
immature animals may still be found after this.

Thus the season of activity is about 1388 days for the females
and 168 days for the males.

a
FATTENING ALL FATTENING

a en |
LACTATION ENTER HIBERNATION
| me | _
HIBERNATION EMERGENCE YG. EMERGE HIBERNATION

re e—
BREEDING BIRTH

4
CARE OF YOUNG

——
GESTATION

10 20 | 0 201 10 201 10 201 10 201 10 2 1 10 201 10 20 3!
JAN FES MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
Fig. 2. Average dates for the various events of the seasonal activity

evele of Arctic ground squirrels near Anchorage, Alaska.

The black bear, in Alaska, emerges from its winter quarters
about mid-April to early May, perhaps due to melt-water from
the snow running into the dens. At this time, there is no avail-
able food, and, as a matter of fact, the bears seem not to be
hunery or very thirsty. Bears eat mostly grass in summer, with
an oceasional tidbit of mouse, ground squirrel, or salmon. They
also dig for roots, and in the fall eat the abundant berries of
many kinds, especially Vaccinium spp. The bears are active
until middle or late October, and it appears to be the first heavy
snow of winter that sends them to the dens.

Breeding occurs in the latter half of June, and the young are
born about mid-January. However, the gestation period is not
precise, as one of my females had young this year about January
25, and dates from December to March may be found in the

158 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

literature (see especially Baker, 1904). The variation is probably
due to the discontinuous development of this species (Hamlett,
1935). The young are born in the winter dens and nursed by
the female on her body resources. The cubs (normally 2) weigh
250-400 grams each at birth, and on emergence 3 months later
about 4 to 5 kilograms each.

Weight Variation

It is generally agreed that the acquisition of adequate fat
reserves IS a necessary preparation for hibernation in most
species that hibernate (Lyman and Chatfield, 1955). The loss
of weight throughout the period of hibernation has been studied

800

700

IN GRAMS

IGHT
ej
fe)
,o)

W
_
[e}
[o)

20 30 10 2 %0 10 2 30 10 20 3 10 2 30 10 2 30 10
APR MAY JUNE JULY AUG SEPT OCT

Fig. 3. Average weights of Arctic ground squirrels for each date. <A
tive-year period is represented without distinction as to years. Data from
uear Anchorage, Alaska.

by several authors, but the weight evele during the active period
has been neglected.

Male adult Aretie ground squirrels emerge in) April with
welghts averaging about 550 em. There is no food available at
this time and, in fact, snow 120 to 240 em in depth may he
uniformly over the ground. In some years, there are patches of
bare ground, and some dead plant debris may be utilized.
Small amounts of food are stored in the burrows in fall (Mayer,
1953; Krog, 1954), and may be used at this time. However, there
is a weight loss at this season, and lowest weight is reached in
mid-May (Fig. 3). Weight of the entire adult population then

1960 MAMMALIAN HIBERNATION 159

remains relatively stable until late July, with the males slowly
eaining weight while the females lose due to lactation and care
of the young. Beginning in the first half of August, the entire
population rapidly fattens until the onset of hibernation. Fe-
males reach weights of about 600 em before disappearance, while
males go to 700 gm or above. Males disappear in early October,
apparently when weights approach 750 em. [| have rarely caught
one over this weight, and maximum weight recorded in this
area is 960 em.

---NO |
150 iq NO. 2
a ——NO. 3
i ae NO. 4
120 ; « MISC. CAPTIVE
° 4 MISC. WILD

KG.

WEIGHT
NS
a

1952 1953 1954 1955

Fig. 4. Weights of black bears plotted against time of year.

Weight loss durine hibernation in eaptivity is normal for
hibernators, so | will not discuss it.

The black bear is perhaps more interesting from the point of
view of weight loss, for the inability to radically drop its meta-
bolic rate as do the true hibernators imposes a more severe
energy demand on the bear during its stay of nearly six months
in the winter den. Figure 4+ shows change in weight of two
bears over a period of nearly 3 years. It will be seen that there
is a loss of about 9 ke during the winter denning season in
1953-54 for bear 1. During the following summer we attempted
to feed the bears as they would find available calories in the
wild, and weight loss continued. About August 1, they were

160 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

provided with all they could eat (12,000 ealories/day ) and they
gained up to 25 kg in two months. It appears that this is not
unlike the weight eyele seen in the wild, for on September 1 I
have seen bears with over 5 em of subcutaneous fat and great
masses of depot fat. Dr. Robert Rausch of the Arctic Health
Research Center has provided data on a four-year old male brown
hear which he collected on Kodiak Island on September 10, 1952.
Total weight was 195 ke, and fat weighing 20 k@ was removed.
It was estimated that total fat weighed about 380 to 32 kg, or
that 16 per cent of the weight was fat. Subcutaneous fat varied
in thickness from 40 to 53 mm over the hind quarters.

x= AVERAGE FOR DATE

7c,

OVERALL
MEAN .

TEMP,

20 30 10 20 30 10 20 30 10 20 30 10 20 30 10 20 30 ile]
APR MAY JUNE JULY AUG SEPT OCT
Fig. 5. Reetal temperatures of wild Aretic ground squirrels plotted

against date. Five years are represented.

It appears that wild black bears may lose up to 20 to 30 kg
during the winter denning period. Assuming the average adult
weight to be 150 ke, this would be a loss of about 15-20 per cent of
their weight. Ground squirrels lose nearly 380 per cent, although
their torper is more profound. This difference in percentage
weight loss is probably due to the great difference in total body
mass of the bear and ground squirrel, and is in itself tribute to
the efficieney of deep hibernation.

Body Temperature Variation

It has lone been stated that hibernators have a greater lability
of temperature while active than that found in homeotherms
(Johnson, 1931; Gelineo, 1938). However, in the course of other

1960 MAMMALIAN HIBERNATION 161

studies [ discerned a seasonal variation in rectal temperature in
active squirrels. Figure 5 shows this variation in wild squirrels
plotted against date. There is no apparent relation to ambient
temperature, sex, or degree of fatness. Figure 6 shows averages
of rectal temperatures of about 100 captive squirrels kept at
10°C, approximately the average temperature of the wild squir-
rel’s habitat. There is a progressive fall in temperature under

38 ae

37 e
oO x
°
x
a 36
=
WW
= x
35
fe) | 10 70 | 30 40 50 60 |70 Bb | 30 100 II0, 120 liso
20 JUNE 15 JULY 31 JULY IIAUG 26 AUG It SEPT 23SEPT BOCT 230CT

DAYS & DATES
Fig. 6. Average rectal temperatures of captive Aretie ground squirrels

plotted against date.

these captive conditions which is not discernible in the wild
squirrels until just prior to the onset of hibernation. his points
up the tact that data on captive animals is not necessarily valid
for extension to the wild or natural state. Therefore, it appears
that wild ground squirrels exhibit four different phases of tem-
perature control during the vear, as follows: (1) the low tempera-
tures found during hibernation; (2) the variable temperatures
found for a short period after emergence; (3) high and relatively
constant temperatures during most of the active season; (4)
falling temperatures just prior to entrance into hibernation.
During hibernation, temperatures close to ambient are re-
corded. Normally, I have kept my animal quarters at 4° ©2°C,

162 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

and have had highly successful hibernation at these tempera-
tures. One winter an ambient temperature of 10°C was main-
tained with shghtly less success.

It has been stated that hibernating Arctic ground squirrels
must endure temperatures below O°C, as they inhabit permafrost
regions. However, they avoid the low-lying ground, and burrow
in river banks and other high sandy or gravelly locations where
permafrost is well below the burrow level. Certainly, on exposure
to O°C the normal reaction is one of arousal from hibernation,
although I have in one case reduced rectal temperature to below

390 es
o e
38.0
+ oo = s 00-0 5
+ a
+ ° .
= 370 s
?
<
«
~
i 360
»
z CAPTIVE BLACK BEAR
<x
2 350
= a WILD BLACK BEAR
°
e
. a CAPTIVE BROWN BEAR
34.0 000 ° s °
WILD BROWN BEAR
330 WILD POLAR BEAR
° * FROM LITERATURE
320
310 6 i 28 68 W268 6 6 6 18 28 8 18 cs 5 18 88 6 16 86 8 18 88 5 is t5

JAN FEB MAR APR MAY JUN JUL AUS sep oct NOV DEC
Fig. 7. Rectal temperatures of bears plotted against date. (Reprinted
from Cold Injury. Trans. 5th Conf. Josiah Macey Found., 1958.)

0° by exposure of a hibernating squirrel to —20°C. The squirrel
recovered, with extensive freezing damage. Other squirrels in
the same situation aroused from hibernation or did not arouse
successfully (Hoek, 1958).

The black bear is able to maintain high constant temperatures
While active in any ambient temperature. Actually, when [ first
measured rectal temperatures in the winter dens, | found to my
surprise that the only recorded bear temperatures were some
taken on the Arctie expeditions of the 1820's, and 380’s on polar
bears. L have since been able to record a number of active bear
temperatures, of black (Ursus americanus), brown or grizzly
(Ursus arctos, ef. Rausch, 1958), and polar bears (Thalarctos
maritimus). Most are close to 38°C, as seen in Fieure 7. Polar

1960 MAMMALIAN HIBERNATION 163

hears seem to be about one degree lower, and perhaps they are
more variable in reetal temperature characteristics.

In contrast, all temperatures taken on lethargic bears are
around 34°C, as seen in Figure 7. On one occasion, a rectal
thermocouple stayed in place for three days, and recorded a
temperature of 31.2°C (Hoek, 1957). This is the lowest recorded
normal temperature for bears.

Dr, Rausch has recently been able to record the rectal tempera-
ture of a wild black bear denning near Anchorage, Alaska, on
February 16, 1959. Air temperature was —16.5°C, and immedi-
ately after it was shot, the bear’s rectal temperature was 33°C.
This point is not shown in Figure 7, but it will be seen that it
is in the range of the captive black bear readines.

Metabolie Rate Variation

It appears obvious that the rationale for the evolution of
hibernation is for purposes of conservation of energy so that the
animal's food supply, either in the form of depot fat or physically
stored food, can be made to support it for the required period.
The metabolic rate reduction durine hibernation has been
admirably treated by several authors, notably Kayser (1940,
1997). T should like to treat another aspect, namely seasonal
variation of metabohe rate of active ground squirrels. Hart
(1957) has recently reviewed such seasonal variation, but no
hibernators were included in his review.

Twelve ground squirrels that had not hibernated in winter
were used for the determination of metabole rate under ambient
temperatures of 30° to —10°C. Figure 8 shows the average
oxygen consumption of these animals. .\ second and larger
group was studied in summer under the same scheme. Both
groups were kept at 10°C, ambient temperature, and the prinei-
pal difference between them was that the winter group had
been captive longer and was under a constant photoperiod,
while the newly captive summer eroup was under a decreasing
photoperiod. Figure 8 shows the average values for the latter
eroup also.

It is first evident that Erikson’s (1956) results on the Aretic
ground squirrel were not duplicated in this study, as he found
no essential change in metabolic rate from 10° to 50°C. Sullivan
and Mullen (1954) found no difference between 5° and 25°C, in
this species. Figure 8 shows a well-marked relationship of
metabolic rate to ambient temperature. Kayser (1957) has

164 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

pointed out that Erikson’s failure to find dependence of meta-
bolic rate on ambient temperature is not in line with studies
on other hibernators in the active condition.

It is also apparent that the ground squirrel does not show
the same lack of seasonal variation in metabolic rate as reported
by Irving et al. (1955) for the red squirrel. Butterworth (1958)
has stated that the Arctic ground squirrel has two annual molts,
although other northern ground squirrels have only one. This
may explain the seasonal variation here shown, as Irving et al.

TEMP °C

Fig. 8. Average metabolic rate of Aretic ground squirrels in relation

to ambient temperature. Winter and summer curves are shown.

(1955) coneluded that the seasonal variation shown by the red
fox was due to better insulation of the winter pelage over the
summer pelage, while that of the red squirrel might be due to
little pelage insulation change. However, it appears that this
reduction in metabolism of the winter group may rather be a
reflection of : (a) lower body temperature, and (b) considerably
reduced general activity. It may also be that endocrine factors
are concerned, but | have no direct evidence on this point.
Metabohe rate during hibernation at 6° *2°C ranges from
0.03 to 0.10 ml 0./gm/hr, with an average of about 0.063
ml/em/hr, which is clearly within the expected range. It thus

1960 MAMMALIAN HIBERNATION 165

appears that the Arctic ground squirrel exhibits no metabolic
or temperature peculiarities due to its hibernation in far north
ern areas underlain by permafrost.

Figure 9 shows the metabolic rate of an active yearling blaek
bear in relation to varying ambient temperature. The normal
seasonal variation of metabolic rate is seen, similar to that shown
by Irving ef al. (1955) for well-insulated Aretie animals. Figure
10 shows the metabolic rate of active bears in winter compared
with that of a lethargic bear, plotted over 24-hour periods.

FALL- xX
0.50 x WINTER-O

CONSUMPTION, ML/GM/HR
te) (2)
o S

°
re)

-30 =20 -10 ie} 10 20 30
TEMPERATURE, °C

Fig. 9. Metabolic rate of active yearling black bear in relation to
ambient temperature. Early fall and late winter curves are shown. (Re-
printed from Cold Injury. Trans. 5th Conf. Josiah Macy Found., 1958.)

These animals were housed in adjacent cages, and their dens were
metabolic chambers. It can be seen that the metabolie rate in
lethargy, where reduction in rectal temperature is only of the
order of 7°C, is about 50-60 per cent of normal. This is greater
than that evident in man under hypothermic anaesthesia ( Virtue,
1955), so the bear apparently is superior to man or dog in this
respect. [ must emphasize that | was unable to determine rectal
temperature during this study, and the fall in den temperature,
found when outdoor temperature fell to ~—40°C and below, may

166 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

indieate that the bear’s temperature fell lower than the 31°C
eviven above. However, previous caleulations on weight loss in
winter indicated that the energy expenditure during letharey
must fall to about 45 to 24 of normal levels when rectal tempera-
ture was assumed to fall no lower than 31°C. It should also be

50
NO.5 OFF SCALE
45
.40
35
30 cere eer eee
NO. 4 ACTIVE
Pesce cee Xevnea ee eel ! ' \
sc3 ' ! '
~. 25 a i NO.1 VERY ACTIVE
£ —-—¥%- — 4 - —-—* —-—--—-—
S re ah [ee Rite tact area Ue ra
~ 20 ial NO. 3 ACTIVE
“J NO. 2 ACTIVE | eal
O-~ fis eels =a oe RRsasas= | 1
<5 | NO. 2
= f----- SGeseacke.
NO.1 ACTIVE nerd
Ona =~ = uae near

NO.| "HIBERNATING"

sins Mos

12 6 2
HOURS

Fig. 10. Approximate 24-hour metabolie rates of black bears in the
active and ‘‘hibernating,’’ or lethargic condition in winter. Values are

provisional only, but are useful for comparative purposes.

emphasized that the data in Figure 10 are rough in nature,
and have not yet been properly analyzed. However, for compara-
tive purposes, they are adequate to demonstrate the reduction
in metabolic rate found under the lethargic state assumed by
the black bear.

Breeding Cycle

It appears that hibernators have made several breeding adapta-
tions to the shortened season they have left available to them
after emergence from hibernation (Hoek, 1958). In this short

1960 MAMMALIAN HIBERNATION 167

season, they must not only carry out the breeding funetion, but
must allow the young animals adequate time for the acquisition
of fat reserves sufficient for the approaching period of hiberna-
tion. My contention that all seasonal activities of hibernators
are geared to the fact that they do hibernate is perhaps most
clearly demonstrated by this curtailed reproductive period.

The Aretie ground squirrel breeds almost immediately after
the emergence of the females, about May 1-10. In- order
for this to happen, gametogenesis must occur, at least in
part, during hibernation. It is apparent that it does so, for the
males emerging on April 21 and for a few days thereafter have
spermatids present in the seminiferous tubules, and the testes are
enlarged and descending, though not vet serotal. By May 1, the
testes are scrotal, and the lumina of the epididymis and semini-
ferous tubules are packed with motile spermatozoa.

It is apparent that this is one of the reasons, at least, why
the males emerge carlier from hibernation than do the females.
The females begin to emerge on May 1, and have ovarian follicles
ready to rupture. Breeding occurs almost at onee, and ends
about May 10-15. Ovulation in ground squirrels oceurs only
when copulation has occurred. The testes soon beeome smaller
and retract to the inguinal position, so that no late breeding is
possible. Spermatogonia and some primary spermatocytes are
the only elements present in the seminiferous tubules during
summer,

In the fall, shortly before the onset of hibernation, the testes
hegin to grow in size and secondary spermatocytes begin to
appear. The situation in the female is more difficult to determine,
perhaps due to their earher entrance into hibernation.

During the winter growth continues, as evidenced in the male
largely by the increasing size of the testes and the large number
of spermatoeytes. It appears that the spermatids are first evident
only just before the emergence from hibernation. The growth
of the ovarian follicles continues slowly through the winter.
Lyman and Chatfield (1955) have postulated that this growth
of gonads occurs during the periodic arousals, rather than during
the actual periods of hibernation. I have no information to shed
hght on this pomt and, in considering the preparation for a
short season, it Is of little significance whether the growth occurs

at high or low body temperature.

168 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Summary

It appears evident that hibernation is a eyelie phenomenon,
both in its occurrence and in the preparation for it. In short,
the whole year’s activities are related to the facet that the animal
will hibernate for a considerable time. Certainly this is true
of the Arctic ground squirrel for, due to the short season imposed
on it by its high northern distribution, it behooves this mammal
to compress its annual ecyele of breeding and the aequisition of
adequate nutritive reserves into a very short period. To this end
its whole evele of activity is geared, and many physiologic fune-
tions have responded by showing seasonal cyeles of various in-
tensities during the active season as well as during hibernation.

REFERENCES
BAKER, A. B.
1904. A notable suecess in the breeding of black bears. Smithsonian
Mise. Coll., 45:175-179.

BUTTERWORTH, B. B.
1958. Molt patterns in the Barrow ground squirrel, J. Mannnatl.,
39:92-97.

KISENTRAUT, M.
1933. Winterstarre, Wintersehlaf und Winterrube. Mitt. Zool. Mus.
Berlin, 19:48-63.

ERIKSON, HH.
1956. Observations on the metabolism of Arctie ground squirrels
(Citellus parryi) at different environmental temperatures. Acta
physiol. scand., 36:66-74.

GELINEO, 5.
1938. Sur la thermogenése de l’hibernant pendant |’éte. C. R. Soc.
Biol., 127:1357-1359.

HAMLETT, G. W. D.
1935. Delayed implantation and discontinuous development im the
mammals. Quart. Rev. Biol., 10:432-447.

ILarr, J. 8.
1957. Climatie and temperature indueed changes in the energetics of
homeotherms. Rey. Canad. Biol., 16:183-174.

Hock, R. J.
1951. The metabolic rates and body temperatures of bats. Biol. Bull.,
101 :289-299.
1957. Metabolic rates and rectal temperatures of active and ‘‘hiber-
nating ’’ black bears. Fed. Proe., 16:440.

1960 MAMMALIAN HIBERNATION 169

1958. Hibernation. In: Cold Injury. Trans. 5th Conf. Josiah Macy
Found., 341 pp. (Pp. 61-133.)

Irvine, L., H. Krog anp M. Monson
1955. The metabolism of some Alaskan animals in winter and summer.

Physiol. Zool., 28:173-185.,

JOHNSON, G. FE.
1931. Hibernation in mammals. Quart. Rey. Biol., 6:439-461.

IXAYSER, CH.
1940. Les échanges respiratoires des hibernants. Théses, Univ. Stras-
bourg, 364 pp.
1957. Le sommeil hivernal probleme de thermorégulation, Rev. Canad.
3i01., 16:303-389.

Kkroa, J.
1954. Storing of food items in the winter nest of Alaskan ground
squirrel, Citellus undulatus, J. Mammal., 35:586-587.

LYMAN, C. P. aNp P. O. CHATFIELD
1955. Physiology of hibernation in mammals. Physiol. Rey., 35:403
425.
MAYER, W. V.
1953. A preliminary study of the Barrow ground squirrel, Citellus
parryt barrowensis. J. Mammal., 34:334-345.
Mayer, W. V. AND E. T. RocHE
1954. Developmental patterns in the Barrow ground squirrel, Spermo-
philus undulatus barrowensis. Growth, 18:53-69.

Rauscu, R.
1953. On the status of some Arctie mammals. Arctic (Montreal), 6:
91-148.

SULLIVAN, B. J. AND J. T. MULLEN
1954. Effects of environmental temperature on oxygen consumption in
arctic and temperate-zone mammals. Physiol. Zool., 27:21-28.

VIRTUE, R. W.
1955. Hypothermie Anesthesia. Springfield, 62 pp.

DISCUSSION FOLLOWING HOCK’S PAPER

MAYER noted that the body temperature of 36.4°C which he
had cited previously was on captive squirrels which are used to
being handled; he believed this temperature substantiated the
data HOCK presented. HOCK noted that his (HOCK’S)
‘“average’’ wild value was 38.8°C.

170 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

MUSACCHIA asked what evidence was available as to the
periodicity of arousals in the wild of Aretie ground squirrels dur-
ing the hibernating season because there is an apparent lack of
experimental data and, therefore, no significant evidence eon-
cerning the nature of arousal, either periodie or sporadic, during
the hibernation of the Arctic ground squirrel. ILOCK said there
is none, but he is currently analyzing data on this subject on
captive animals accumulated over a nine-year period. For this
purpose he notes the periods of activity, but he does not claim
this method is precise.

PENGELLEY inquired as to how one can be sure the animals
arouse every two or three weeks. HOCK said he could not be
sure, but this was the longest normal period for this species.
MAYER remarked that he could substantiate this particular
periodicity from his laboratory observations, and that he could
substantiate the fact that animals aroused in the wild during
the winter.

PENGELLEY then asked if they eat when they become active
in this way. HOCK said they do store food and, if food is
available, they will eat. PENGELLEY said Citellus lateralis
nay or may not eat or drink during an arousal, but certainly
they do not need to. HOCK said that in the captive squirrels he
did not provide them with water, but with lettuce, and that no
water would seem to be available to wild hibernating squirrels.
MUSACCHIA added that if these animals eat at the time of
their arousal, they must clean mucus out of the alimentary canal
so that an initial or limited feeding may be of little nutritional
benefit. MAYER referred to his own experiments, noting that
for studying histological changes he did not provide food. Ani-
mals without food simply arouse from hibernation and streteh
or move around before re-entering hibernation. If animals come
out of hibernation and run around, this active state may last for
several davs. MAYER further pointed out that after 24 hours of
sueh activity, the metabolism of the animal is running at ‘‘ full
blast.”” HOCK eited the example of an animal which had never
heen seen in the active state over more than a three-month period,
hut it turned out that although it had not been seen active in
this period, nevertheless it had aroused since it had disturbed
the activity indicator several times.

WIMSATT asked if HOCK had calculated the maximum time
an animal could remain active durine the hibernating season
without prejudicing its survival through the season until spring.

1960 MAMMALIAN HIBERNATION 17]

HOCK rephed that he did not know exactly, but if one takes
the hibernating metabolic rate at M4, of the active rate, then
an animal which has enough food stored to hibernate 180 days
can survive six days of activity fasting at the same environmental
temperature with the same weight loss.

MORRISON remarked that the figure given on fat deposits
in the bear seemed very low. He (MORRISON) had data on
one denned bear in which the adipose tissue was 40 per cent of
the body weight. He indicated that his figures showed that if the
bear had gone on without a reduction in metabolism and body
temperature, it could have lasted 120 days. MORRISON stated
that in this bear the temperature (heart) was in the normal
range. He asked if it might not be possible to obtain low rectal
temperatures in a bear with a normal deep body temperature
due to the dense feeal plug that closes the reetum during hiber-
nation. HOCK rephed that he made deep rectal insertions whieh
gave him figures of 38.0° £1.0°C for aetive bears and a low of
31.0°C for bears in the ‘‘hibernating’’ or lethargic state.

PROSSER asked if there were any seasonal figures available
on rectal temperatures of polar bears — for those in water and
out of water. HOCK replied that he had taken one rectal meas-
urement of a polar bear that had been shot in the water and
the body recovered. The temperature drop observed was small,
although a more labile body temperature is indicated in this
species. He had also taken the rectal temperature of a youne
polar bear he had outside his laboratory just before he came to
this meetine: the Tp was 37° to 38°C. The latter is a maximum
for this species.

OBSERVATIONS ON A COLONY OF CAPTIVE
GROUND SQUIRRELS THROUGHOUT
THE YEAR’

By Barpara R. LANDAU
Department of Physiology
St. Louis University School of Medicine
St. Louis, Missouri

and

Auperr R. Dawe
Office of Naval Research

Chicago, Llinois

Some general aspects of hibernation and manifestations of a
torpid condition have been considered in several species, includ-
ing some in the natural (wild) state. For purposes of intensive
investigation, however, it is desirable to have these animals in
captivity. Bringing them into the laboratory may affect their
behavior and aetivity, but it permits better control and closer
observation. People have been observing hibernators for several
centuries, but their studies usually involved one or two animals
rather than a colony.

One who is interested in hibernation is interested in how and
why it occurs. Throughout the years attempts to identify the
factors controlling hibernation have followed either of two
general approaches. One of these has dealt with characteristics
that are umque to hibernating species, and which enable, or
cause, these animals to shift from the homeothermic to a seem-
ingly poikilothermie state. It is well known that the tendency
to hibernate is a evclic phenomenon, associated with the season
of the year. It is also well known that there are other phenomena
which show seasonal cycles in most hibernating species, such as
body weight, endocrine gland function and = sexual activity.
Numerous attempts have been made to relate the occurrence of
hibernation directly to one or more of these seasonal eyelic
phenomena.

1’This investigation was supported in part by a grant from the Wisconsin

Alumni Research Foundation (WARE), and in part by a grant trom the Na-
tional Institutes of Health, National Heart Institute, H-2095(e).

174 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The other approach to hibernation is concerned with environ-
mental conditions which favor, or are necessary for, entrance into
the hibernating state. Among these conditions, cold and tempera-
ture, confined air and carbon dioxide, and food or laek of it,
have been frequently mentioned, as well as light, sound and
mechanical disturbances. A consideration of the hibernating
state must take into account both seasonal eyche activity and
the factors in the environment, because the effectiveness of either
in inducing’ hibernation will be modified by the other. The
material to be presented is based upon observations of the
behavior and responses of a colony of ground squirrels, and at
times of individual animals. The data were collected from April,
when the first animals were caught, throughout the year until
the following summer. There were daily observations of the
behavior of the colony in regard to certain cyche phenomena, and
in response to the environment. The effect of specifie altera-
tions in the environment were also determined.

The colony. The colony consisted of 109 ground squirrels
(Citellus tridecemlineatus). OF these, 21 were caught in April and
early May (sprine caught), 63 in mid-June (summer caught),
and 25 in late September and early October (fall caught). The
eround squirrels caught in spring and summer were housed in
pairs. In the fall all animals were transferred to individual cages
in the general animal quarters, where they were kept until they
were placed in the cold. The temperature of this room ranged
between 20° and 29°C; hence it was designated the ‘Swarm
room.”’

To encourage hibernation, animals were placed in a ‘‘cold
room.’’ The temperature was not held constant, but was allowed
to fluctuate somewhat with that outside. It never fell below

-1°C, and it exceeded 12°C twice before April 1. Individual
hibernacula were provided, which consisted of an insulated metal
box, with a detachable screened lid. Each box contained cotton
waste for nesting. An attempt was made to have food and water
available at all times for those animals in the cold, even though
hibernation is said to be more likely if the animals have been
deprived of food and water (Johnson, 1930; Kayser, 1950). It
was felt that, beeause of additional disturbances, these animals
would be aroused more frequently than in nature, and would
probably need the extra energy supply. During the summer,
and while in the warm room, the animals were fed and watered
on alternate days. The diet consisted chiefly of sunflower seeds.

1960 MAMMALIAN HIBERNATION 175

Accommodations in the cold room were lmited, and since
these animals were being used in another series of experiments,
the same animals were not in the cold room all winter. Indi-
viduals were taken to the laboratory, sometimes for several
days, and often were aroused from hibernation. Every ground
squirrel was in the cold room part of the winter.

Irom late November through April the ground squirrels in the
cold room were checked daily (except for 2 days), usually
between 8S and 10 a.m. The warm room animals were checked
daily at first, and later on alternate days. Checking the colony
consisted of noting: (a) room temperature, (b) barometric pres-
sure, and (c) condition of each animal, as to health and state of
activity.

Body weight. Among the eyelice phenomena which were ob-
served was body weight. It has lone been asserted that the
weight of the animal contributes to the tendency to hibernate
(Horvath, 1881; Wade, 1930). Johnson (1930) reported that
heavy ammals (146-185 ems) hibernated 62 per cent of the time
in the cold, while light animals (86-125 ems) hibernated only 39
per cent of the time in the cold. Johnson’s animals, however,
weighed considerably less than those studied here, for after mid-
summer, all of our animals weighed more than 100 ems, and
some exceeded 250 ems.

The animals were weighed when caught, and at the beginning
of many experiments. Among the spring and summer caught
animals, males were heavier than females, but by mid-summer
this difference had disappeared. Young ground squirrels gained
weight rapidly, and by September they had caught up with the
fattening adults. Animals caught in the fall weighed approxi-
mately the same in October as the spring caught animals had
weighed in April. They gained weight slowly after capture, but
remained lighter than animals caught earlier. The spring and
summer caught animals, as groups, hibernated 28.6 and 23.4 per
cent of the time in the cold, while the lehter weight fall caught
animals hibernated 41.4 per cent of the time in the cold. So,
in general, heavy animals hibernated more than light ones, but
as Shown in Figure 1, high percentages of time in hibernation
were attained at much lower weights amone the fall caught
animals. Thus, size is not an absolute eriterion for hibernation.

At least two factors could have contributed to this difference
between catches. Many spring and summer caught animals were
not placed in the cold room until late in the season, when hiberna-
tion was less likely to oceur, whereas more fall caught animals

176 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

were placed in the cold room early in the winter. Secondly, a diet
of sunflower seeds, which the summer caught animals received
for a longer period of time, may simply result in obesity, with-
out enhancing biochemical preparation for hibernation.
Gonads and sexual activity. The endocrine glands of most
hibernating species go through an annual cycle, returning to a
relatively dormant state before the hibernating season (Kayser,
1950, 1953; Foster, Foster and Meyer, 1939). This involution

280
¢ SPRING & SUMMER CAUGHT ANIMALS

°FALL CAUGHT ANIMALS
260 e

240 ,

iar)

ine)

(e)
e

ipo)
(e)
(e)
8
°

WEIGHT IN GRAMS
@
i)
e
e
e

NN tL 1 = 1 te
(0) 20 30 40 50 60 70 80
PER CENT OF TIME IN HIBERNATION
Fig. 1. Relationship between body weight and the percentage of time that
an animal hibernated when kept in the cold room,

is generally considered to be associated with the time of year,
rather than with hibernation, since it occurs prior to the hiber-
nating season. For example, the testes of male ground squirrels
are serotal only during the few brief weeks of the mating season
in the spring. However, by mid-December most of the males
in the warm room showed some degree of scrotal enlargement.
Four of these animals were transferred to the cold room, and

3 hibernated after 21, 33 and 52 days in the cold, respectively. It

1960 MAMMALIAN HIBERNATION 177

was determined at autopsy that the testes of these animals had
become abdominal. The fourth ground squirrel did not hiber-
nate, and the testes were still scrotal when it was sacrificed after
62 days in the cold. By early March there were also signs of
scrotal enlargement in several males in the cold room. Johnson
and Wade (1931) have also observed scrotal testes in January
and February among ground squirrels kept in the laboratory.

It is said that ground squirrels will not live together except
during the mating season (Johnson, 1917). However, our spring
caught animals were housed in pairs (male and female) from
mid-April to as late as mid-September without incident. In view
of the signs of gonadal descent in January in warm room males,
attempts were made to encourage mating in two different pairs.
There was some excitement when the animals were first placed
together, but no fighting, and both pairs lived together peace-
ably for a month or more during the winter.

General activity. The daily cheek of the colony included a
rating of the voluntary activity of each animal at the time of
the check, according to an arbitrary seale, as follows:

‘1?’ — Very active. Running about, excited.

**2”’ — Active. Out of the nest, eating or moving about.

‘*3’’?— Alert. In the nest, but awake.

“4°? _ Asleep, or nearly so. Drowsy, eyes closed.

‘5°’ — Borderline. Sleeping soundly, not awakened by cheek-
ing. Breathing slowed, but body not cool to the touch.
Also ineludes animals in the process of arousing from
hibernation,

“6°? — Hibernating. Characteristic position, respiration very
slow or not apparent. Body cool to the touch.

Although this activity rating was subjective, it provided an
indication of the activity of the ground squirrels that were
not hibernating. The results of this tabulation are shown in
Figure 2. All animals were relatively inactive in late autumn
and early winter. Cold room animals were considerably less
active than those in the warm room. After mid-March the cold
room animals became increasingly active. This coincided with
the scrotal enlargement noted in the cold room males. It is of
interest that, unlike non-hibernating species, ground squirrels in
the cold room tended to be less active than those in the warm
room. This parallels the lack of endocrine response to cold,
shown by Deane and Lyman (1954), and supports the concept
that cold is not stressful to these animals.

178 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Several processes are known to diminish measurably as the
hibernating season approaches, such as metabolism (Kayser,
1953) and body temperature (Dubois, 1896; Benedict and Lee,
1938), as well as the endocrine involution previously mentioned.
Ileart rate and breathing rate are functions that are related to
metabolism, and they also show seasonal variation. Table |
contains data for resting heart and breathing rates obtained from
unanesthetized ground squirrels. In spite of a rather small
sampling, the restine rates tended to decline progressively from
relatively high levels in April. Some of the animals in the

FING
on

al
@

>
°

ACTIVITY R

i5 20 {12 26 9 23. 7 2a 6 20° 3. lv 2. i6- 30°13 (27
Sept. Oct Nov. Dec. Jan Feb Mar. Apr.

Mig. 2. General activity of all animals that were not hibernating. Animils
were rated according to an arbitrary scale (see text), in which smaller

numbers indicate greater activity.

spring exhibited very rapid rates, suggesting that the values
elven might not be true resting rates. Ilowever, it is quite
possible that the animals were more excitable at this time. The
slugeishness in autumn is probably one facet of the broad overall
preparation for seasonal hibernation.

Depth of hibernation. In the course of experiments on ani-
mals in hibernation, it became apparent that arousal was more
easily triggered in some animals than in others. For example,
if an animal had lost one of the electrodes (implanted previously
for other experiments), a wound clip could sometimes be applied
Without initiating arousal, vet at other times merely bringing

1960 MAMMALIAN HIBERNATION 179

a box containing an animal into the laboratory would set off
the arousal process. On one occasion, thermocouples and elec-
trodes were imbedded subcutaneously in 9 hibernating eround
squirrels. All of the animals were aroused by this procedure, but
their reactions differed markedly. Some began to squirm imme-
diately and continued to do so, while others stirred only when
the skin was pierced, and then but slightly. An hour later the
greatly disturbed animals were quite alert, while the others had
burrowed down into the nesting in typical hibernating position,
although their breathing was rapid. It was felt that these ground
squirrels must have been in different depths of hibernation, and
hence they were said to be in ‘‘shallow’’ and ‘‘deep’’ hiberna-
tion, respectively.

TABLE |

Heart and Breathing Rates (Range and Average) of Quiet
Unanesthetized Ground Squirrels at Different Times of the Year

Number of Resting Rate per Minute
Date animals Heart Breathing
April 16-30 12 284(188-444 ) 180 (126-255 )

May 1-15 12 2°77 (180-456 ) 169 (108-276 )
May 16-31 ? 7 256 (240-276 ) 207 (136-348 )
June 16-30 ih 223 (168-246) 169(108-276)
September 28-

October 20 5 212 (180-260)
January 17-

February $ 6 190 (120-260 ) 81(45-113 )

lollowing this observation, the response of hibernating animals
to the handling necessary for recording experiments was noted
Whenever possible, and the animals were classified as ‘‘shallow’’
or ‘‘deep.’’ Of the 46 occasions in which this was done, 19
ground squirrels were considered ‘*‘deep’’? and 27 were judged
“*shallow.’’ ‘‘Deep’’ animals were found to be in the first to
eleventh day of consecutive hibernation, an average of 4.9 days.
“Shallow”? animals had been in hibernation for from 1 to 5 days,
an average of 2.0 days. It seemed that by the fifth day of hiber-

(fa

nation, an animal was likely to be ‘‘deep,’’ so 5- or 6-day ani-

180 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

mals were used for experiments whenever possible. In this way
fairly good success was obtained in connecting an animal for
recording without initiating arousal. Later in the season, after
about the first of March, 1- and 2-day animals occasionally were
found to be ‘‘deep.’’ It appeared that in the early part of the
season a longer time was required to reach ‘‘deep’’ hibernation.
The only previous references to depth of hibernation have
been those (Horvath, 1881; Wade, 19380; Johnson, 1931) con-
cerning ‘partial hibernation,’’ at relatively high environmental
temperatures, and the observation of Lyman and Chatfield

Cc oo
a
ie}

——

PRESSURE mm. Hg

wes PERCENT
————- BAROMETRIC PRESSUPE
AMBIENT TEMPERATURE

TEMPER ATURE

BAROME TRI¢

PERCENT IN HIBERNATION
<} uw

Pig. 3. Daily tabulation of the percentage of the colony hibernating in
the cold room, with barometric pressure and ambient temperature.

(1955) that hibernating hamsters may be more sensitive to
externally applied stimuli on one day than on the next. These
data suggest that the depth of hibernation does vary, and that
several factors may be involved. Among these factors are: (1)
The number of days in consecutive hibernation, since animals
which had been hibernating for 5 or 6 days were more likely
to be in what was termed ‘‘deep’’ hibernation. (2) The
number of entrances into hibernation, or the season itself, for
late in the hibernating season animals were found to be ‘‘deep’’

1960 MAMMALIAN HIBERNATION 18]

after a shorter period. This may in some way be related to the
phenomenon of successively lower test drops, described by
Strumwasser (1959a). (3) The depth of hibernation is probably
also a species-related characteristic, for Lyman and Chatfield
(1955) noted that various species differed in their sensitivity
to externally applied stimuli while hibernating. Marmots and
hedgehogs are relatively insensitive to stimulation, while ham-
sters are notoriously easy to arouse.

The depth of hibernation does not seem to be eyelie in quite
the same sense as the phenomena previously discussed, but it does
modify the effect of environmental conditions upon the tendency
to hibernate, and it may be eyeclic.

The tendency of an animal to hibernate is modified by environ-
mental conditions which do, or do not, favor the hibernating
state. We were able to observe the effect of a number of these
factors, both incidentally and experimentally. Information
gathered in the daily check of the cold room animals provided
much of the data, which is summarized in Figure 3. The per-
centage of the colony in hibernation fluctuated widely, but de-
clined from about February 1. Ambient temperature and baro-
metric pressure varied randomly with no noticeable change until
April 1, when temperature tended to rise.

Ambient temperature. Figure 4 relates the percentage of
the colony hibernating on a given day to the ambient tempera-
ture. Because of a decreasing tendency to hibernate after about
the first of February, data from early and late winter have been
handled separately. In early winter an ambient temperature of
between 5° and 10°C was most favorable for hibernation, but
in late winter a lower temperature (0-2.5°C) was optimal, a
range of 0-10°C for the entire hibernating season. High tempera-
tures (12.5°C) were more apt to arouse animals than tempera-
tures near zero, even in early winter. The ground squirrels
apparently had a different range of response to cold in early
and late winter, for they required a lower temperature as winter
progressed.

Large daily fluctuations in percentage of the colony hibernat-
ing led to the suspicion that perhaps sudden temperature changes
might be partly responsible. When the change in environmental
temperature from that of the previous day was plotted against

182 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

the percentage of the colony in hibernation, there was little
correlation, due to the wide scatter.

In late autumn and well into the winter, animals in the warm
room hibernated briefly. Perhaps the term ‘‘ partial hibernation’’
is desirable, because the ambient temperature was at least 20°C,
which automatically precluded the extremely low body tempera-
tures ordinarily attained. Torpor and body cooling were observed

cop)
oO

Oo oOo

PERCENT HIBERNATIO
yo WwW pH w
ro)

O
“2:9 O 25 SO 7S 10.0 I25 6D

AMBIENT TEMPERATURE °C.

Fig. 4. Relationship between ambient temperature and percentage of the
colony hibernating. Crosses are values obtained from 21 Nov. - 30 Jan.
Closed cireles are values obtained from 1 Feb. - 30 April. Short-dashed lines
with crosses are averages for 21 Nov. - 30 Jan. Long-dashed lines with
closed circles are averages for 1 Feb. - 30 April. Solid lines with open
circles are averages for the entire winter, 21 Nov. - 30 April.

fl times in 18 different animals in the warm room between
mid-September and early February. There were undoubtedly
other occurrences, since part of this time the warm room animals
were checked on alternate days.

In this colony, hibernation was seen to occur at temperatures
ranging from O°C in the cold room to a maximum in exeess of
20°C in the warm room. This is essentially in agreement with

1960 MAMMALIAN HIBERNATION 183
the work of others, on this and other species. The ground squir-
rels hibernated most when the ambient temperature was be
tween 5° and 10°C.

Barometric pressure. Barometric pressure, like temperature,
showed no obvious relationship to the percentage of the colony
in hibernation (Fig. 3), but Figure 5 shows that the hieher
pressures favored hibernation, especially in late winter. The

I<) 730 T3 740 745 750 (350, 160
BAROMETRIC PRESSURE mm.Hg.

Fig. 5. Relationship between barometric pressure and percentage of the
colony hibernating. Symbols as in Figure 4.

optimum atmospheric pressure was above 750 mm He. Day to
day fluctuations were great, tending to mask any effect of daily
pressure variations, but if such a relationship existed, it was
with a shghtly rising pressure. This is supported by Lindemann
(1951) who found a relation between the onset of hibernation
and a rising barometric pressure. The effect of pressure, how-
ever; was less apparent than that of temperature.

Several hibernating animals were placed in a specially con-
structed cold chamber, where they were subjected to pressures
ranging from +32 to —60 mm He for from 5 minutes to 5
hours, followed by sudden return to atmosphere. Arousal was
not initiated in any of these animals.

184 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Season. Season usually is considered a factor contributing to
the hibernating state, mainly as winter versus summer. It has
already been noted that the tendeney to hibernate diminished as
the winter progressed. Figure 6 shows the hibernation trends
of the animals in the cold, as well as the temperature and pres-
sure, averaged by 2-week intervals. In all but the spring caught
group, the percentage of the animals hibernating began to decline
about February 1, although ambient temperature did not rise
until April 1. Thus the tendency to arouse was not a simple
reflection of elevated environmental temperature. The spring

12a— 2
J Tenties
ies}
a
=
a = |
& 2-80-7409 ie
g Fa ‘< \ Poarometric
WwW O-7Ot-—
2
5 60-735
Ww WW
a 50-& ----- Spring-caught
= QD —«— Summer-caught
ae 3 Fall- ealght
fo) a 4 2 qm Whole colony
E3e a <<
=z 20+ Sa"
WwW
2 io
x oO 3 | | | | =i

Nov 2!-30 Dec. !-I5 Dec,16-3! Jan |-6 Jani6-31 Feb!-'5 Feb-29 Mar FIS Macl6—3! Ap I-I5 Apr, 6-30

Fig. 6. Two-week averages of the percentage of the colony in hibernation
throughout the hibernating season, shown by groups and as a whole.

caught animals hibernated very little until they had been in the
eold for about 6 weeks, and thus hibernated very little until
quite late in the season. Animals kept in the warm room until
late in the winter showed little tendency to hibernate when
they finally were put in the cold, but those animals which had
been hibernating well all winter also hibernated less in the
spring. This is best seen among the fall caught animals, which
as a group, were in the cold longest and hibernated most. Ground
squirrels placed in a cold chamber (5°C) in June and July
frequently hibernated after several days, and lethargy was
observed in the warm room from August throughout the fall.
This leads to the conclusion that, although natural hibernation

1960 MAMMALIAN HIBERNATION 185

may oeeur throughout most of the year, the tendency to hibernate
is greatest from November to February.

Kxternally applied stimuli. Rotating an animal in its nest
on the turntable of a record player was used as a motion stimulus.
Speed of rotation was varied by changing the inertia of the
turntable, and ranged trom 7 to 70 rpm. Stimulation lasted
for from 5 to 15 minutes. Acceleration was gradual, but de-
celeration Was usually brought about abruptly by hand. All rota-
tion was in the horizontal plane, but with the animal’s head
curled under, it was not possible to tell which semicircular canals
had received primary stimulation. In 28 trials arousal was never
initiated, either by acceleration or abrupt deceleration.

It is often assumed that a satisfactory hibernaeculum must be
quiet but this has not been adequately substantiated. It might be
mentioned, however, that the cold room used in this study was
not particularly quiet, but the noise level was fairly constant.
kor auditory stimulation, a hibernating animal in its box was
put in a small refrigerator (with the door open), which was
plaeed squarely in front of a loud-speaker in a sound-proof room.
Sound of any intensity and frequeney within a wide range could
be delivered through the speaker from an adjoining control room.
Iixperimentally, 7 animals were subjected to the auditory stimu-
lation, applied for 2 minutes every 10 minutes. The stimulus
consisted of various combinations of frequency and intensity as
well as random noise. One animal showed absolutely no response
to the successive two-minute periods of sound and noise, even
at maximal intensities. Several attempts to dupheate the experi-
ment resulted in aroused animals. These arousals were at least
partially due to the difficulty in maintaining a cold environment,
as it Was necessary to leave the refrigerator door open during
auditory stimulation. It is of interest that the animal that was
not disturbed by sound had been hibernating for 7 conseeutive
days, whereas 5 of the 6 that were aroused had been in
hibernation for 1 to 4+ days.

On several occasions it was necessary to leave a light shining
through the transparent cover of the experimental cold cham-
ber, directly on a hibernating animal. This never initiated
arousal, and at least one animal is known to have entered the
hibernating state while the ight was on. Aroused animals in the
light tend to seek out dark corners of the nest, and bury them-
selves under the nesting. By doing this, and curling up as they
do, it is not likely that hght could exert a very strong effect.

186 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

This does not preclude a possible effeet of light upon the tendeney
toward seasonal hibernation. The animals in the eold room
in this study were kept in darkness 24 hours a day, whieh would
resemble the condition in a sealed burrow during the winter.

There were several indications that a hibernating animal has
an elevated pain threshold. It has been mentioned that wound
clips could occasionally be applied without initiating arousal.
Intraperitoneal injections of cold solutions of physiological saline,
epinephrine and atropine have been given without causing
arousal. This was not the rule, however, for such procedures
usually initiated arousal.

The inconstant results that have been obtained upon stimula-
tion of sensory receptors of animals in hibernation ean no doubt
be explained at least partially on the basis of the concept of
differences in the depth of hibernation, i.e. ‘‘shallow’’ and
‘‘deep.’’ Mild stimulation readily initiates arousal in ‘‘shallow’’
animals, whereas ‘‘deep’’ animals show but a shght response
of short duration. The relative insensitivity of ‘‘deep’’ animals
to stimulation could be due to inability of the peripheral nerves
to econduet, or to depression of the ascending reticular activating
system. Excised tibial nerves of hamsters function at tempera-
tures as low as an average of 3.4°C (Chatfield et al.. 1948). The
auditory nerves of hamsters probably do not conduet below
18°C, and they are said to be functionally deaf while hibernating
(luavman and Chatfield, 1955). Cortical activity has been evoked
at cortical temperatures as low as 9°C in the hamster by sciatic
nerve stimulation (Chatfield ef al., 1951), and 7°C in the wood-
ehuek by auditory stimulation (Lyman and Chatfield, 1953).

Thus, it seems that there is variation in the ability of
nerves to function in the cold, both between species and between
different nerves in the same animal. These differences probably
are not related only to temperature of the nerve. Since the
sensitivity of an animal varies over a period of time, during
which the temperature may vary but slightly, there must either
be other factors governing the ability of fibers to conduct, or
peripheral nerve conduction is not the whole story.

‘The reticular activating system, which is associated with con-
sciousness and wakefulness, is thought to be particularly sersi-
tive to cold. In the arousing hamster, electrical signs of activity
in the reticular activating svstem (high frequency, low voltage
deflections of the electrocorticogram) were not found until the
cortical temperature had reached 29°C (Chatfield et al., 1951).

1960 MAMMALIAN HIBERNATION 187

More recently Strumwasser (1959b) reported spontaneous activ
itv in Crtellus beecheyi at cortieal temperatures near 6°C, but
they were reduced about 90 per cent in amplitude. It may be
that these central mechanisms are more depressed after several
days at low brain temperatures. It would be of interest. to
compare the electrical activity of ‘‘shallow’’ and ‘‘deep’’ ground
squirrels with that of hamsters, since the latter are known to
be very easily aroused,

It appears that animals which are capable of hibernation dif-
fer im several respects from animals which are not capable of
hibernation. These differences are associated primarily with the
response to cold, and involve not only survival of the whole ani-
mal at low temperatures, but also functioning of the individual
tissues, such as peripheral nerves and the heart. For a triggering
mechanism to imitiate induetion into hibernation in a species
which is capable of it, at least two conditions must obtain. One
of these is that the environmental temperature must be such
that the animal’s body temperature can fall to low enough levels
to result in lethargy. The other is that the endoerine e@lands,
which show seasonal activity eveles in most hibernating species,
must be in the inaetive phase. A major role has been assigned
to the endoerine system by some (Kayser, 1950), but it seems
more likely that the state of the endoerines, as well as the pres-
ence of a coldt environment are permissive rather than causative
Factors. iA certain degree of biochemical preparation is very
likely also essential.

Environmental conditions, such as the degree of cold, atmos-
pheric pressure, season and external stimuli, modify the oeeur-
rence of hibernation in a group of ground squirrels, but these
conditions do not actually cause hibernation.

Acknowledgments

Acknowledgment is gratefully made to Dr. J. E. Hind for
making possible the experiments on auditory stimulation, and
to Miss Nona Klapproth for her technical assistance.

188 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

REFERENCES
BENepIcT, F. G. AND R. C. LEE

1938. Hibernation and marmot physiology. Carnegie Inst. Washing:
ton Publ., 497:1-239.

CHATFIELD, P. O., A. F. Battista, C. P. LYMAN AND J. P. GARCIA
1948. Effects of cooling on nerve conduction in a hibernator (golden
hamster) and non-hibernator (albino rat). Am. J. Physiol.,
155:179-185.

CHATFIELD, P. O., C. P. LyMAN aNpD D. P. PURPURA
1951. The effects of temperature on the spontaneous and induced
electrical activity in the cerebral cortex of the golden hamster.

EEG Clin. Neurophysiol., 3:225-250,.

DEANE, H. W. anno C. P. LYMAN
1954. Body temperature, thyroid and adrenal cortex of hamsters dur-
ing cold exposure and hibernation with comparisons to rats.
Mndoerinol., 55:300-315,

Dusois, R.
1896. Physiologie comparée de la marmotte. Ann. Univ. Lyon. Paris,
268 pp.

Foster, M. A., R. C. Foster AND R. K. MEYER
1939. Tlibernation and the endoerines. Endocrinol., 24:603-612.

HlorvatrH, A.
1881. Einfluss versechiedener Temperaturen auf die Wintersehlafer,
Verh. phys.-med. Gesellsch., 1§:187-219.

JOHNSON, G. FE.
1917. The habits of the 13-lined ground squirrel. Quart. J. Univ. N.
Dakota, 7:261-271.
1930. Hibernation of the 13-lined) ground squirrel. V. Food, light,
confined air, precooling, castration and fatness in relation to
production of hibernation. Biol. Bull., 59:114-127.
1931. Hibernation in mammals. Quart. Rev. Biol., 6:489-461.

JOHNSON, G. KE. and N. J. Wade
1931. Laboratory reproduction studies of the 13-lined ground squirrel.
Biol. Bull., 61:101-114.

KAYSER, C.
1950. Tesommeil hibeinal. Biol. Rey., 25:255-282.
1953. L’hibernation des mammiféres. Ann, Biol., 29:109-150.

LINDEMANN, W.
1951. Zur Psychologie des Igels. Zschr. Tierpsyehol., 8:224-251.

1960 MAMMALIAN HIBERNATION 189

LYMAN, C. P. AnD P. O. CHATFIELD
1958. Hibernation and cortical electrical activity in the woodchuck
(Marmota monaa). Seience, 117:5383-534.
1955. Physiology of hibernation in mammals, Physiol. Rev., 35:405
425,

STRUMWASSER, F.
1959a. Factors in the pattern, timing and predictability of hibernation
in the squirrel, Citellus beecheyi, Am. J. Physiol., 196:8-14.
1959b. Regulatory mechanisms, brain activity and behavior during deep
hibernation in the squirrel, Citellus beecheyi. Am. J. Physiol.,
196 : 23-30,

Wapbk, O.
1930. Vhe behavior of certain spermophiles with special reference to
aestivation and hibernation, J. Mammal., 11:160-188.

DISCUSSION FOLLOWING LANDAU’S PAPER

FISHER asked whether the consecutive hibernation period
spoken of began with an arousal period, or began with entrance
into hibernation. LANDAU replied that it was timed from
entrance into hibernation, and that usually by the sixth day an
animal was in ‘‘deep’’ hibernation. In contrast, animals in
March were in ‘‘deep’’ hibernation after only one or two days.

STRUMWASSER asked how many animals died in the cold
room. LANDAU rephed that very few did so. Several animals
that were exposed to rather severe weather conditions died, but
after the colony was moved into the cold room only 3 or 4 of
about 60 animals were lost.

POPOVIC asked if, when animals died in the cold room, they
died in the hibernating state. LANDAU said she believed this
to be the case. HOCK then noted that such animals fit Maneili’s
definition of ‘‘morbid hibernators.”* Ile (ILOCK) had observed
that small individuals captured in the fall of the year stay in
hibernation almost continuously as if they are unable to
arouse. The ‘*morbid hibernator’’ is a small or improperly nour-
ished animal that ‘Shas to hibernate.’’ HOCK also mentioned
the state of the gonads in hibernation —~- he noted that in the
wild Alaskan ground squirrel the testes are not scrotal at the
time of emergence on April 21, but that spermatozoa are present
on May 1, and on that date the male is ready for mating.

190 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

ZIMNY asked two questions: (1) had LANDAU ever put a
female she was sure was in estrus in the cold room to see if
it would hibernate; (2) had she noticed higher food consumption
on the part of animals in the cold room which would not
hibernate. LANDAU replied that an experiment of this type
involving an estrous female had not been tried; she stated that
food was kept available ad libitum at all times in the cold room,
and that after mid-March they ate more and had to be fed every
other day. They also drank more water and were more active.

SMITH said he would like to emphasize the matter of diet.
He stated that several discussions had been concerned with the
length of hibernation, and also with the time animals must
remain in a cold room before goine into hibernation. He indi-
cated that diet could be an important factor here, especially in
considering which dietary factors animals may be able to pick
up in nature during the summer (pre-hibernation period) in
contrast to what an animal is given in the laboratory in the pre-
hibernation period.

MAYER asked if the obesity LANDAU observed might not
be due to a sunflower seed diet. LANDAU said she did not
think sunflower seed diet was the only cause of weight increase,
but it was one factor in the relatively heavy weights of some
of the animals. MAYER asked if LANDAU thought hiberna-
tion would oeeur on any diet, and pointed out that 800 em
animals he had caught in the field would increase their weight
to 1000 em on an ad libitwin laboratory diet. Food had to be
forcibly withheld from such an animal in order to maintain its
body weight near 800 gm. LANDAU replied that she wondered
at this because her lighter weight fall-caught animals hibernated
on a sunflower seed diet.

PENGELLEY asked if it were not true that animals with
scrotal testes would not hibernate, but when testes became ab-
dominal hibernation would take place. LANDAU rephed that
this apparently was true and that the one animal she maintained
for a lone period in the cold whose testes did not become
abdominal, did not hibernate.

PENGELLEY then asked as to the exact eriteria used to
determine that an animal had remained in continuous hiberna-
tion for as long as 7 days. LANDAU replied that animals
were checked once each day in the morning. She conceded it

1960 MAMMALIAN HIBERNATION 19]

Was possible that animals might have gone into hibernation and
aroused again during the interval between checks. She said she
had no other evidence than her subjective impression, but felt
strongly that the number of times she erred in not noting this
were few — simply because it takes a while for the animal to
pertorm the cyele of entrance into and arousal from hibernation.
PENGELLEY noted, on the contrary, that he had observed, in
keeping hibernating ground squirrels, that they will come out
of hibernation and go into hibernation in a 13-hour period.

DAWE then asked LANDAU if she would describe the ob-
servations she made of three animals that died in hibernation.
LANDAU stated that when these animals were autopsied, no fat
deposits were to be found.

POPOVIC then noted that a hibernator hibernates in accord-
ance with the season. When, during hibernation, the ambient
temperature drops below O°C most ground squirrels die. LAN-
DAU remarked that lowering the external temperature sutf-
ficiently (to 0°C) results in an increase in metabolic rate im
order to maintain body temperature at 3°-4°C. If an animal
is compelled to do this over a lone period without arousal, it may
lack sufficient energy reserves for a proper arousal.

xX
AESTIVATION IN THE
MOHAVE GROUND SQUIRREL
CITELLUS MOHAVENSIS

By Georce A. BARTHOLOMEW and JAacK W. Hupson
Department of Zoology
University of California

Los Angeles, California

Physiological information on aestivation is extremely scarce.
This is in part due to the facet that most physiologists live and
Work in areas where adaptation to scasonal cold rather than to
seasonal drought represents the major adaptation of mammals
to environmental stress. In desert areas aestivation offers an
effective mechanism for survival during the periods when food
and water are most scarce. A miumber of rodents have developed
this ability to aestivate and offer attractive opportunities for
the investigation of naturally occurring hypothermia at rela-
tively high ambient temperatures. The Mohave ground squirrel
is a diurnal desert rodent that undergoes prolonged periods of
both hibernation and aestivation. It has an extremely restricted
range in the Mohave Desert of California and is sympatric
with the much wider ranging Antelope ground squirrel, Citellis
leveurus, which neither hibernates nor aestivates.

Methods

The animals used in the present study were captured during
the spring and summer of 1957 and 1958 in the Antelope Valley
of the Mohave Desert about three miles east of Palmdale, Los
Angeles County, California. They were housed individually in
elass terraria in a windowless room with a twelve-hour photo-
period and were given commercial rat food, sunflower seeds
and water ad libitum. Under these conditions the animals were
extremely docile, and survival was excellent. The temperature
of the animal room varied between 22 and 27°C. Field observa-
tions on seasons of activity and hehavior of C. mohavensis were
made incidental to a year-round program of study of C. lewcurus.

Measurements of oxygen consumption were made by placing
the squirrels in an air-tight two-liter container equipped with
a thermocouple and ports for the introduction and removal of
air. Dry air was metered through the container at a rate of

194 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

400 ce per minute and then delivered to a Beekman paramagnetic
oxygen analyzer, which, used in conjunction with a recording
potentiometer, gave a continuous record of oxygen consumption.
The determination of oxvgen consumption during torpor, entry
into torpor, and arousal were obtained by putting the animal into
the respirometer and placing it in a constant temperature cham-
ber. Oxygen consumption was then recorded continuously until
the animal became torpid. Some of the arousals were spontane-
ous, others were induced by the disturbances incidental to meas-
urement of body temperature during torpor.

Continuous records of body temperature during arousal were
obtained by inserting a vinyl-sheathed copper-constantan thermo-
couple through the rectum to a depth of five or six centimeters,
securing the leads to the tail with adhesive tape and then attach-
ing them to a recording potentiometer. Oral temperatures were
determined manually either by thermocouple or by quick-acting
mercury thermometer.

Results

Natural history and behavior. In the Antelope Valley, Mohave
evround squirrels are active above ground from early March to
August. They remain in their burrows throughout the rest of the
vear and presumably are dormant much of this time. The young
are born in the early spring. In our experience they are solitary.
Under natural conditions the animals are quite tame and can be
readily approached; in captivity, they are extremely lethargic
and spend much of their time asleep or torpid. Despite their
placid behavior, captive Mohave ground squirrels are so intoler-
ant of members of their own species that they must be housed
separately.

In eaptivity the animals became extremely fat. They re-
mained fat at all seasons but tended to lose some weight during
the spring. From Mareh to August, in the laboratory, the ani-
mals were active and showed no signs of dormancy. During the
remainder of the year they were intermittently torpid at room
temperature despite the continuous availability of food and
water and despite the frequent disturbances associated with the
maintenance of other experimental animals in the same room.

Oxygen consumption and body temperature during normal
activity. Continuous records of oxygen consumption obtained for
many hours in the present study offered favorable opportunity for
the determination of standard metabolic rate. It was possible
to seleet from many hours of recordings those intervals showing

1960 MAMMALIAN HIBERNATION 195
a nunimal uniform oxygen consumption. Standard weieht-rela
tive metabolism at 23 to 26°C averaged shehtly more than 0.8 ce

0.o/gm/hr (Table I).

TABLE |

Wet. in ce Oo/gm/hr Air
Sex Grams Temp. °C
228 1.0 26.0
i 232 0.9 23.0
4 270 0.8 25.0
3 306 0.7 24.0

Standard metabolism of alert C. mohavensis. The figures for oxygen
consumption are rates maintained for 40 to 60 or more minutes by post-
absorptive animals. All the animals were extremely fat. The oxygen
consumption figures shown are based on total weight and would be at least
one-third larger if they were calculated on the basis of fat-free body weight.

Although the body temperature of C. mohavensis varies with
environmental temperature and activity, it is quite uniform at
room temperature in the absence of disturbance. Reetal tempera-
tures from five different animals measured on each of four
consecutive days at 8:30 a.m. during early July, a time when
the animals rarely aestivate, fell between 35.2 and 36.1°C (mean,
5, £0.13).

Entry into torpor. Animals placed in the respirometer at room
temperature during fall and winter and left undisturbed some-
times became torpid. Since oxygen consumption was being
continuously measured, it was possible to measure metabolic
rate while the animals were entering torpor. Satisfactory records
were obtained from five animals. Representative records are
shown in Figures 1 and 2. At ambient temperatures of 22 to
26°C entry into torpor is completed in three to four hours.
During this period oxygen consumption may deeline smoothly
(Iie. 1) or it may show irregular excursions, presumably associ-
ated with body movements incidental to changes in posture
(Fig. 2). In two of the five instances, the decline in oxygen con-
sumption was preceded by a brief but conspicuous inerease in
metabolism.

Under the conditions of measurement of oxygen consump-
tion it was not possible to observe behavior during entry into
torpor. Tlowever, aestivatine animals were observed almost
daily in the laboratory during late summer, fall and winter.
While entering torpidity, they assumed the usual sleeping pos-
ture with feet and head tucked under the body.

OU eae ory
Od

196 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Torpor. Aestivating animals were most readily distinguished
from sleeping animals by then respiratory pattern. Torpid
animals showed prolonged periods of apnea, while sleeping ani-
mals did not. Oxygen consumption during torpor was usually
extremely uniform and fell between 0.1 and 0.2. ce/gm/hr.
There were, however, occasional increases in rate to as much as
0.4 ce/em hr. Under the conditions used for the measurement of
oxygen consumption the duration of torpidity was variable,
extending from eight hours to several days. Aestivating ground
squirrels observed in the laboratory occasionally shifted position
or changed posture without arousing.

3.0
2:9
c 2.0
al
>
=— £5
on)
oO
o)
oO 1.0
RECTAL RECTAL
0.5 TEMP TEMP
ZS? CX 212° C5
\
we AT a =| ree) ear — tl
2 4 6 l2 l4 te 18 20 22 24 26 28 30° 32 142

HOURS

Fig. 1. Oxygen consumption during two entries into torpor, one induced

arousal, and normal activity in an adult ¢ C. mohavensis, weighing 232

erams. Ambient temperature varied between 21 and 23.7°C.

During torpidity body temperature varied directly with air
temperature and oral and rectal temperatures did not differ
significantly from each other. Torpor was observed in animals
at body temperatures ranging from 10.6 to 27.1°C. We do not
know whether or not these represent the limits of body tempera-
ture at which animals of this species can remain torpid.

Arousal. When an aestivating Mohave ground squirrel starts
to arouse, its OXveen Consumption rises rapidly and may increase
10 to 20-fold in less than 15 minutes (Figs. 1 and 2). The peak
of oxveen constimption is usually reached within 20 minutes of

1960 MAMMALIAN HIBERNATION 197

the start of arousal. (As discussed below, body temperature
increases much more slowly.) Oxygen consumption then declines
to the normal resting level during a period of two hours or more.
Thus, during much of the period of arousal from aestivation,
oxygen consumption is actually deereasing from its initial peak
while body temperature increases. As pointed out above, during
aestivation the breathing of these animals is characterized by
prolonged periods of apnea. With the onset of arousal, the
breathing of an animal aestivating at 20°C or more immediately
becomes continuous and within five to ten minutes reaches the
normal rate of about 80 to 90 per minute; thereafter, it usually

2.5

2) -220
x
x
~
=
= 1s
~N RECTAL
a RECTAL TEMP
2 RENE 34.6°C |
re) 36.0° C :
SEO 7
0.5

2 4 6 8 [0 l2 14 6 I8 20 22 24 26: 28

HOURS
Fig. 2.) Oxygen consumption during normal activity, entry into torpor,
and spontaneous arousal in an adult & C. mohavensis, weighing 306 grams.
Ambient temperature varied between 23 and 26°C,

remains relatively constant in rate but may increase markedly
in amplitude. At body temperatures below 20°C, periods of
apnea continue to oceur even while the animal is arousing.
Toward the end of arousal the breathing rate sometimes becomes
conspicuously depressed and the respiratory movements become
very deep and heavy.

The time required for body temperature to rise to levels char-
acteristic for normal activity depended largely on initial body
temperature, but the rate of increase showed no significant cor-

198 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

relation with body temperature at start of arousal. Rate of
temperature increase of the same animal during arousal varied
from time to time (Fie. 3). At ambient temperatures between
2? and 27°C the maximum rate of inerease in body tempera-
ture above ambient temperature was 0.4°C/min while the
minimum rate was 0.1°C/min. The body temperatures at the
termination of arousal showed considerable variation, extending
between 33 and 38°C. In general, rectal temperature was the
same as oral temperature at the beginning and end of arousal and

35 L
Lo a at

. oO)
oO
aR A
Ww Ay
w 25
x
Oo
oe B
Q

30 60 90 120 150 180
MINUTES
Fig. 3. Increases in body temperature during arousal in C. mohavensis.

Lines A and Ay represent different arousals by the same animal. Each of
the other lines is for a different animal. Line B shows an arousal in which

shivering was barely perceptible.

rarely was more than 0.5°C lower than oral temperature during
arousal.

Characteristically, these ground squirrels shivered strongly
during arousal. Occasional sheht quivering of the anterior parts
of the body were observed at body temperatures as low as 16°C,
Strong, sustained shivering characteristically did not begin until
body temperatures of 23 to 24°C were attained. Shivering ap-
peared first anteriorly and then spread to the posterior parts of

1960 MAMMALIAN HIBERNATION 199

the body. Strong shivering did not invariably oeceur during
arousal although some shivering was always observed. Those
animals showing least shivering had the slowest rates of inerease
of body temperature (lig. 3). In no ease did shivering continue
after body temperature had reached 35°C.

Mohave ground squirrels with body temperatures as low as
10°C respond to touch by withdrawal. (Lower temperatures were
not tested.) No vocalization could be elicited at body tempera-
tures below 21°C, but it occurred in response to all disturbances
at body temperatures above 25°C. At body temperatures below
15°C the animals were unable to right themselves when placed
on their backs. With a body temperature of 20°C, the animals
were capable of poorly coordinated crawling and slow, jerky
walking. At this temperature they often attempted to dig. At
27 to 28°C their walking and digging activities appeared normal
and coordinated. By the time body temperature reached 32
to 33°C behavior appeared normal in all respects.

Diseussion

Aestivation versus hibernation. Lone usage has established
the word *‘aestivation’’ for summer dormaney in mammals as well
as in other organisms. Although there ean be no doubt that aesti-
vation occurs naturally in mammals, particularly in regions of
seasonal drought, the evidence for it is largely cireumstantial —
many species of rodents are not active above ground during parts
of the dry season, and it is assumed that they are dormant. (See
Kalabukhoyv, 1956, for a detailed review.) However, detailed
physiological data on aestivation in placental mammals are lim-
ited to two genera, Crtellus and Perognathus. Studies on Perog-
nathus longimembris (Bartholomew and Cade, 1957) led to the
conclusion that in this species aestivation and hibernation are
the same physiological phenomenon, the only difference between
the two being the level of body temperature, and this is depend-
ent on ambient temperature. A similar conclusion seems justified
in the one bird for which data are available; the Poor-will,
Phalaenoptilus nuttalli, will become torpid over a range of
environmental temperatures extending at least from 2° to 19°C
(Howell) and Bartholomew, 1959).

In the present study we have found that in the Mohave ground
squirrel aestivation shows the classical criteria of hibernation :
(1) body temperature within a degree or less of ambient tempera-
ture; (2) oxyeen consumption markedly redueed; (3) prolonged

200 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

periods of apnea; (4) a torpor more pronounced than deep sleep ;
(9) arousal, either spontaneous or induced, accompanied by acti-
vation of the major heat producing mechanisms. Furthermore,
the physiological and behavioral performance of this animal is
qualitatively similar over body temperatures ranging from 10°C
to 27°C. The physiological and behavioral differences between
aestivatine and hibernating Mohave ground squirrels are matters
of degree and appear to be simply functions of body temperature,
the level of which is determined by environmental temperature.
We, therefore, suggest that the terms aestivation and hiber-
nation should be used only in the context of natural history to
deseribe the summer and winter dormaney of warm-blooded
animals. We feel that some other word or phrase, such as *‘faeul-
tative hypothermia,’’ should be used for the physiological aspects
of these phenomena in birds and mamimals. Such a change in
terminology would have the advantage of distinguishing between
the performance of warm-blooded animals and all other organ-
isms, would emphasize the close physiological similarity of sum-
mer and winter dormancy in a variety of mammals and birds,
and could in addition encompass the ‘‘ partial hibernation’’ of
bears and the daily torpor of bats and hummingbirds.
Physiology. The capacity of various species of Citellus to be-
come dormant at temperatures between 20 and 30°C offers the
opportunity for further insights into the mechanisms of seasonal
hypothermia in mammals. In mammals dormant at low tempera-
tures (i.e. in the ‘‘deep hibernation’’ of Lyman, 1948, p. 56) the
peak of oxygen consumption is not reached for more than an
hour after the onset of arousal. However, in Mohave ground
squirrels dormant at temperatures above 20°C, oxygen consump-
tion may reach its maximum within 15 or 20 minutes from the
onset of arousal. We interpret this to indicate that initiation of
arousal is under the control of the central nervous system and
that the rate of heat production is limited by cell temperature
and, of course, modified by the condition of the animal. In an
animal clormant at relatively high temperatures, oxygen con-
sumption during arousal ean increase more rapidly than in an
animal dormant at low temperature because the metabolically
depressing effects of low cell temperature are less. The behavioral
responses of Mohave ground squirrels at a body temperature of
20°C are sufficiently complex and coordinated that one can infer
a complex level of cortical activity at this temperature even in
the absence of direct measurement. Our data did not show peaks
of oxygen consumption during arousal as high as those reported

1960 MAMMALIAN HIBERNATION 2())]

for some other species of Citellus (see, for example, Popovie,
1957) ; the general pattern, however, of an overshoot in oxygen
consumption followed by a slow decline was the same in our
animals as in ground squirrels arousing from body temperatures
near O°C,

In most hibernating mammals during arousal the increase in
rectal temperature lags far behind the increase in oral tempera-
ture. This is usually not the case in the arousal of aestivatine
Mohave ground squirrels. They have, however, the necessary
cardiovascular mechanisms for the establishment of this tem-
perature differential. During an arousal from 16°C one indi-
vidual had a rectal temperature of only 25°C when its oral
temperature reached 35°C. The failure to maintain a marked
antero-posterior temperature difference may be associated with
the relatively high body temperatures at the onset of arousal
in the present experiments.

In captivity with food continuously available, Mohave eround
squirrels show intermittent periods of dormancy in all seasons
except spring and early summer. We assume, therefore, that
for months at a time they are in condition to aestivate or
hibernate and that during this period no intervals of transition
or physiological adjustment between normal body temperature
and marked hypothermia are necessary. Entry into torpor is
rapid and essentially unbroken, although the process may some-
times be interrupted. The rapidity with which this species can
reduce its rate of oxygen consumption and allow its body tem-
perature to drop to ambient temperature leads us to assume
that initiation of the reduction of metabolism like the initiation
of arousal is under central nervous control. Once metabolic
rate has been reduced, body temperature declines with the end
point being determined by ambient temperature.

Since the behavior of these animals is essentially normal at a
body temperature of 32°C and sinee they can become dormant
at a body temperature at least as high as 27°C, the difference
between the minimum temperature of normal activity and the
maximum temperature of aestivation is no greater than the
range of body temperatures observed in normally active animals.
This difference may in fact be even less than 5°, for Popovie
and Popovie (1956) report that C. citellus ean become dormant
at 30°C. Viewed thus, the transition from normal activity to
dormancy represents only a minor change in body temperature,
but represents a profound change in metabolic state. As pre-
viously discussed, the transition from aestivation to hibernation

PO? BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

is unbroken. Therefore, for months at a time this species is a
facultative poikilotherm (or facultative homeotherm) and can
have the metabolic advantages of both poikilothermy and homeo-
thermy, as needed, to meet the demanding circumstances of its
desert environment.

Ecology and Sympatry. Citellus mohavensis occurs only in
the Mohave Desert of California, a region characterized by
extremely hot, rainless summers and mild winters with light
undependable precipitation. Although in winter nighttime tem-
peratures may fall below freezing, the days are almost invariably
mild and a diurnal rodent need never be exposed to prolonged
low temperatures. Seasonal dormaney in the Mohave ground
squirrel does not appear to be an adaptation for the avoidance
of low environmental temperatures, but appears to be an adap-
tation to seasonally restricted food and water.

The range of C. mohavensis lies completely within the distri-
bution of the Antelope ground squirrel, Citellus (Ammospermo-
philus) leucurus. The two species occur together in an extremely
simple desert plant association. Their maintenance of svmpatry
appears to be most readily explieable in terms of the marked
differences in their patterns of metabolism. C. lewcurus neither
aestivates nor hibernates but remains active above ground at all
times of the year; we have been unable to induce dormaney in
the laboratory. C. mohavensis stays underground and presum-
ably dormant except durine the most favorable part of the
year — spring and early summer. Thus, in the area of syvmpatry
during the more demanding and difficult parts of the vear — late
summer, fall, and early winter — only C. lewcurus is active. Con-
sequently, the two species compete for water (in the form of
insects and sueeculent vegetation) and food only when supplies
are maximal and presumably adequate for both. Thus, from
the point of view of energetics, during the more difficult parts
of the year only one species is present. It seems, therefore,
reasonable to postulate that between these two sympatric ground
squirrels competition, in the sense of utilization of a common
resource which is in short supply (Birch, 1957, p. 6), is minimal
and perhaps does not exist, except in very poor years, because
of the differences in the seasonal patterns of their metabolism.

Patterns of Seasonal Dormancy in Citellus. The genus Citellus
has a cireumpolar distribution. Ellerman and Morrison-Scott
(1951) recognize seven species in the Palaearctic: more than a
score occur in the Nearetie and several occur in the Neotropics
(Miller and Kellogg, 1955). Howell (1938) recognizes eight

1960 MAMMALIAN HIBERNATION 203

subgenera. To date no demonstration of seasonal dormancy is
available in the five species of the western North American sub-
genus Ammospermophilus, but hibernation, and less commonly
aestivation, occur in all of the other subgenera with the possible
exception of the neotropical subgenus Notocitellus. If one ex-
cludes Ammospermophilus, a general pattern for the occurrence
of seasonal dormancy in ground squirrels suggests itself: (1)
they hibernate where the winters are cold; (2) they aestivate
in regions of prolonged seasonal drought; (3) aestivation
merges into hibernation in northern arid regions where precipi-
tation is seasonal and restricted to winter and spring; and (4)
aestivation does not occur in areas of regular summer rainfall.
Viewed in this perspective the seasonal dormancy of ground
squirrels offers a primary physiological key to their abundance
and success in a variety of habitats ranging from the tundra to
subtropical deserts and covering over 50 degrees of latitude. The
fact that this dormancy can occur from near 0° to 30°C and,
therefore, is not closely dependent on environmental temperature
allows various species of this genus to avoid those periods of the
year during which drought, high temperature, low temperature,
or availability of food is locally limiting. Thus, a single physio-
logical capacity adapts this genus to the Arctic, to high moun-
tains, and subtropical deserts.

The capacity for seasonal dormancy is of no a prior? advantage
in tropical regions in which seasonal drought is not severe. It is
of imterest that this genus has had very limited success in
occupying the humid tropics.

Summary

The Mohave ground squirrel, which is confined to the Mohave
Desert of Califormia, is normally active above ground only during
spring and early summer. Under laboratory conditions it spon-
taneously enters torpor at room temperature and spends much
of the summer, fall, and winter in a dormant condition. During
periods of normal activity its standard metabolism averages
shehtly more than 0.8 ce 0o./gm/hr and its body temperature
averages slightly less than 36°C. The animals may become
torpid over a range of ambient temperatures extending at least
from 10 to 27°C. When entering torpor at ambient temperatures
between 22 and 26°C they assume the usual sleeping posture, and
their oxygen consumption declines rapidly and body temperature
approximates environmental temperature within three or four
hours. Thereafter, both oral and rectal temperatures vary

POF BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

directly with ambient temperature. During torpor oxygen econ-
sumption is less than 0.2 e¢/gm/hr.

Following the onset of arousal oxygen consumption increases
10 to 20-fold and usually peaks within 20 minutes. Body temper-
ature increases more slowly, and the levels of body temperature
characteristic of normal activity are usually attained in 45 to 60
minutes. Typieally, rectal and oral temperatures are within
0.5°C of each other during arousal. The behavioral capacities of
the animal imerease steadily as body temperature rises and
appear normal at 30°C,

Since aestivation and hibernation are physiologically similar
in those few mammals and birds in which they have been com-
pared, it is suggested that the two words be used only in a
natural history context and that another term, perhaps faeculta-
tive hypothermia, be used when dealing with the physiological
aspects of the phenomenon.

The ecological roles of seasonal dormancy in the genus Citellus
are surveyed and the role of dormaney in distribution and
sympatry is discussed.

REFERENCES

BARTHOLOMEW, G. A. AND T. J. CAbr
1957. Temperature regulation, hibernation, and acstivation in the little
pocket mouse, Perognathus longimembris. J. Mammiatl., 38:60
72.
BircwH, L. C.
1957. The meanings of competition. Amer, Nat., 9125-18.

HLLERMAN, J. R. anD T. C. S. MorRISON-ScotTr
1951. Cheeklist of Palaearetie and Indian mammals. London, 810 pp.

HoweEL.LL, A. H.
1938. Revision of the North American ground squirrels. U. 8. Dept.
Agric., N. Amer. Fauna, No. 56, 256 pp.

HoweE.u, T. R. AND G. A. BARTHOLOMEW
1959. Further observations on torpidity in the poor-will. Condor,
61:180-186.

IXALABUKHOV, N, I.
1956. Animal Dormancy. Kharkov, 268 pp. (Russian. )

LYMAN, C. P.
1948. The oxygen consumption and temperature regulation of hiber-
nating hamsters. J. Exp, Zool., 109:55-78.

1960 MAMMALIAN HIBERNATION 2O5

MILLER, G. S. AND R. IXKELLOGG
1955. List of recent North American mammals. U.S. Nat. Mus. Bull.,
205: XII+954 pp.

Popovic, V.
1957. La calorification du réveil de lhibernant. Areh. Sci. Physiol.,
11:29-35.

Popovic, V. AND P. PoPovic

1956. Sur les limites de température du sommeil hibernal. C. R. Soe.
Biol., 150:1489-1440.

DISCUSSION FOLLOWING BARTHOLOMEW’S PAPER

SOUTH ‘‘heartily endorsed’? BARTHOLOMEW’S point of
view except for one matter: he noted that in dealing with
clinicians there already is confusion in the use of the word
‘hibernation,’’ that he would anticipate still further difficulties
when scientists tried to make their interests understood by
chnicians if the phrase ‘‘facultative hypothermia’’ were used.
BARTHOLOMEW noted that, since temperature is the most
obvious parameter measured, the phrase ‘‘facultative hypo-
thermia’’ would be logical.

SOUTH agreed that BARTHOLOMEW’S approach made
sense. BARTHOLOMEW added further that the use of this
phrase would allow scientists to get away from many problems;
for mstance, it would permit seasonal falls in body temperature
change to be related to ‘‘hypothermia’’ and not to ‘‘hiberna-
tion;’’ it would give a convenient phrase for referring to condi-
tions such as partial or ‘‘shallow’’ hibernation and daily tor-
pidity.

FOLK asked BARTHOLOMEW to discuss the oxygen con-
stunption of a lizard, frog, and mammal of the same size at
22°C in terms of the ‘‘thermostat’’ concept. BARTHOLOMEW
noted that the word ‘‘thermostat’’ should be used as a symbol
for a complex physiological mechanism. ‘‘ Facultative hypo-
thermic’? mammals have oxygen constunptions at body tempera-
tures of 22°C which are not greatly different from those of
medium-sized lizards or bie frogs at that temperature. What
seems to be essential, BARTHOLOMEW observed, is as MORRI-
SON said — that such a mammal has reset its ‘‘thermostat.”’
This, of course, does not explain the process; it is possible that
muscle activity is completely, or almost completely, cut off and
that this relaxed muscle is simply not producing heat. This, he

206 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

said, was as far as he could go with this explanation. The
‘thermostat’? concept does not demand an elaborate reorienta-
tion of body chemistry or reinterpretation of what goes on in
the central nervous system.

PEARSON asked if it was not true that an ‘‘obseure physi-
ologist named LYMAN”’’ had shown that heart rate decreased
first, and that then the body temperature went down as an
animal went into hibernation. He noted that this does not. fit
into the ‘‘thermostat’’? concept. LYMAN disagreed by pointing
out that the heart could be ‘‘turned off’’ first. BARTHOLOMEW
noted that as long as the phenomenon is characterized by a word
like ‘‘thermostat’’ one does not have to say what the ‘‘thermo-
stat’’ is.

SMITH then pointed out that ‘‘setting’’ the thermostat at a
lower level was a valid proposition, but when one considered
the resting bat, the body temperature of which follows the en-
vironmental temperature, difficulty arose. BARTHOLOMEW
said that he did not feel the difficulty was great, but there was
“ift’’ in the system if the animal were not regulating its
temperature closely. MORRISON remarked that this was not
too easy for non-hibernators. BARTHOLOMEW. generalized
by saying that the ‘‘thermostat’’ concept allows one to relate
many phenomena to the same mechanism.

HOCK then indicated that he believed the ‘‘thermostat’’ con-
cept was a simplification of a mechanism about which we were
fairly ignorant, and it may very well be equally feasible to con-
sider that the animal is not ‘‘turnine off’’ anything whatever,
but rather ‘‘turning something on.’’ Turning off a thermostat
or ‘‘drifting down’? may be incorrect. Observed phenomena
might not represent passive abandonment, but rather an active
additional mechanism. This, of course, may vary with species.
Animals which move from one physiological state to another
generally do so in a positive way. BARTHOLOMEW remarked
that this may apply to animals that go to low temperatures.
The Mohave ground squirrel, on the other hand, showed virtually
normal movements at 32°, but could become dormant at 27°C.
He said he felt there is a passive drift once this condition
(dormaney) is reached. What happens below 10°C, he could
not say, but each genus and possibly each species has its own
particular pattern.

1960 MAMMALIAN HIBERNATION 207

PROSSER stated that all of these concepts of a thermostat
are as broad and perhaps more vague than ‘‘centrencephalic
control of autonomic function.’’ He indicated he would not sup-
pose all of this could occur in one small bit of the hypothalamus,
but rather in whole portions of the brain. BARTHOLOMEW
said that if one could find a ‘‘trigger’’ which set off the whole
mechanism, everyone would be happy.

WIMSATT asked if there were any cases in which body tem-
perature dropped below ambient temperature. BARTHOLO-
MEW said the only way this could be done would be through
evaporative water loss, and this would be metabolically expensive.

MORRISON stated that one cannot speak of a positive regula-
tion in the thirteen-lined ground squirrel in which the body
temperature follows the varying ambient temperature within
a degree. Obviously some animals such as the hedgehog do
regulate when the temperature becomes too low.

HOCK commented that this animal is not passively turning
off anything, but just resetting his thermostat. BARTHOLO-
MEW said that he agreed with MORRISON ; if an animal is not
regulating, it drifts to the ambient temperature. This idea of
allowing body temperature to drop to the environmental temper-
ature encompasses bats, hummingbirds, ete.

LANDAU asked why the heart does not slow before the
breathing rate. BARTHOLOMEW replied that this question
will be answered when one knows what is triggered, and what
is doing the triggering. MORRISON pointed out that where
heart rate slows first, it can be due to the faet that hibernation
may first be preceded by sleep, at which time the heart slows,
and that then the animal goes into the hibernation stage. It is
usually a requirement that sleep precede hibernation.

POPOVIC then noted he had done experiments which would
be in line with BARTHOLOMEW?’S observations on correla-
tions between hibernation and aestivation. He (POPOVIC) said
they kept ground squirrels in a Herter apparatus, and the ani-
mals hibernated when they could choose the temperature of
the room. The animal chose a 28°-30°C room to hibernate during
the winter. Also, the eolor-marked male and female e@round
squirrels in the field showed that the males disappeared first,
entering hibernation at the end of August, which is the hottest
month in Yugoslavia.

208 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

DAWE returned to the question of the ‘‘thermostat.’’ He
noted that if the hibernating ground squirrel is decapitated, and
the heart removed and placed in cold saline, it continues to beat.
If the heart is removed from an active animal (which normally
hibernates) in a non-hibernating ‘‘season,’’ it usually stops
quickly in cold saline. This difference in the tissue is not ex-
plainable on the basis of a simple reset of a thermostat. BAR-
THOLOMEW replhed that he thought the entire animal changed
truly enough, but the critical thing in the present discussion, it
seems, 1s to consider how he does get from one state to another,
and what adjustments he is making.

MORRISON concluded the discussion by stating that he agreed
entirely with BARTHOLOMEW’S concept of the identity of
the dormant states of hibernation and aestivation, but he felt
the term ‘‘facultative hypothermia,’’ although quite proper,
would be difficult to substitute for the older terms. He suggested
as an alternative that the terms ‘‘hibernation’’ and ‘‘aestiva-
tion’’ continue to be used, but with general recognition of their
physiological identity. In most cases, he said, the respective
word could describe the animal or circumstances and there would
be a negheible number of cases in which the choice would be
difficult.

XI
DAY-NIGHT RHYTHMS AND
HIBERNATION?

By G. Epaar Fouk, Jr.
Department of Physiology
State University of Iowa

Towa City, Iowa

Introduetion

Biologists have recognized for many years the presence of
24-hour rhythms of physiological activity or movements in
plants and animals. These rhythms appear to have originated
because of the day-night hght eycle. Many animals and plants
utilize the light cycle by accepting dawn or sunset as a clue to
keep their rhythms synchronized with solar time (Pittendrigh
and Bruce, 1957). When many plants and animals are placed
in continuous darkness, the biological rhythms persist, sometimes
on a 24-hour basis but also at times with a ‘‘period’’ which is a
few minutes (up to an hour) more or less than 24 hours. Many
investigators beheve that a physiological interval-timer, or in-
nate physiological periodicity of about 24 hours, is as much a
fundamental -characteristic of animals as certain oft-repeated
organ patterns such as nervous systems and brains, or circulatory
systems and hearts. A characteristic of these rhythms is their
temperature independence. Such a phenomenon demands the
attention of biologists, because any presumed biological clock
Which controls periodic recurrence of physiological functions
with the accurate timing observable in biological rhythms, must
depend for its mechanism upon the activity of enzymes. One
would predict that just as the activity of brain, heart and muscle
tissue is reduced at cold temperatures, likewise the biological
clock would run more slowly. The problem encompassed by the
present report is a search for marked persistent 24-hour rhythms
of mammals when their body temperatures are reduced to about
oC. Since there is admittedly little information on mammals,
let us first consider temperature independent rhythms as found
in plants, one-celled organisms, fiddler crabs, and lizards. An
illustration of a rhythm of growth in plant tissue is found in the
work of Ball et al. (1956) (Fig. 1). When seedlines are trans-

1This research was supported by the National Science Foundation.

210 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

ferred from red light to darkness they do not continue to
erow at a constant rate but show two or three 24-hour rhythms
of high and low growth rates. The amplitude of this rhythm
changes at a cold temperature, but the timing of the peak of
activity remains essentially unchanged. The second example is
seen In Huglena, as studied by Bruce and Pittendrigh (1956)

22°-23°C

O
On

24 36 48°

ee
LAS AE eee

S90 760 70 80 90 100 II0 120
Age of Seedlings (hours)
transferred

Growth (mm/hr)
oO

26°-28°C

1S” =17°C

30 60 70 80-90 100 110

Fig. 1. Mean growth rate of Avena coleoptiles in darkness. Seedlings

transferred from red light to darkness at the 50th or 56th hour from soak-
ing. Times of emergence of primary leaves are indicated by dots. Three
experiments demonstrating endogenous rhythms are shown. At the lower
temperature (15°-17°C) the amplitude of the rhythm is low, but the period
of the rhythm is the same. Diagrams reproduced from work summarized in
Ball, Dyke and Wilkins (1956).

1960 MAMMALIAN HIBERNATION 211

(Fig. 2). These organisms orient to h¢ht for about 12 hours and
fail to respond for about 12 hours, showing a 24-hour rhythm in
this respect. One might expect that when the environmental
temperature is changed from 33°C to 23°C the 24-hour rhythm
might become one of about 28 or 380 hours. However, there is no
change at 23°C and very little change at 17°C. Turning now
to the Crustacea, we find that a central hormonal mechanism
darkens the color of fiddler crabs for a period of about 12 hours,
so that the shell of these animals will more nearly match their

24
¢
518
ae
6
= 2
ro Line with slope
corresponding \
6 toa Qj), of 2

15 20 25 30 35
Temperature (°C)

Fig. 2. Temperature-independence in persistent rhythm of phototaxis 1
Euglena gracilis. From Bruce and Pittendrigh (1956).

surroundings in the daytime and will provide protection against
the sun’s rays. Brown and Webb (1948) showed that this cen-
tral mechanism is independent of temperature (Fig. 3). As usual
the amplitude of the response is lowered but the timing remains
approximately unchanged. Kayser (1952) has observed the same
phenomenon in the locomotor activity of the lizard, when either
oxygen consumption or total activity is recorded (Fig. 4). The
quantity of activity follows the law of Arrhenius, and shows a
reduction at low temperature, but the fime of the activity in
continuous darkness is unchanged by low temperature. When
the lizard must ‘‘meet a friend at the bank’’ at 12 0’clock on a
hot day he trots vigorously and briskly to arrive on time at the
destination. If the day is cold he sluggishly and laboriously lifts

212 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

one foot at a time, but arrives at the destination as usual at the
appointed hour.

When the mammal is considered, a special problem arises.
Hibernators and other mammals in hypothermia (including
man) experience a reduced metabolism and body temperature.

INDE X

CHROMATOPHORE

Be WN Me We FA To OU ae alt ae a) OU ea? al eal
AM PM AM PM AM PNM AM PM AM PM AM. PM. AM

TIME

Fig. 3. Daily variation in the average indices of black chromatophores in
the fiddler crab, Uca, kept in constant darkness at 26°C (A), 16°C (B), and
6-C (CC). The temperature independence of the rhythm of color change is
apparent. From Brown and Webb (1948).

When body temperatures are normal some of the most accurate
and regular of biological rhythms are found in mammals. Com-
pare the running wheel of thirteen-lined ground squirrels (day-
active), and rats and hamsters in Figure 5 remembering that two
of these rodents are hibernators. Mammals and birds must re-
quire a narrow range of variation in their blood temperature,
evidenced by the fine and sensitive control found in their physio-
logical thermostat (the hypothalamus). Perhaps as a result of

1960 MAMMALIAN HIBERNATION 213

this requirement, the biological clock of the mammal cannot fune
tion when it is cold. On the other hand, there may be the same
mechanism for temperature compensation in the mammal that
has just been illustrated in plants, one-celled organisms and
other eectothermie animals. Some experimental data will be
presented in support of the seeond of these two alternatives.

Time 4:30pm. 12 noon 12:30p.m. | l2 noon 12 noon
Day / 2 I 4 5

Time 12:30pm. 12:'30p.m. 12:30p.m 2:30p.m
Day / 2 3 4

Fig. 4. Day-night rhythm of activity of the lizard. From Kayser (1952)

Methods

Studies on ground squirrels. A colony of ground squirrels
(Citellus tridecemlineatus) was maintained for three years with
intermittent periods of 8 months at a warm temperature and
f months of hibernation in the cold. The detailed treatment
of the 11 experimental ground squirrels and the 14 control
ground squirrels (no hibernation) has been presented earlier
(Folk, 1957). During the periods of hibernation each winter,
observations were made twice a day. The animals were marked
with oats or sawdust which fell off or was cleaned off when the
animals awoke from hibernation. Actographs were not con-
sidered appropriate here because animals have been known to
remain awake but unmovine for reasonably long periods even

214 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

in darkness. In another series of experiments, body temperatures
were taken by hand at selected times of the day using a ther-
mistor and a telethermometer (Yellow Springs Instrument Co.,
Yellow Springs, Ohio). Skin temperatures were measured with
calibrated copper-constantan thermocouples. Heart rates were
measured with a Burdick electrocardiograph. or 24-hour meas-
urements the instrument was turned on automatically every half
hour for two minutes.

Up - stroke or Down - stroke of pen=/00revolutions
of running wheel

LIGHT DARKNESS LIGHT DARKNESS

Ground Squirrel | | C2 miles
es ey
Hamster 8.1

tI aoe

— UN te

_—_—_—_—_—_______ oo
6am 6pm 6a.m 6pm 6a

Kig. 5. 48-hour records of spontaneous running of three species of rodents.
The times of starting of activity are very regular. The ground squirrel is
day-active but continues to run after the dark period begins. The hamster
is nocturnal but usually is through running by 3 a.m. Activity was measured
with Welsh recorders.

The 1957 series of squirrels was sacrificed to obtain physiologi-
cal and anatomical data (11 hibernators, 14 controls). The 1958
series (heart-rate series) consisted of five ground squirrels
studied in a two-door refrigerator rather than in a cold room.
The test chamber was in a water bath in the refrigerator, which
was Uluminated from 9 a.m. to 9 p.m. (2 foot candles). During
four months of cold exposure, one animal did not hibernate;
of the others, three were light hibernators with short periods
of hibernation, and the remaining animal was a deep hibernator

1960 MAMMALIAN HIBERNATION

215

with extremely long bouts of hibernation lasting weeks at a time
(Fig. 6).

Studies on bats. A series of studies was done on 16 hibernating
bats (Hptesicus fuscus), since they have been shown to have at
normal temperatures a 24-hour rhythm of activity, both with
a light evele and in continuous darkness.

Actographs are not
always satisfactory, and so a stimulus test and index, described

e--e HEART RATE
7 of o— ABDOMINAL BODY TEMP.

e x 6°
el2t \ : :
= A. Deep Hibernation
Sa "GC
se \
a Bt \
5 \ @ ------- : e 3°
* 4 Animal ‘poccces’ 0 cso ---- 00

moved FO 2
] fo Chamber
O TOT

B. Light Hibernation
Chamber- 5.1% O.1°C

8 1012 2 4 6 8

lo 12 2 4 6 8
Noon

lola 24 6 a
Midnight Noon
Fig. 6. Heart rate and temperature of ground squirrels in light and
deep hibernation. The deep-hibernator (A) usually had a heart rate of
4/minute (4.5°C), and the light-hibernator (B) had a rate of 7/minute
(5.0°C). Hibernation before these readings for animal A was 13 days,
after these readings, 8 days; hibernation before readings for animal B was
3 days, after readings, 2 days.

earlier, were used again (Folk, 1957). The method involved a
stimulus consisting of a standardized, mechanical puff of air at
6 inches. This was applied daily or hourly to test the hibernating
condition of each bat. Series A consisted of giving the stimulus
every 25 hours so that a 24-hour rhythm would not be estab-
lished; Series B consisted of testing each member of the colony
every 2 hours over a 24-hour period (both series new, 1959). The
animals, each in a separate cage, were given the stimuli quickly

216 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

when the refrigerator door was opened briefly. Toenails and
hibernating position were marked with yellow paint. Each animal
also received the stimulus of the weak light of a flashlight used
for observational purposes. The animals were maintained in
continuous darkness in hibernation for three winter months.

Results

Regularity in periodic awakening of ground squirrels from
hibernation. Most of the awakenines during hibernation, in the
second and third winter, of 11 hibernating ground squirrels
occurred during the simulated daylight. This pattern of awaken-
ing was more marked during the second winter (72 per cent of
the time during the periods corresponding to the original light
eyele). The awakening pattern was also evident during the
third winter of hibernation, in observations on the same squir-
rels. The animals were illuminated in the cold room daily from
9 am. to 9 pan. and only at these times were observations made.

TABLE I.
Effect of Three Winters of Hibernation
on 13-Lined Ground Squirrels.

ma
Females J and ¢
cova covet] wae, [come] wae
Weight (gms.)
Body Temperature (°C) 36.4° | 37.1° | 36.3°| 36.6"
O2 Consumption (mi/am//nr)| 0.769 | 1.027 | 0.980 | 1.535 | 0.874
Spleen (qms.) ‘
Heart (gms.) , ‘ | .776 | 876 |
Testis (gms.)
Hematocrit (%) Pp -- | -- |
Lipemia Index [ee ee
aged rea" |

Coagulation Time (°%)

Body temperatures and O»2 consumption were measured at 30°C. Note the
loss in weight of spleens and gain of hearts and kidneys. Other studies have
shown that this effect on the last two organs is a true hypertrophy.

1960 MAMMALIAN HIBERNATION 21'

At the end of the winter the animals were taken from hiberna-
tion, permitted to warm for six hours and sacrificed. <A series
of measurements was compared with those of controls (Table 1).
All eleven animals had hibernated intermittently for the four
months, awakening after intervals of dormancy lasting from
one to 26 days; 164 periods of dormaney were observed (Table
Il). Again, for the second season, more squirrels (63 per cent)
awoke from the dormant condition when the light was on (Table
IIl). If the hypothesis is tested that hght or some hght assoei-
ated phenomenon caused the awakenings in heht, then they are

TABLE II

Hibernating Data for 13-Lined Ground Squirrels
During the Third Winter. N= 11. Ambient: 6 £1°C

Range of period

_, rotal, Total. length in days
hibernating hibernating - - —— -— -
days periods (bouts) minimum maximum
October 218 51 1.0 17.0
November 297 37 1.0 22.5
December B38 By 3.5 26.0
2.0 22s5

January 294 35**

* This maximum hibernating period is tbe longest recorded in three years of

study on these squirrels.
** There were 9 additional periods in February.

significantly different from the awakenings in darkness. One
individual always awoke during the illuminated period, while a
few awoke most of the time in darkness. Two interpretations
of this regular awakening would be: (1) that the animals, in
spite of a reduced metabolism and body temperature, still re-
ceived some stimuli from the external environment which could
act as clues to cause the periodic awakening; or (2) that some
24-hour rhythm was still expressed in the animals in such a way
as to influence their awakening from hibernation. One might
postulate ight and deep hibernation corresponding to the regu-
lar periods of sleep and wakefulness which occurred before
hibernation. If this were so then the usual or typical stimuli
which cause awakening from hibernation such as hunger or a
full bladder, might be more apt to take effect during the period
of light hibernation. The experiments to determine whether
the regular awakenine was due to external stimuli will be
described first. It appeared unlikely that any artificial light

218 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

could penetrate to the eves of a hibernating ground squirrel
sinee the head was invariably buried beneath the body with the
eyelids closed. In addition, there is no evidence that the optic
nerve can conduet when maintained at a temperature near
»°C. It was considered most profitable to first design experi-
ments on hibernating ground squirrels with ‘‘white noise’’ as a
stimulus.” Four series of tests were tried with three hibernating

Tape III

Distribution of Awakenings of Dormant 13-Lined Ground
Squirrels During a 4-Month Hibernating Period.

DARKNESS LIGHT ot )
12 hours ae Statistical Analysis*®

5 8 rID=35
[| 8 D=+3.18

4 t XD*= 413

3 9 (3D) = 111.364
5 lO ¥d2= 301.636
Sp = 27.42!
6 14 Sp = 5.24
4 lO G= 1.66

D
- lO 3.18.) go

ee mea

| |.66—

4 t 50, (df=I0) = 1.8

96, (One-ended Test)
a7

* Courtesy Prof. P Blommers, State University of lowa.

Animal

“

-OWO ON OO HhAND —
oO

3
me
6|

Note that one animal always awoke during the illuminated period. Long
and short bouts of hibernation were found with most animals, and length

of dormancy was unrelated to time of awakening.

2 These experiments were made possible by the generous assistance of two staff
members in the Department of Otolaryngology: Dr. J. Tonndorf designed the
experiment and loaned the equipment; Dr. R. Voots engineered the electrical
circuits.

1960 MAMMALIAN HIBERNATION 219

ground squirrels (Table 1V). The experiment was planned to
produce repeatedly a loud noise such as to cause a ‘‘startle re-
sponse’’ in non-hibernating ground squirrels. In each series all
animals had hibernated for one day or longer, before the noise
was applied in the refrigerator. In the first experiment, two
animals were not hibernating. They were observed through a

TABLE IV

Experiments Applying White Noise as a Stimulus to
Hibernating Ground Squirrels. N =:

Length of hibernation

. Stimulus after ‘white noise”
Experiment characteristics Duration Animal #
1 90 db. 140 mins. Seer over 24 hrs.
2 sec. on (2.3 hrs.) 2..... over 24 hrs.

2 see. off

2 90 db. GO mins. Tt paps over 24 hrs.

2 see. on (1 hr.) 2...... over 24 hrs.

2 see. off 3......over 24 hrs,

3 90 db. 240 mins. 1......over 24 hrs.

2 sec. on (4 hrs.) 2 over 24 hrs.

2 sec. off 3 _.. Awoke 2 hrs. after
noise stopped. (This
response within
control awakening times,
1-3 days.)

4 97 db. 320 mins. | over 24 hrs.
5.0 Sec. on (5.3 hrs.) 2 over 24 hrs.
9.0 see. off 3 _Awoke 20 minutes

after noise started.
Had hibernated unusually
long (4 days).

thermopane in the door. The expected startle response was easily
observed, demonstrated by a characteristic head jerking when
the noise first came on which was not continued throughout the
applieation of the noise. The other two animals did not awaken
from hibernation. In the second and third experiments, the
hibernators appeared to be uninfluenced by the sound. During
the third experiment heart rates were measured on one animal

220 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

(Fig. 7). The heart rate did not change from control values.
In the last experiment the intensity of the sound was inereased
from 90 db to 97 db. Although one animal awoke 30 minutes
after the noise began (respirations 18/minute), it is impossible
to know whether this awakening was spontaneous or not. This
animal had been hibernating an unusually long time. The other
two animals were undisturbed by the noise. The conelusion is
that these ground squirrels are seldom, if ever, awakened from

"WHITE NOISE" applied:
db=90
Cycle = 2sec.ON-—2 sec.OFF
Genre

22
i Be,
21 Po ts ie

cy /
: |
i ‘
g 204—Heart Rate H /
n” 1 ,
2 ymin i a
$19 ! ® -Mean of 3readings
& ! ° -Meanof 2 readings
= 1
~ 18 eet
fo) ane ia
wy a an |
a or ae
5 174 ii
a 1 Vv
= é é
16) Heart Rate
4/min
vw
15 VW AAW Ae oe VV a oe oe FAIA
ee ee
456 12 6 78 9 10 ll 12 | 6 7 8 9 10 ll 12 6 7
Midnight Noon Midnight
Fig. 7. Study of hibernating ground squirrel exposed to light and ‘‘ white
noise.’?’ The heart rate during the application of noise stayed within
Later, over 24 hours, the beat gradually became slower.

control values.
There was no evidence of any effect of noise upon the hibernating animal.

hibernation by noise. It could hardly be a factor in influencing

the observed regular awakening from hibernation.
Experiments on variation in depth of hibernation. Heart rates
on the ground squirrel which showed deep hibernation were re-
peatedly measured over 24-hour periods (1958 series). There
was slight evidence of any change in heart rate during day and
night (Fie. 8). Kew measurements could be obtained on the
hight-hibernators since they usually awakened when electrodes
were attached to the chronicaliv implanted surgical clips. When
measurements were made on these animals, hibernating heart

1960 MAMMALIAN HIBERNATION 221

rates were higher than those of the single animal in deep hiberna-
tion. One series indicated a heart rate level around seven per
minute with a skin temperature of 5°C for the light-hibernator,
and a heart rate level of 4 per minute and a skin temperature of
4.5°C for the deep hibernator (Fig. 6). Since it was possible
that there might be a difference in the heart rate interval in day
and night, this was studied on one animal in deep hibernation.
The evidence indicates that there are frequent occurrences of

beats /min.

6. 8 JO-I2 <2 4-6-8 0 I2 2 456.8
Noon Midnight

Fig. 8 Heart rates of hibernating ground squirrel sampled each half hour
for three consecutive days. There was no evidence of a persistent 24-hour
rhythm of heart rate, nor of any similar rhythm of approximately 23 or 25
hours. The entire 24-hour day was scanned, but in some cases instrument

failure prevented 3 repeat readings.

regular and irregular heart beats distributed equally over day
and night (Fie. 9). There was lttle evidence from the overall
series of experiments for regular variations in depth of hiberna-
tion. Only in one experiment was there any clue at all; in this
case body temperature readines showed spontaneous partial re-
warming. Temperatures were taken day and night for six days,
although no heart-rate measurements could be made at this
time (Fig. 10). On the 7th day there was a partial temporary
rewarming, following which the animal returned to deep hiberna-
tion. The fact that spontaneous rewarming may have begun in
the light is not important in a single occurrence of this sort.

222 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

6
° 3 beats/min

2
oO
200 al | {8 I " '
Cc
[e)
eb)
3
ce)
3 2}
E 40
4 beats/min.
30

St tr St a
6 7 tl2 + 23 4 5 6 7 4 l2 1-2-3) 4°35 6
Noon Midnight

Fig. 9. Consecutive heart beat intervals of ground squirrel in hibernation.
This analysis was made to reveal a 24-hour rhythm in heart beat regularity,
There was no apparent difference between day and night readings. Like-
wise, the mean heart rate was unvarying over 24 hours.

l15 im ] }
/ Chamber-Animal /
1 Fi a 107 Relationship 2
3 104 / 94 During Rewarming ,’
° / /
— | / 84 /
(Tp) / /
a 94 Ff 7] ef
a. 47 6 /
re 8 4 / 5 1 Pi sere
= J _--””” CHAMBER
a | 4) bee AR TEMP.
, 74 ee ee ee
2 bare 12M 6 12N 6 12M
az 64
=
=> 4
oO
am 7
SHS ae SR ee
V+WV1
44
a nn I 01) Sa Pe
6 8 lOl2°2 4 °°56°8° 10 12. 2 4-6 8 lO 12) 2 $46.48) 1012
Noon Midnight Noon Midnight

Fig. 10. Spontaneous partial rewarming (Day VII) from hibernation.
This animal was studied for eight consecutive days, using skin temperature
as un index to reveal day-night differences. For five days the temperature
index showed a constant level of hibernation. There was one partial re-
warming, during which the animal did not awaken from hibernation. Visual
observations were made prior to temperature records on Day VII. No heart

rates were taken,

1960 MAMMALIAN HIBERNATION 223

Kxeperiments on bats. Regular observations on 16 hibernating
bats (1959 series) showed no particular time of the day when
these animals arose from dormancy to drink or become active. As
an earher experiment showed (1957 series), the dominant effect
was one of constancy. Some individual animals remained dormant
for periods of days (Figs. 11, 12). Three did not even move their

RECORDINGS -25 hr. intervals
AMBIENT - 5.2°+ 41°C

5A ACTIVITY INDEX

—NMWD

{
2
3

Animal Number
ito)

16 | ws _ - =|
Tolls helhleled, tate pae

f SOM: We 2S Si TOS aNPe i Se Se eh Bellic’

am pm am

Fig. 11. Activity index of hibernating bats (Hptesicus). Recordings were
made at 25-hour intervals. An animal with an index of ‘‘1’’ does not move
when stimulated; with an index of ‘‘5’’ it is moving about the cage. An
inherent rhythm of about 22 to 26 hours would have been apparent. Some
evidence for such a trend was present, but the higher readings after 15

days could also have been due to lateness of the season.

feet for at least 39 days. Other individual animals remained
semi-dormant for the total period of time and appeared to come
down from their hibernating positions at irregular intervals.

224 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Over any selected 24-hour period for each individual animal
there was no obvious tendency for the animals to be in lighter
hibernation for a portion of this period. The colony as a whole
appeared to be in hghter hibernation when readings were made

RECORDINGS-2 hr. intervals
AMBIENT -5.2°#1°C
5 ACTIVITY INDEX
4
13
2
a re ee ao eee
re
ee ee
ce ee
Cc ee a
7 =,
S25 RA RSS

Animal Number
=dowo

. OOM less Ve oes SS aes Sill
a.m p.m a.m

Fig. 12. Activity index of libernating bats (Lptesicus). Recordings were
made at 2-hour intervals, after the long series (25-hour intervals) was
completed. As in the other experiment, there is some evidence for increased

activity in the evening (see Table V

at 4 a.m. (mean index 3.00) (5 p.m., index 1.85), as if a number
of individual rhythms of activity had come into phase during
the night period (Table V). If this were so, it would be a per-
sistent rhythm, since there was no heht evele in this experiment.
Althoueh it can be said that there is little evidence of a clock
mechanism in bat activity at a reduced body temperature, at a

1960 MAMMALIAN HIBERNA'TION 225

warmer temperature this phenomenon must be important. Hock
(1951) has shown that the bat is the only mammal in which body
temperature is a direct function of environmental temperature in
the resting animal. It will be noted from the actograms of
Griffin and Welsh (1937) (Fig. 13) that for about half of the
day, with either the hght eyele or in darkness, the animal is
inactive. We have shown in our laboratory that if the animal
is maintained at 19°C its esophageal temperature during activity
may be 29.1°C, but when at rest its temperature drops to 20.5°C.

TABLE V

Day-Night Differences in Mean Colony Dormancy Index
of Hibernating Bats (lM ptesicus). N = 16

Me i as F
ee a Time Mean index p Value
Exp. 1 5 p.m. 1.81
Every 25 hours : 1%
4 a.m. 3.00

for 29 days

Kap. 2

: = er
Every 2 hours 0 p.m. 2.506
for 28 hours. > 5%
vertormed ere
a : 9 p.m. 3.06

after Exp. 1)
Thus, during its resting period any hypothetical biological clock
was perfused by blood at a temperature 8.6 degrees lower than
that found in the active bat.’ Apparently from the regularity of
the actograms of Griffin and Welsh, the bat behaves in the same
way as Kayser’s lizards (1952). The clock corrects for the drop
of 9°C.

Other observations of mammalian physiological activity inde-
pendent of temperature. The case for the mammalian biological
clock at low body temperatures receives supporting evidence
from other experiments. If we ask whether the performance of
some mammalian nerves remains unchanged at different tempera-
tures, we can cite the experiments of Kahana ef al. (1950), who
found that the neural component of the cochlea potential of the

3’The drop in body temperature of the bat mentioned above must influence
many experiments. In the experiments on bat rabies being conducted in_ the
U.S). it is not commonly recognized that the rabies virus is not being maintained
by the animal at a constant temperature of approximately 38°C. Instead, for half
of the day the virus is “incubated” at whatever the animal-room temperature
happens to be. Perhaps this phenomenon helps to explain why some individual
bats are symptomless transmitters of the rabies virus.

226 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

hamster in the range 39°C to 25°C showed no change in its
amplitude. More evidence for the temperature independent
biological clock was also contributed by Rawson (personal com-
munication) who showed that when activity records were made
for bats and mice, and then the animals were kept under anes-
thesia and a reduced body temperature for several hours and
returned to the activity recorder, their activity occurred at the
appropriate time as if body functions had not been reduced or
altered by the period of anesthesia. Finally, Strumwasser
(1959) found that Citellus beecheyi ‘‘ measured preferred times’’
for arousal from hibernation at body temperatures lower than
23°C,

l2 4 8 12 4 8 12
Noon Midnight Noon

Fig. 13. Periods of activity of Myotis l. lucifugus in normal cyele and in
continuous darkness, to illustrate a 24-hour rhythm of activity of bats which
persists in darkness. After activity the body temperature of this animal
probably decreased. Its biological clock would have to be temperature com-
pensated. From Griffin and Welsh (1937).

Discussion

There is a regular pattern of awakening from hibernation in
ground squirrels, but there is no clue in the study of heart rates
as to a persistent regularity in physiological function during
deep hibernation. The body temperature under these circum-
stanees (5°C) may be too low (there are no other animal rhythms
at this temperature). Either the changes in approximately 12-
hour units during hibernation are not reflected in heart beat or
else there is a relatively steady state in all functions. At the
moment, the most plausible theory for the regular awakening of

1960 MAMMALIAN HIBERNATION 227

ground squirrels from hibernation appears to be as follows: the
animals start to come out of dormancy; if they are in a period
of darkness, then progress is slow but if they have a dim light
shining on the cage, then the awakening from dormancy is more
rapid. Some of the slow-awakeners will delay their progress
until the Hight period. Few of those in the hight will postpone
their awakening until the dark period occurs.

The regular day-night activity of cooled lzards and bats still
must be explamed. It is apparent that animals other than
mammals correct for the effects of cooling, and some cooled mam-
mals show normal activity of nerve tissue within at least the
upper part of the body temperature range. There must be en-
zymatie adjustments which permit this cold compensation, Some
theoretical considerations associated with this hypothetical en-
zyimatie adjustment are now presented. Attempts to explain the
temperature independence of biological rhythms cannot be sep-
arated from the search for the nature and location of the biologi-
eal clock. This ‘‘strueture’’ can be visualized as either a localized
area or a diffuse collection involving many types of tissues. It is
of interest that lack of agreement has occurred previously con-
cerning the nature of the causes initiating some specific physio-
logical processes. A localized origin was postulated by Cannon
(1918) to explain thirst in mammals and by Lorand (1912) to
explain the aging process in vertebrates. Others now believe in
diffuse sources to explain both of these phenomena. In _ the
present case of the biological clock we must look not only for this
clock but for a ‘‘strueture’’ which by definition also has a very
unique biological characteristic, that of temperature independ-
ence. This characteristic is so unusual that our credulity is less
tried by supposing a discrete localized clock which has a special
mechanism to permit performance at low temperatures. If we
assume the alternative (a diffuse series of clocks in each animal )
there must be a broader distribution than we have supposed of
the rare biological material which can display temperature inde-
pendence. The clock-identification is complicated, as usual, by
the fact that one-celled animals have temperature-independent
endogenous rhythms.

Some scanty evidence exists of two types of physiological
mechanisms which increase their activity as temperature is
lowered, including (1) enzymatie activity, and (2) action poten-
tials of brain tissue (EEG). There is not direct evidence of
enzymes which increase their activity as temperature is lowered,

228 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

but Precht (1958) has shown that some oxidative enzymes of
fish show a decrease in activity after acclimatization to a raised
temperature. Presumably these enzymes also show the opposite :
an increase in enzymatic activity when temperature is lowered.
A clue to this behavior is given by Swartz ef al. (1956) who
studied enzymes which were activated by heat. This occurred
due to the fortuitous coincidence of a heat-stable enzyme and a
heat-labile inhibitor. In a similar fashion a temperature-inde-
pendent biological clock needs only to possess a collection of cold-
stable enzymes and cold-labile inhibitors which release more of
the enzymes as the temperature is lowered. Bunning and Lein-
weber (1956) provide a shghtly different theory: ‘‘It is by no
means impossible to imagine a chemical mechanism that also
has no temperature dependence. If part of the mechanism were
concerned with supplying a particular substance while a second
process destroyed it and both processes were equally temperature
dependent, then the substance would accumulate at the same rate
irrespective of temperature.’’ Perhaps such enzymes are in-
volved in the observation of Suda ef al. (1956) who showed
that the EEG of the cat shows an imecrease in amplitude as tem-
perature is lowered from 37°C to 24°C.

Other widely different approaches to explaining temperature
independence remain to be discussed. The first is described as
part of Pittendrigh and Bruce’s (1957) oscillation model of the
hasi¢ biological rhythm in any animal. They refer to this as an
endogenous self-sustaining oscillation (ESSO), but believe that
the control or clock presiding over this oscillation is a complex
system with constituent oscillatory processes. The control system
is not a single temperature insensitive process. Then they ex-
plain, ‘The mutual entrainment (svnehronization) of constituent
oscillators would result in temperature independence over a
limited range provided that key members of the system had re-
ciprocal temperature coefficients.’ They note further that tem-
perature has been used to replace lght for reversing rhythms
by 12 hours. If this is so, it is difficult to conceive of a process
(the clock) which responds to temperature but is also insensitive
to temperature. Pittendrigh and Bruce’s argument supports
their description of the clock as a system of compensating proe-
esses (some of which respond to temperature, while others being
resistant to temperature, can restore svnehronization ).

The second and final approach to explaining the temperature
independence of persisting rhythms concerns the possible re-
sponse by cold animals to subtle environmental clues. As Brown

1960 MAMMALIAN HIBERNATION 229

(1957) expresses it: ‘*The main difficulty is to explain how a
metabohe clock ean maintain such uneanny precision over a
temperature range of more than 20 degrees C. An alternative
hypothesis which fits all the known faets equally well is that the
mechanism is one which ean perceive some kind of physical force
in the environment hitherto not known to affect living orean-
isms.” If any physical time-giver other than light affeets the
cold animal in darkness, there is no reason to suppose it cannot
respond to this, since the cold animal still responds to a heht
eyele. (The rhythm of a cold animal can be reversed by 12
hours of hght.) One particular merit to the hypothesis of Brown
is that it does not require that any tissue or tissues of the body
behave biochemically in an atypical fashion.

To summarize: we have been considering what mechanisms
could be responsible for the regular rhythm of aetivity im ani-
mals lke the lizard and bat in darkness, when their blood is
cool or cold. One theory includes a chemical mechanism without
temperature-dependence, another depends upon a system of con-
stituent oscillators, and the third refers to the possibility of
subtle environmental clues. This theorizing at the moment should
be applied only to bats and probably ground squirrels at moder-
ate temperatures (15-18°C) where they both show a drop in body
temperature. Although ground squirrels may awaken from deep
hibernation as if influenced by a 24-hour rhythm, no physiological
evidence of this rhythm was found with ground squirrels 17
deep hibernation; likewise, little evidence was found in hiber-
nating bats. Future experiments should be done with hiberna-
tion at a warmer temperature (10°C) and without a light evele in
the cold chamber. The regularity of awakening from deep hiber-
nation by ground squirrels is best explained as due to possible
speeding of the awakening process by the environmental clue of
light. An alternative theory is less plausible until some evidence
is obtained of a physiological rhythm in deep hibernation.

Acknowledgments

The author eratefully acknowledges the illustrations and edi-
torial assistance provided by Mary A. Folk. Technical assistance
was provided by James M. Lipp, M.A.

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1952. Le rythme nycthéméral des movements d’énergie, Rev, Scient.,
3:173-188.

LORAND, A.
1912. Old Age Deferred. Philadelphia, 486 pp,

PITTENDRIGH, C. S. AND G. V. BRUCE

1957. An pon model for biological clocks. Jn: Rhythmic and

synthetic processes in growth. Princeton, Pp. 75-109.
PrecHT, H.

1958. Concepts of the temperature adaptation of unchanging reaction
systems of cold-blooded animals. Jn: Physiological Adaptation.
Washington, 185 pp. (Pp. 50-78.)

STRUMWASSER, F.

1959. Faetors in the pattern, timing and predictability of hiberna-
tion in the squirrel, Citellus beecheyi. Am. J. Physiol., 196:8-14.

1960 MAMMALIAN HIBERNATION 93]

BROOKS

Supa, I., K. KoizumMi Anp C. M.
Fed.

1956. Effeets of cooling on central nervous system responses

Proc., 15:182.

Swartz, M. N., N. O. KAPLAN AND M. E. FRecH
1956. Significance of ‘‘heat-activated’’ enzymes.

Science, 123:50-53.

‘a Base line~9 8°C
6pm '

2 6am 12 noon
Continuous record of the rectal temperature of a female Myotis lucifugus in

hibernation at 910 degrees C. Successive days are plotted under each other

Fig. 14

DISCUSSION FOLLOWING FOLK’S PAPER

MENAKER showed a slide (Fig. 14) indicating that rhythmic
fluctuations in body temperature persist in the little brown bat,

232 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Myotis lucifugus, down to body temperatures of 10°C in constant
darkness without awakening of the animal. He eited this as evi-
dence that the **clock’’ of bats continues to funetion at this low
temperature,

SOUTH indicated that in studyine ‘‘cloeck’’ mechanisms in
rats almost any cue would reset a ‘‘biological cloek,’’ for ex-
ample, a hole in a chamber admitting a small amount of heht.
FOLK answered by stating that it is his belief that an animal
hibernating or in an essentially stable environment is in a sense
‘“hunery for cues’’ to assist its own ‘* clock’? mechanism. SOUTH
asked if a 25144 hour cue could be used to ‘‘confuse the eloek.’’
FOLK replhed that Pittendrigh reports that a range of cues
viven between 22-27 hours could be followed, but outside of
this range the animal would resist resetting its ‘‘cloeck’’ meeh-
anism (C. 8S. Pittendrigh in Symposium on Perspectives im
Marine Biology, Berkeley, 1957, p. 255).

XI]
BROWN FAT AND ITS POSSIBLE
SIGNIFICANCE FOR HIBERNATION

By Brenatr JOHANSSON
Cardiological Laboratory, Department of Medicine
Malmo General Hospital
Malmo, Sweden

Generally, the term ‘‘fat’’ is used to designate the yellowish
or white fat occurring mainly in the subeutaneous fat depots.
There is, however, another type of fat; one of its names is
‘brown fat.’’ It is said to have been described first by Velsch
(see Auerbach, 1901-02) in 1670 or perhaps by Gressner (see
Bole et al., 1951) as early as 1551. In spite of this, it took many
years before brown fat was generally considered as a separate
entity ; it was often mistaken for thymus tissue. At the turn of the
century, however, excellent gross and microscopic descriptions
of brown fat appeared such as those by Hammar (1895) and
Auerbach (1901-02). Later more detailed studies, especially on
metabolic aspects and physiological function, have appeared. It
is the aim of this paper to give a short summary of our knowledge
about brown fat and to discuss, especially, two things:

(1) Are the differences between brown and white fatty tissue
anatomically and functionally so great as to allow a definite
separation between these two tissues ?

(2) How valid is the evidence of a connection between brown
fat and natural hibernation in mammals, evidences which have
created such synonyms for brown fat as ‘‘hibernating gland,’’
‘*masse hibernale,’’ and ‘‘ Winterschlafdruse’” or ‘‘ Winterschlat-
organ’’?

Gross and Microscopic Appearance

In the animals investigated, including both hibernators and
non-hibernators (Rasmussen, 1923; Wegener, 1951), brown fat
has been found in essentially the same places in all species.

Hammar (1895) has given a detailed report of the appearance
of brown fat in rats. In the thorax, the brown fat is situated
as a strip on the ventral part of the spine surrounding the de-
scending part of the aorta. This dorsal mass proceeds ventrally
through the mediastinum towards the dorsal side of the sternum
where it forms two strips with small extensions on the costal

934 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

cartilages. The brown fat which is situated on the vertebrae
continues into the abdomen, always surrounding the aorta and
the inferior vena cava. At the aortic bifurcation one part follows
the external iliae vessels ending just above the inguinal lga-
ment; another smaller part may follow the internal iliac vessels.
Sometimes a small amount is found in the groin. Brown fat is
most abundant in the back, especially interscapularly, extending
between the muscles ventrally and laterally. The axillae can give
accommodation for rather large amounts of brown fat which is
also found in the ventral part of the neck — here, however, to a
much lesser extent. Thus the brown fat is mostly situated in
the inner part of the body, while most of the white fat is situated
more peripherally (Feyrter, 1945-48).

The microscopical picture of brown fat differs principally
from that of white fat in havine’ smaller cells with a round
nucleus, and multiple fat droplets within the cell in contrast
to the bigeer white fat cell with a flattened peripheral nucleus
and a single big fat droplet. Furthermore, the evtoplasm in
brown fat is much more abundant than in white fat. Lachanee
(1953) points out that brown fat contains 15 per cent non-fatty
dry material and 46 per cent water. Corresponding figures in
white fat are 1 and 11 per cent. The lipids, however, are more
abundant in white fat — 8&8 per cent, against 39 per cent found
in brown fat. Schierer’s (1956) value for non-fatty dry ma-
terial in white fatty tissue in the hamster is somewhat higher,
3.3 per cent, while the value found in brown fat, 14.9 per cent,
is very close to that reported by Lachance (1953).

In the rat, a non-hibernator, the brown fat is arranged in
lobules and these are separated by connective tissue, into which
the blood vessels extend (Hammar, 1895). The brown fat cells
are polygonal and contain many fat droplets all of about the
same size. The cytoplasm is rather coarsely granular. One,
sometimes two, nuclei are found, most often situated somewhat
eccentricaliy but not as far toward the periphery as in white
fat. The vascular supply is rich. The black and pale grey color
after injection of a black dye into brown and white fat, respec-
tively, indicates that the blood supply is more ample in the
brown than in the white fatty tissue.

In extreme emaciation of the rat, the brown fat has a dark
reddish-brown color. The vascular content is abundant with
dilated vessels. In some cells a few small fat droplets remain.
On the other hand, in fattened rats the color is pale whitish-
brown but still stands out distinctly against the white fat. The

1960 MAMMALIAN HIBERNATION 235

fat droplets are bigger but there is no essential difference from
the picture found in a medium nourished animal. In the peri-
phery of the lobules, however, the fat droplets may sometimes
fuse to form one single drop.

In the woodchuck, a hibernator, the appearance of brown fat
differs in certain respects from that found in the rat although
the essential characteristics remain (Rasmussen, 1923). The
cell contains one and occasionally two large fat globules and
many smaller ones of various sizes. Because of this, the nucleus
is more eccentric than in the rat. However, it is never flattened
much, if at all, no matter how fat the animal is. The amount of
brown fat shows a decrease during hibernation accompanied by
an increase in the intensity of the brown color. The fat droplets
disappear but only to a certain extent. The brown fat in man
has recently been described by Wegener (1951). This author
points out the varying size of the cells and of the fat vacuoles
of different cells. No essential differences are, however, found
microscopically in human brown fat compared with the above-
mentioned forms,

Biochemical Observations in Brown Fat

Different components of brown fat have been investigated with
chemical analyses and also microscopically with histochemical
methods. (See Johansson, 1959, for a review of the literature. )

The carbohydrate content has been found to be higher in brown
fat than in white, at re-feedine after starvation. The lipids,
however, are less abundant in brown fat. Significant differences
in iodine number have not been found (Shattock, 1909; Coninx-
Girardet, 1927) although histochemical methods indicate that the
hpids in brown fat are more saturated than in white. Durine
hibernation, the weight and lipid content decrease and the
amount of water shows an increase. According to Carlier and
Evans (1903) the lipid decrease in brown fat from hedgehoes
hegins rather late during hibernation (in the month of January ).
After injection of labeled phosphorus in milk to rats, the specific
activity has been found to be eight times less in white than in
brown fatty tissee where the activity is the same as in gland
tissue (Littrell ef al., 1944; Favarger and Gerlach, 1955). The
concentration of different enzymes has been determined. As
for glycogenesis, Shapiro and Wertheimer (1956) claim that all
enzymes necessary for this process occur in brown fat. The

236 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

chemical properties of hexokinase, phosphoglucomutase and phos-
phorylase from brown fat resemble those found in muscle (Creasy
and Gray, 1952).

KFaweett (1952) has made a comparison of the cytochemical
reactions of brown and white adipose tissues in rats and mice.
Both brown and white fatty tissue contain neutral fats; in the
former, however, these are more saturated and less abundant.
The eytoplasm of brown fat appears richer in phospholipids,
acetyl phospholipids and glycogen. Both tissues contain esterase,
succinic dehydrogenase and alkaline phosphatase, but they
seem to be more ample in brown fat. Cholesterol and ascorbic
acid were found neither in brown nor in white fat. Mensechik
(1953) has performed a similar histochemical study in guinea-
pigs. In contrast to Faweett (1952) he found a conspicuous
amount of succinic dehydrogenase. Furthermore, Mensehik
(1953) claims that brown fatty tissue contains more z-amino
acid groups, mucoproteins, watersoluble polysaccharides, e@luco-
lipids, phospholipids, cholesterol and its esters, amine oxidase,
znaphthol oxidase and cytochrome oxidase but less neutral fat
than white fatty tissue. Rémillard (1958) has recently presented
a detailed paper on the histochemistry of brown fat in a bat.

When measured by the Warbure technique the oxygen con-
sumption has been found to be higher in brown fat than in
white. It shows a seasonal variation, being highest around Sep-
tember and October. When using a substrate like pyruvate or
succinate, no difference in oxygen consumption of brown fat
was obtained between hibernating and non-hibernating animals
at the same temperature. In contrast, the oxygen consumption im
kidney slices was diminished by 12 per cent in the hibernating
animal (Hook and Barron, 1941).

The cause of the brown color has not been satisfactorily ex-
plained. The rich vascularization probably contributes to it in
part. Seasonal variations also play a role, for during hibernation
the brown fat becomes darker. The brown color has, among other
things, been aseribed to oxidation products of phospholipids, to
hemoglobin, hemosiderin, hemines and lpochrome.

Influence of Various Hormones

ACTH produces an increase of water, lipids and fat-free dry
material, and the fat droplets fuse to form a large single drop,
the cells assuming the character of white fat.

bo
we
~l

1960 MAMMALIAN HIBERNATION

Brown fat takes part in the reaction to stress (Lemonde and
Timiras, 1951). There is a close parallelism between the reac-
tions of the adrenal cortex and of the brown fatty tissue. Suoma-
lainen and Herlevi (1951) have studied the brown fat durine
the arousal process of hibernating animals and conclude that the
arousal represents a stress to the animal.

Injections of adrenocortical hormones have given somewhat
different results. These are, partly at least, caused by ineon-
sistency in dose, length of observation time and age of the ani-
mals. Aronson et al. (1954) describe a hypertrophy of the brown
interscapular fat after cortisone injection, more pronounced in
hamsters than in mice. Lachance (1953) has found an increase
of the lipid content after cortisone; cortisone exerts a ereater
influence upon the weight of the interscapular body than desoxy-
corticosterone in adrenalectomized rats.

Thyroxine injected into rats results in hypertrophy of the
interscapular brown fat; it is due to an increase in the lipids,
whereas water and fat-free dry material remain unchanged
(Lachanee, 1953). Thiouracil treatment splits up the fat drop-
lets so that they decrease in size and increase in number (Littrell,
1948; Fawcett and Jones, 1949).

After starvation and refeeding, glycogen can be demonstrated
in both brown and white fatty tissue, comparatively more, how-
ever, in the former. Insulin causes a deposition of glycogen in
both forms of fat.

Discussion

KMarlier authors have discussed the importance of brown
fat for hibernation and many have stressed a causal relation-
ship. Recently, however, Herter (1956) and Schierer (1956)
have emphatically denied such a connection, and other authors
have refuted a definite position.

To solve this problem it is necessary to have an answer to at
least three questions :

/) Are the differences between brown and white fat ereat
enough to characterize them as two different tissues?

2) Does brown fat exist in all hibernators?

3) Tlow valid are those experiments showing a causal relation-
ship between brown fat and hibernation?

1) Schierer (1956) vigorously claims that brown fat is
only a fat deposit, that the biochemical differences between the
two sorts of fat are all due to the greater amount of evtoplasm in

238 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

the brown fat, and that brown fat has no connection with
hibernation. In her experiments she has used wild rats and
hamsters but the number of animals is very small.

The histochemical studies by Faweett (1952) and Menschik
(1953) were unfortunately performed only on non-hibernators.
Schierer (1956) has compared rats and hamsters histochemically
and found no differences between these two animals. Histochemi-
cal studies are often more qualitative than quantitative, and,
although Menschik (1953) tries to take into consideration the
different amount of cytoplasm, it is possible that many of the
differences in amount found by these two authors are not real.
Mirski (1942), however, using the ability of converting fructose-
1-phosphate (Cori ester) to fructose-6-phosphate (Robison
ester) as a test of the phosphoglucomutase content, claims that
white fat lacks this enzyme in contradistinction to brown fat.

The Oo-consumption is mueh higher for brown than white fat ;
judging by the figures given by Hook and Barron (1941) it
seems as if the difference is greater than can be explained by the
different amounts of eytoplasm. It has also been shown that brown
fat retains a comparatively high metabolic activity during hiber-
nation as reflected in oxygen consumption (Hook and Barron,
1941) and redox potentials (Klar, 1941). Many authors have
stressed the glandhike appearance of brown fatty tissue in con-
trast to white. Schierer (1956), too, admits that the cells of
brown fat have an epithelioid arrangement and that the vascular
supply is abundant. These properties are found in endocrine
elands also. She points out, however, that brown fat lacks certain
essential qualities characteristic of endocrine glands.

Some important facts have not been considered by Schierer
(1956) — for example, different reactions of brown and white
fatty tissue to various hormones and vitamins. Cortisone causes
a hypertrophy of brown fat (Aronson eft al., 1954) as does
thyroxine (Lachanee, 1953), while white fat shows no change
and a decrease, respectively. The response to stress is much more
pronouneed in the brown fat (Selye and Timiras, 1949). So is
the response to hypophysectomy, adrenalectomy and thiouracil
feeding (Faweett and Jones, 1949). Vitamin E deficiency causes
pigmentation and cellular developmental changes in white fat,
while brown fat remains unchanged (Mason ef al., 1946). The
amount of androgens is said to be very high (Sweet and Hoskins,
1940), and hormones of the corticosteroid type have been de-
seribed in brown fat (Nigeon-Dureuil et al., 1955; Ratsimamanga

1960 MAMMALIAN HIBERNATION or

et al., 1958; Zizine, 1958). Certain types of virus show pre-
dilection for brown fat (Pappenheimer ef al., 1950; Godman
et al., 1952; Aronson and Shwartzman, 1956; Sulkin e¢ al.,
1957). In starvation there are nearly always some fat droplets
left in brown fatty tissue, which retains its characteristic as-
pects, while the white fat shows a total depletion and adopts
the character of fibrocytes. In fattening, some of the brown fat
cells show a fusion of the fat droplets. This, however, occurs
only in the periphery; in the central parts the cells retain their
characteristic appearance. According to Hammar (1895), brown
fatty tissue shows an embryological development different from
that of white fatty tissue and the blood supply is richer. Faw-
cett (1952) states that white fat can be found in all places where
connective tissue abounds while the distribution of brown fat
is confined to certain definite localities in the body. It seems,
then, that brown fatty tissue possesses so many properties dif-
fering from white that they can be looked upon as two different
tissues. This, of course, does not exclude the fact that brown
fat can act as a fat deposit, too. From a teleological point of
view, however, it 1s remarkable that it contains so much eyto-
plasm if its only task is to store lipids.

2) If brown fat has a close connection with hibernation, it
should be found in all hibernators. There seems to be a general
agreement among workers in this field that brown fat is found
in all hibernators.' In Rasmussen’s (1923) review he mentions
two ‘‘hibernators’’ that lack brown fat, the badger and the
‘raccoon. The badger, however, is no real hibernator. During the
winter it enters a state of winter torpor, but like the bear
(Hock, 1958) the animals are not poikilothermic. Personal in-
vestigations on the badger show that this animal during the
winter does not behave biochemically in the same way as a real
hibernator like the hedgehog, and, most important of all, when
being cooled the heart stops beating at about +13°C; the hedge-
hog’s heart still beats a few degrees above zero (Johansson,
1957a). Cushing and Goetseh (1915), in a footnote, characterize
the raccoon as a hibernator without brown fat. Later authors,
however, are of the opinion that this animal is not a hibernator
(Hisentraut, 1956).

As stated earlier, brown fat is found not only in all hibernators
but also in many non-hibernators. This has been taken as an

1In response to my query, Drs. Eisentraut, Herter, Kayser and Lyman all
assure me that they know of no hibernators which do not have brown fat.

240 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

argument against a connection between brown fat and hiberna-
tion, There are, however, examples in the realm of animals that an
organ remains although its original task has disappeared or
changed. It seems as if no extensive investigation has been
performed to find out whether hibernators contain more brown
fat than non-hibernators. Preliminary personal investigations
indicate that hibernators like the hedgehog have definitely more
brown fat than non-hibernators and that among non-hibernators
the rat contains more brown fat than, for example, the g@uinea-
pig. The brown color in rats seems to be less pronounced than
in hedgehogs.

There is a tendency for the hearts of rats at least to withstand
cooling better than, for example, guinea-pigs which have less
brown fatty tissue. Furthermore, rats and mice have an ex-
tremely short S-T’ interval in the electrocardiogram, which has
been found to be typical of many hibernators (Johansson,
1957b).

3) There are at least two different ways of studying the func-
tion of brown fat. One way is to extirpate or destroy the brown
fat, another to extract the possible active substances.

Extirpation experiments are very difficult because brown fat is
so widespread that it is impossible to perform a complete re-
moval. The results of sueh experiments have been divergent.

KKayser (1953) could not find any differences in the resistance
of rats and hamsters to cold after extirpation of the ‘‘hibernat-
ing eland.’’ Vienes (1913) states that removal of the brown fat
in rats causes a decrease of the body weight and the animals
eradually die. Trusler et al. (1953) conelude that in marmots
extirpation of the brown fat causes a decrease of the resistance
to cooling. Zirm (1956a) has found that hedgehogs die upon
exposure to extreme cold during hibernation after extirpation
of about 50 per cent of the total amount of brown fat.

Wendt (1948) has published the results of experiments with
an extract from brown fat which was injected iter alia into
rats. He found that such injections depress the basal metabolic
rate, pulse rate, blood pressure and body temperature. Re-
cently Zirm (1956a,b) has shown that implantation of pieces of
tissue from the ‘hibernating gland’’ of hibernating hedgehogs
into mice eauses a decrease of the body temperature of the ani-
mals in proportion to the size of the implant. In addition,
within two weeks of the implantation the animals increased in
body weight. Zirm also succeeded in preparing a yellow-green

1960 MAMMALIAN HIBERNATION 241]
substance from the ‘‘hibernating gland.’’ Injection of this prep-
aration into mice was followed by a drop in the temperature,
respiratory frequency and blood pressure. Extracts from brown
fat from non-hibernating hedgehogs and extracts prepared in
the same way from the liver, lungs, spleen or kidneys of hiber-
nating or non-hibernating hedgehogs produce no such effect.

TABLE I

Laeogenous factors Endogenous factors Inborn qualities

1. Temperature 1. Inhibition of temper- Ability of the tissues
2. Rest ature regulation, (especially the cardio-
3. Light 2. Increase of fat depots. vascular and the nery-
4. Deprival 3. Hypertrophy of brown ous systems) to fune-
5. Composition of food fat. tion at temperatures

(especially decrease 4. Polyendocrine involu- just above 0°C.
of water content). tion.
6. Confined air. 5. Decrease of sympa-
thetic and increase of
parasympathetic tone.

HIBERNATION

The importance for hibernation of experiments with extracts
of brown fat has been denied by Wertheimer and Shapiro
(1948), among others, who believe that the retarding effect of
brown fat on metabolism is non-specific.

There is one point in connection with these extraction expert-
ments that I should like to point out. As is shown in Table I, it
is not only the exogenous factors and the endogenous change in
the autumn that are necessary for the entrance of hibernation.
The organism must also be able to maintain at least some cir-
culation during hibernation, i.e. the heart must be able to beat
at the low temperatures that obtain during hibernation. This is
a property that is not restricted to the hibernation period but
one which the hibernators show during the whole year and which

242 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

adult non-hibernators never show. Preliminary experiments on
a small number of rats indicate that in animals given a diet with
a high percentage of corn oil, containing a high amount of un-
saturated fatty acids, the hearts keep beating at a lower tempera-
ture than in rats given coconut oil which contains a high per-
centage of saturated fatty acids. It is thus possible to change
the heart’s resistance to cooling by exogenous means, but |
think this is rather far from stating that a single injection of
some material, as has been done in the extraction experiments,
could give a non-hibernator’s heart the ability of performing
work at a temperature just above zero. The conclusion is, then,
that the extracts of brown fat should be tested also on hibernators
in a non-hibernating state to get a real appraisal of the ability
of the extracts to mduce hibernation. The problem of how hiber-
nators can stand very low temperatures must, I think, be solved
by comparative biochemical studies, especially a comparison of
the properties of different enzyme systems. Studies of this type
have just started at the Malmo General Hospital.

Krom the above it is apparent that no valid objections have
been given against a connection between brown fat and hiberna-
tion. On the other hand, although some interesting facts point
in this direction, no definite evidence has been eiven stating
that there really is such a connection. It seems to me that the
experiments that are most interesting and that in the future will
contribute the most conclusive evidence to the solution of this
problem are of the type that have been performed by Wendt
(1943), Hook (1940) and recently, by Zirm (1956b).

Summary

A short review of some facts on brown fat is given. The
author discusses the possible connection between brown fat and
hibernation. It is coneluded that brown fat occurs in all hiber-
nators and that the occurrence of brown fat in many non-
hibernators must not be considered evidence against a possible
connection between brown fat and hibernation, for there are
examples that an organ ean remain even after having lost its
original task. The differences existing between brown and white
fatty tissue are considered so great that there are reasons to look
upon them as different tissues. This does not exelude the fact
that brown fatty tissue can also act as a depot for lipids. Dif-
ferent ways to show experimentally a connection between brown
fat and hibernation are discussed. Experiments with extracts

1960 MAMMALIAN HIBERNATION 943

of brown fat seem to give the most interesting results. The
necessity of using the extracts on hibernators in a non-hibernat
ing state in order to induce hibernation is stressed.

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MENSCHIK, Z.
1958. Histochemical comparison of brown and white adipose tissue
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DISCUSSION FOLLOWING JOHANSSON’S PAPER

SMITH reported that he had removed substantially all the
brown fat from bats and had discovered that such animals (less
brown fat) also lowered their body temperatures in the face of a
cold stress. JOHANSSON indicated that all brown fat could
not be removed by gross surgery, but if the brown fat got its
blood supply from one or a few main vessels, ligation of these
might cause a complete loss of the brown fat from the entire
hody.

PEARSON said he had carried out brown fat injection ex-
periments using the interscapular gland of Myotis, and found
that this tissue was very toxie and would kill white mice when
injected.

JOHANSSON believed it should be established whether
brown fat, taken from animals in the hibernating state and in-
jected into animals of the same species during the non-hibernat-
ing season, makes it easier for such animals to enter hibernation.

MORRISON pointed out that there may be difficulty in study-
ing these effects in an animal (white mouse) not constitutionally
arranged for hibernation. He noted that his group tried without
success to lower the body temperature of white mice by implants
or injection of brown fat homogenates from hibernating 13-lined
ground squirrels.

KAYSER said the best experiments to this end have been
done by G. A. Trusler et al. (Surgical Forum of 1953, 4:72, 1954).
The body temperature at which respiration will cease is a lower
temperature when sufficient brown fat is present, than when it is
in short supply. He indicated, however, that he had not been able
to repeat Zirm’s experiments with the hedgehog; he felt he may
have destroyed the principle in preparation. On the other hand,

248 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

he beheved that hypothermia depended on the amount of brown
fat. He indicated that he has totally reversed his former opinion,
and he now believes brown fat is important in hypothermia and
hibernation.

JOHANSSON asked HOCK if brown fat was found in bears
or badgers, although these animals are not true hibernators.
ILOCK indicated bears have some, but of an unimportant quan-
tity comparatively. JOHANSSON added that brown fat is pres-
ent in a comparatively large amount in small children.

ZIMNY called attention to the study of brown fat reported
by G. Rémillard (Ann. N. Y. Acad. Sei., 72:1, 1958).

BALL remarked that in the rat the brown color of the fat
scems to be entirely due to the high content of evtochromes which
presumably act as an energy supply. With reference to eyto-
chromes and tissue metabolism, ZIMNY indicated that biochemi-
eal studies of glycogen, lactate and pyruvate showed variations
in these compounds during hibernation.

XITT

SOME PROBLEMS OF REPRODUCTION IN
RELATION TO HIBERNATION IN BATS’

By Wiuuiam A. Wimsatt
Department of Zoology
Cornell University
Ithaea, New York

Introduction

Many physiological aspects of hibernation are no doubt com-
mon to all mammalian hibernators, but there are a few which
are peculiar to individual species or groups. My purpose here
is to review one of these less generalized phenomena, the pro-
found influence of hibernation on the physiology of reprodue-
tion in bats. I intend not so much to provide answers, few of
Which have as yet been forthcoming, as to indicate the nature
of some of the more important problems still unsolved.

It is characteristic of nearly all hibernating mammals that the
annual periods of hibernation and reproduction do not overlap,
at least not significantly, and it is doubtful that the profound
metabolic depression of hibernation has any important influence
on reproductive activity, except possibly to delay its onset.
These species, which include all the more familiar hibernators
such as the woodchuck (Marmota), hedgehog (Hrinaceus) and
ground squirrels (Citellus), are typically spring breeders. They
enter, and pass through hibernation in a state of virtual sexual
quiescence, or anestrum, and both males and females become
sexually active only after awakening and emerging from hiber-
nation in the spring. There appears no reason to suppose that
the factors which condition reproductive activity in these species
are qualitatively any different from those operative in other
spring-breeding mammals whieh do not hibernate.

By contrast, it is now widely recognized that hibernating bats
present a unique departure from this general pattern, for in them
hibernation falls fairly astride the reproductive season, and
influences to a marked degree the reproductive physiology of both
sexes. It should of course be emphasized that not all bats
hibernate, or even necessarily possess the capacity to do so.

L'The original work reported berein was supported by research grants (G-2188
and G-7474) from the National Science Foundation.

250 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Bats are tropical in origin and primary distribution, and an-
nually recurring periods of hibernation are observed in only the
few species (belonging to but two families, Vespertilionidae and
Rhinolophidae) which have become adapted to living in the
cooler temperate latitudes. Significantly, these alone manifest
the reproductive pecularities for which bats are noted (ef.
reviews of Wimsatt and Trapido, 1952, and Herlant, 1953).

Background: The Reproductive Cycles of Hibernating Bats

The chronology of major reproductive events in relation to the
annual evele of hibernation in bats is summarized schematically
in Figure 1. Differences between species oceur in the timing
and degree of expression of some of the phenomena shown, but
at least as far as the female is concerned the basie picture is
similar in all hibernating bats, with the single exception of the
Kuropean vespertihonid, Miniopterus schreibersvi, in which the
eyele is depicted separately. In contrast to other hibernating
mammals, bats initiate reproductive activity in the fall, but
typically enter hibernation very soon thereafter. Dormancy
appears to arrest the normal progress of reproductive events,
and postpones their completion until after the animals per-
manently emerge from hibernation in the spring.

The reproductive status of males and females before, during
and after hibernation may be summarized as follows. In males
the spermiogenic phase (of spermatogenesis) 1s achieved in late
summer. When copulations begin, just prior to hibernation, the
cauda epididymides are congested with mature spermatozoa,
which continue to reside here throughout the period of dormaney.
However, the spermatogenie activity of the testis has by now
spent itself, and at the time of the fall copulations the semini-
ferous tubules are rapidly reverting to the quiescent state charac-
teristic of the hibernating period, consisting in the main only
of spermatogonia and Sertoli cells. The sex accessory glands
remain small and unstimulated throughout the summer sperma-
togenie phase, but suddenly and rapidly hypertrophy over a
short interval in the fall which coincides with pairing and
copulation. When the males enter hibernation soon after copu-
lating the sex accessories are still in a maximally hypertrophied
state, and they remain more or less enlarged throughout the
period of dormancy. Involution occurs suddenly, however, fol-
lowing emergence from hibernation in the spring (Courrier,
1927 ; Miller, 1939; Pearson e¢ al., 1952; Krutsch, 1956; personal

1960 MAMMALIAN HIBERNATION 95

observations). In the foregoing respects the cycles of all male
hibernating bats except Miniopterus are complementary: the
latter differs in that the sexual accessories involute completely
during the fall. There is an apparent paradox, however. in
respect to the changes in the interstitial cells in various species.

FIGURE |
REPRODUCTIVE CYCLES IN HIBERNATING BATS
MALE
SPECIES JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN| AUTHORITY

7 7 Copulation
interstitial ae

——— Se
weeere view ad
Eptesicus ser

Courrier 1927
Pipistrelivs pip

Sex Accessories

Ce ee es

Miller 1939
Pearson et al. 1952)

Myotis sp
Corynorhinus raf
Interstitial Cells

74-97 & Copulation

HIBERNATION

Miniopterus schr Courrier !927

Inferstitia] Cells
Sex Accessories \_ ry

: ;
itieatshiase F nes Bae a
Berane Er D8).6 (0:68 6) 6160 * Pee eer eer eewre

Eptesicus s

= : d
r Courner 1927 |
(3) : = Christian 1956 | |
p Preov cr Courrier 1927
Follicular Growth | Su Guthrie 1933

Guthrie &

Jeffers 1938 |
Wimsatt 1944 |
Wimsatt & |
Kallen 1957 |

Pearson et al. 195%

Pipistrellus p

Myotis so

HIBERNATION eS ee ae HIBERNATION

Corynorhinus rof| Uferus

2 ee a oe eb oe ae ee o_o ee ee oe

Rhinolophus fereq} § Proestrum — Diest (Freq) m= = = = Proasicum Matthews 1937|

Ft a nee ¥—~ Copulation

Miniopterus schr. Courrior 1927

Uterus we emmy

l
iz t
Lact Anes —___Proestr ung Estrum Diest,

Diest

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN!

i,
bo

BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

According to Courrier (1927), in the European Pipistrellus and
Mptesicus the Leydig cells are maximally developed at summer’s
end when the sex accessory glands achieve maximum develop-
ment, but reeress somewhat before the males enter hibernation,
and then experience profound involution at hibernation’s end
in the spring. In closely related American species, on the other
hand (Myotis, Miller, 1939; Corynorhinus, Pearson ef al., 1952;
Pipistrellus, Krutseh, 1956), the interstitial cells are involuting
during the time that the sex accessories are enlarging, and ap-
pear most active in summer, when the accessories are small and
show no evidence of endocrine stimulation. In Corynorhinus, at
least, they are also large in sexually immature males. The
histological picture in the European bats is consistent with
the view that the interstitial cells produce the male hormone,
but the situation in the American species is more difficult to
reconeile with this concept. It is perhaps an anticlimax to record
that in many species, the males, when temporarily awake during
hibernation, demonstrate the continuing capacity to copulate
throughout this period (Guthrie, 1933; Wimsatt, 1944a; Guilday,
1948; Pearson ef al., 1952). Summarizing, the cyele in the
male bat is peculiar in at least three respects: 1) there is an
apparent exaggerated asynchrony between the spermatogenic
and endocrine eyeles of the testis; 2) viable spermatozoa persist
in the epididymis for an unusually prolonged period after the
cessation of spermatogenesis; and 3) the apparent functional
cycle of the Leydig cells of the testes, as judged by the usual
histological criteria, does not coincide completely with the
periods of libido and functional hypertrophy of the sex accessory
olands in American species, but appears to do so in some Eu-
ropean ones.

The situation in the female is more constant, but no less strik-
ing. All known species of hibernating bats are monestrous, but
there is no true period of anestrum. In adults a new estrous
cycle is inaugurated shortly after lactation ends in late summer,
and growth and atresia of vesicular follicles occurs in most
species during the weeks between Jactation’s end and entrance
into hibernation. Copulation occurs near the end of this period,
at which time the number of vesicular follicles in the ovaries
has been reduced, and the animals enter hibernation with one
or more follicles (species differences) destined to survive through-
out the winter. Young females of some species are inseminated
in their first fall, but copulation in these instances usually occurs
in the absence of vesicular follicles in the ovaries (Guthrie, 1933 ;

1960 MAMMALIAN HIBERNATION 253

Wimsatt, 1944b). At the time of the fall inseminations and
entrance into hibernation, the oviducts, uterus and vagina show
all the characteristics of proestrous stimulation, a condition
which they maintain throughout the hibernating period. Sper-
matozoa from the fall and later inseminations are stored either
in the uterus in the Vespertilionidae, or in the vagina in the
Rhinolophidae (Matthews, 1937), and it has been demonstrated
that they remain viable throughout the period of dormancy
(Wimsatt, 1942; 1944a). The surviving large follicle, which
develops a unique structural and chemical organization, remains
unchanged throughout hibernation (Wimsatt, 1944b; Wimsatt
and Kallen, 1957). Preovulatory growth and rupture of the
surviving follicles occur in the spring, within a few days after
the females leave hibernation (Wimsatt, 1944b; Sluiter and Bels,
1951). Summarizing, the unusual features of reproduction in the
female bat are: 1) the prolongation of the proestrous phase of
the eyele through hibernation, 2) the maintenance of viable
spermatozoa in the reproductive tract throughout the period of
dormaney, and 5) the marked delay in ovulation involving the
prolonged survival through hibernation of the follicles destined
to rupture in the spring.

As mentioned earher, the only known exception to this general
pieture in hibernating bats is the vespertilionid bat Miniopterus
schreibersi. As shown in Figure 1, there is no delayed ovulation ;
folheular rupture follows soon after the fall copulations, and
pregnancy ensues immediately. Embryonic development is al-
leged to be retarded, but not actually arrested during hiberna-
tion, and the young are born some time after hibernation ends,
in the early summer (Courrier, 1927). The gestation period is
some months longer than in tropical members of the same genus,
and paradoxically, the breeding season is reversed. In the tropics
the Miniopterinae breed at a time of vear which corresponds to
the northern spring (Baker and Bird, 1936), whereas the Euro-
pean species breeds in the fall.

Nature of the Problem

Up to the present day attention has been focused primarily on
the chronological and morphological aspects of reproduction in
hibernating bats, together with attempts to deduce on an ana-
logical basis the underlying endocrine mechanisms. Experimental
analyses have been sporadically undertaken, mostly in respect to
the cycle in the female, but for the most part they have been

254 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

limited in scope, and have contributed little toward the formula-
tion of an integrated picture of the controlling factors of repro-
duction such as we possess for other mammals. Likewise, scant
attention has been paid to the immediate physiological bearing
of dormancy per se on the mode of action of the agencies control-
ling reproductive phenomena in bats, and indeed there seems to
have been little appreciation that the latter might be of any sig-
nificance! For these reasons it seems worthwhile to outline a few
of the more important gaps in our knowledge of these matters in
male and female bats, and to review recent experiments of my
own and others which relate to some of them.

The greatest deficiencies are found in our knowledge of the
intrinsic endocrine and neural mechanisms which in bats as in
other mammals presumably regulate reproductive processes, and
the possible ways in which hibernation directly or indirectly
modifies their action. We are also essentially ignorant of the
possible influence of external environmental stimuli on regula-
tion of reproductive periodicity in bats, and of the sensory path-
ways through which their effects might be mediated.

The male bat. I shall begin with the male which, though less
studied from an experimental point of view than the female,
seems from the timing of its evcle to present fewer problems, and
can therefore be more rapidly dealt with.

Possibly a significant problem for investigation exists in those
American bats (e.g. Corynorhinus) in which libido, viable epi-
didymal spermatozoa, and hypertrophied sex accessories, are
established and maintained over a long period during which the
interstitial cells of the testis appear to be in a functionally in-
voluted state. If one assumes that these phenomena in bats are
conditioned by testosterone as in other mammals, he must con-
clude that in these American species, in contrast to the Euro-
pean ones described by Courrier, the usual histological criteria
are of little value in assessing interstitial cell function, and that
an apparent involuted condition of the interstitial tissue is com-
mensurate with a maximal hormone release. If this proves cor-
rect, then we are still faced with the problem of reinterpreting
the different conditions in Courrier’s bats. The picture in the
American bats is perhaps further complicated by the facet that
an attempt by Pearson et al. (1952) to demonstrate steroidal
compounds in the interstitial tissue of hibernating specimens of
Corynorhinus by a histochemical test (2-4 dinitrophenylhydrazine
reaction) failed to reveal any, in either interstitial tissue or

1960 MAMMALIAN HIBERNATION 255

tubules, and under the same conditions of testing in which con-
trol mouse testes reacted admirably. A more thorough histo-
chemical and biochemical study of the androgen content of the
testis during hibernation is obviously indicated, for it has been
demonstrated by both Courrier (1927) and Pearson ef al. (1952)
that the sex accessories of male bats are indeed responsive to
androgen, at least in active animals (wide infra).

There is an alternative possibility which to my mind has
neither been adequately considered nor effectively ruled out.
It is the possibility that the testes no longer actively produce
hormone during hibernation, but that torpidity itself retards
the regressive changes which at more elevated body temperatures
quickly follow hormone withdrawal. There is some suggestive
evidence in support of this hypothesis, and some which militates
against it. In support, are the observations of Courrier (1927)
to the effect that in all species examined by him some involution
of the sex accessories has already begun in the fall, but in a
graded fashion in different species; it is extreme in Miniopterus,
intermediate in Myotis, and minimal in Pipistrellus and Eptes-
icus. Also suggestive is the sudden involution which in all species
overtakes the sex accessories immediately after hibernation term-
inates in the spring. On the other hand, the hypothesis is ap-
parently contraindicated by the results of some experiments of
Courrier and.Pearson et al., both of whom were able to demon-
strate, in Eptesicus and Corynorhinus respectively, that the sex
accessories involute more rapidly in males removed from hiber-
nation and bilaterally castrated, than in non-castrated controls.
Also more rapid natural involution during the fall of the sex
accessories of Miniopterus than those of other species would
militate against the hypothesis, if Miniopterus enters hiberna-
tion when the others do.

However, none of the above experiments preelude the further
possibility that small but effective quantities of androgen pro-
duced during the period of breeding activity preceding hiberna-
tion might persist in the testes or elsewhere, e.g. in’ brown
fat, unmetabolized in the torpid animal, and be available to
retard involution of the sex aceessories in non-castrated ae-
tivated males. It is significant in this regard that the hiber-
nating state alone will prevent involution of the sex acces-
sories in the face of abrupt androgen withdrawal, for Cour-
rier (1927) and Wimsatt (unpublished observations) have
observed that if hibernating specimens of ptesicus are
bilaterally castrated, and returned immediately to hibernation,

256 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

no detectable involution of the glands occurs, even over a period
of several months. The possibility of a retarded utilization of
‘‘residual’’? hormone under the influence of hibernation would
seem not to be essentially different in concept from this demon-
strated retarding effect of hibernation on the involution of the
male glands, and furthermore it receives indirect support from
other evidence to be presented shortly. Obviously, many aspects
of the endocrine cycle of the testes in hibernating bats require
further experimental study.

A second, potentially significant, unsolved problem in the male
concerns the physiological mechanisms (endocrine and neural)
which condition the apparent asynchrony between the spermato-
genie and endocrine cycles of the testis. The word ‘‘apparent”’
is emphasized beeause the asynchrony will be real only if it
turns out that the testis continues to elaborate androgen during
the hibernation period, when the seminiferous tubules are quies-
cent. If this proves te be the case, it would appear likely that
the primary cause may reside in the asynchronous release of
hypophyseal gonadotrophins (FSH and LH) known in other
mammals to be separately responsible for stimulation of the two
gvonadal functions. An indication that separate release of go-
nadotrophins can occur is provided by an observation of Courrier
(1927). He deseribes a ‘‘eunuchoid’’ Pipistrellus, collected in
September, in which spermatogenesis had been normal (epi-
didymal sperm) though now terminated, but im which the ses
accessories, which should have been fully hypertrophied, were
totally undeveloped; the interstitial tissue was likewise fully
involuted.

It is conceivable that an asynchronous production of gonado-
trophins might be reflected by alterations in the cytology of the
hypophysis. To date only a single definitive study of pituitary
eytology in the male bat has been carried out, by Siegel (1955),
who worked on Myotis lucifugus in my laboratory. He was the
first to demonstrate that in the bat, as in other common laboratory
mammals, the classical ‘‘basophiles’’ are divisible into two fune-
tionally-specialized groups, ‘‘thyrotrophs’’ (producing TSH)
and ‘‘eonadotrophs’’ (producing gonadotrophins). He was un-
able to subdivide the ‘‘gonadotrophs’’ further, however, and his
study produced no evidence, either for, or against, the concept of
an asvnehronous production of different gonadotrophic hormones.
The problem is one which requires further study, particularly in
conjunction with the working out of the true endocrine status of
the testis in the hibernating male. We shall turn next to condi-
tions in the female.

1960 MAMMALIAN HIBERNATION 257

The female bat. Among the many problems of reproductive
physiology in female bats, two seem to me to be most provocative,
namely, the mechanisms of ovulatory delay, and of sperm sur-
vival in the female tract. I shall be able to consider here only
the first. Several questions might conceivably be asked concerning
the characteristic delay of ovulation: What endocrine mechan.
isms condition follicular growth and ovulation in bats? Are these
subject to neural influences, and if so, of what kind? Does
hibernation per se have any direct effect on the operation of these
factors? And, finally, what is the mechanism for reactivation
of the interrupted evele at the end of hibernation ?

A priort it appears unlikely that the immediate factors con-
trolling reproductive phenomena in bats differ in fundamental
quality from those operative in other mammals, for the repro-
ductive organs of bats respond in the same ways to gonadotrophic
and sex hormones. It seems most reasonable to suppose that the
prolongation of the proestrous phase through hibernation results
from the interplay of at least three factors: the depressing effects
of hibernation itself on cellular metabolism and reactivity; a
probable dissociation in time of hypophyseal gonadotrophie fune-
tions which in other mammals are more closely synchronized ; and
a neural mechanism which may regulate hypophyseal (g@onado-
trophic) function and itself be triggered by environmental and/
or internal stimul. The timing of the cyele in the female and
experimental results to date indicate the possible involvement
of all of these.

As mentioned earlier, the ovary of the hibernating bat typically
contains throughout the period of dormancy a viable vesicular
follicle, or follicles, which are destined to experience preovula-
tory growth and to rupture shortly after hibernation ends in
the spring; no other large follicles are present. The prolonged
survival of these follicles is unique, and it is perhaps not re-
markable that they should demonstrate a chemical specialization
not observed in the follicles of other mammals. It consists in a
pronounced vacuolation of the discus cells surrounding the
ovum (Plate, fig. 1) which is brought about by the deposition
within them of enormous quantities of glycogen (Plate, fig. 2).
Pearson et al. (1952) have adduced evidence to suggest that in
Corynorhinus this characteristic vacuolation of the discus cells
is dependent upon copulation, but Wimsatt and Kallen (1957)
were able to show that in Myotis lucifugus, at least, it oceurs in
the absence of a copulatory stimulus. They postulated that it
is an adaptive response to hibernation itself, the glycogen repre-
senting a readily available source of energy providing for the

258 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

survival of the follicle under conditions of a drastically reduced
metabolism in the hibernating animal; experiments are currently
in progress to test the validity of this concept.

But our immediate problem is to determine the reasons why
this follicle does not complete its maturation and go to rupture
before spring. Experimental results indicate that at least two
interrelated factors may be involved, and perhaps more. The
first is an inability of the follicle to respond to hormone stimula-
tion while the bat is in a torpid condition, and the second is the
probable absence during hibernation of sufficient gonadotrophic
hormone (LH) to precipitate ovulation. These conelusions are
based on the results of personal experiments partially summarized
in Table I, which records the effects of various hormone injections
on induction of ovulation in Myotis under different conditions
and at different times during the winter. First it can be seen
that ovulation was elicited by all pituitary hormones used except
ACTH and oxytocin, but it was effected most easily and con-
sistently by the FSH preparation. One may perhaps suspect
that the TSH and Growth hormone preparations contained some
vonadotrophin contaminant. Secondly, it should be noted that
ovulation was induced only in animals which were maintained at
room temperature during the experiments. In the group of FSH-
injected animals maintained in a torpid condition throughout
the experiment not a single ovulation was induced, which demon-
strates that the ovary is unable to respond to circulating hormone
in the torpid animal. Significantly, removal of injected torpid
animals to room temperature was promptly followed by ovula-
tion induced by the residual hormone previously administered.
Thirdly, thyroxine (Tx) and cortisone were ineffective in elicit-
ing ovulation in animals maintained at room temperature, al-
though thyroxine did induce some follicular enlargement and
elycogen discharge from the discus cells, but without maturation
changes in the ovum.

On the basis of these findines the followine reasoning seems
plausible: Sinee ovulation can be quickly precipitated by pitu-
itary gonadotrophins at any time during the hibernation period
(provided a surviving follicle is present), and hormones which
merely mfluenece the general level of metabolism are ineffective,
there is at present no reason to assume that preovulatory e@rowth
and ovulation are not normally under direct hypophyseal control.
urthermore, it is well known that hibernating bats brought to
room temperature during the early months of hibernation will
not ovulate spontaneously (Guthrie, 1933; Wimsatt, 1944b),

HIBERNATION

MAMMALIAN

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260 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

which suggests that ordinarily no ‘‘residual’’ gonadotrophin is
present, and that an increased metabolism, per se, does not stimu-
late the hypophysis to produce any. In view of the effectiveness
of injected hypophyseal extracts in promoting ovulation at this
time, it seems reasonable to conclude that insufficient gonado-
trophin is one condition of ovulatory delay in hibernating bats.
It is also known (loc. cit.) that the natural refractory status (to
ovulation) of the fall and early winter periods is gradually lost
during the later months of the hibernating period, for artificial
arousal in the later months often results in spontaneous ovulation,
and this occurs with increasing frequency as the end of the
hibernation period is approached. This phenomenon might be
explained by assuming that later in the hibernation period ovu-
lating hormone slowly produced by the pituitary of the torpid
animal has accumulated to the point where it exceeds the fol-
licular threshold of response and can therefore precipitate ovu-
lation in the awakened animal. A possible objection to this as-
sumption is the question as to whether the pituitary is even
capable of carrying out secretory functions at the low body
temperatures of the torpid bat. It seems more reasonable to
consider that the ovulatory function of the hypophysis of the bat,
as In many other mammals, may be dependent upon a stimulus
from the hypothalamus which in turn is activated reflexly by
intrinsie or environmental stimuli. The very periodicity of re-
production in bats suggests, @ priori, the existence of such a
neural mechanism, sensitive perhaps to as vet unknown environ-
mental stimuli.

Two further questions remain in respect to the mechanism of
delayed ovulation which [ should like to discuss briefly. One
concerns the probable identity of the specific gonadotrophic
hormone which is presumably lacking during hibernation, and
the other concerns further aspects of the neural mechanism
through which its release may ultimately be effected. While ovu-
lation was readily elicited with a commercial FSH preparation,
it does not follow that FSH is the ovulating hormone in bats any
more than the similar results obtained with Prolactin, TSH or
Growth hormone indicate that any of these are the physiological
ovulator. The common denominator in all of these pituitary
preparations could well be contaminating amounts of luteinizing
hormone (LH), the agent which is known in other mammals to
precipitate ovulatory responses in conjunction with FSH. The
very presence of the large follicle of hibernation presupposes
that FSH has already exerted its effects before the bat enters

1960 MAMMALIAN HIBERNATION 261

hibernation, and in all probability some ‘‘residual’’ FSH is still
present in the system of the hibernating female. Herlant (1956)
has recently produced some indirect evidenee which sugeests that
LH may be the speeifie ovulating hormone in the bat, and whieh
lends strong support to the notion that the ovulating hormone
is normally produced only at the end of hibernation, or in bats
prematurely aroused in the spring. Herlant’s work is a mae-
nificent study based on a correlation between marked seasonal
changes in pituitary cytology and the annual evele of reprodue-
tion in the female bat Myotis myotis. His results are summar-
ized in Mieure 2. He was able to demonstrate that the anterior
hypophysis contains five distinet types of chromophilic cells:
transitional types were not discerned. Three of these, basophils
1, 2 and 3 respectively, are distinet subtypes of the classical
‘‘basophiles’’, and two, acidophils 1 and 2, belong to the category
of classical ‘‘acidophiles.’’ All three of the basophils are PAS-
positive, and ‘‘basophil 3’’ is, in addition, positive to aldehyde-
fuchsin. On the basis of evelie changes in the number and stain-
ing intensities of these cells correlated with the reproductive
eyele on the one hand, and the annual ecyele of activity on the
other, ITerlant was able to deduce the probable functional specif-
ity of all five chromophilie cells. The aldehyde-fuehsin cell (baso-
phil 5) secretes TSH as in other mammals, and acidophil 2 prob-
ably secretes: ACTH and Somatotrophin. The remaining three
cells are ‘‘gonadotrophs’’, basophil 1 secreting LH, basophil 2
FSH, and acidophil 1, whieh is erythrosinophilie, luteotrophin
(prolactin). In the figure the cyche changes in these cells are
shown to be correlated with the various events of the reproductive
evele known in other mammals to be conditioned by these gonado-
trophic hormones. Thus, basophil 1, which is presumed to secrete
LH, hypertrophies at the time of ovulation, attains maximum
development during gestation, and undergoes massive involution
following parturition, coinciding with the degeneration of the
corpus luteum. Basophil 2 is presumed to secrete FSH. It hyper-
trophies at the time of the autumn rut, remains well developed
through hibernation, but involutes during early pregnaney. The
functional specificity of this cell is further attested by the solu-
bility of its granules in trichloroacetic acid (ef. Ladman and
Barrnett, 1955). The erythrosinophil, which is presumed to se-
crete luteotrophin, shows two phases of activity ; the first coincides
with ovulation, and is followed by a brief involutionary phase,
while the second which begins in late pregnancy, is contempor-
aneous with lactation. In females failing to lactate, this second

MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

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1960 MAMMALIAN HIBERNATION 263

peak of erythrosinophils is not observed. Herlant’s beautiful
study should be eonfirmed in other species of hibernating bats,
and if such confirmation is forthcoming it will provide strong
presumptive evidence that the lone sought endoerine basis of
delayed ovulation in bats has been found.

It is common knowledge that in many vertebrates seasonal
reproduetive periodicity is conditioned by external environmental
stimuli, the responses being mediated through a neural mechanism
involving the activation of a ‘‘sex center,’’ presumably in the
hypothalamus, by incoming sensory impulses. It has also been
demonstrated that specific reproductive events may be dependent
upon appropriate ‘‘psvehie’’ stimuli, as for example the depend-
ence of ovulation on a coital stimulus in the so-called ‘‘induced
ovulators.’’ The basie mechanism is presumably the same im
both instances, activation of the gonadotrophie function of the
hypophysis either by neural, or neure-humoral stimuli from the
hypothalamus. Recently, evidence has been presented (Sawyer,
et al., 1949; Everett, 1952) which indicates that even in spon-
taneously ovulating mammals, such as the rat, the release of
ovulating hormone (lH) may be elicited by a neuro-chemical
mediator arising in the hypothalamus, and that estrogen is an
effective stimulus for its release. The mechanism in the rat
appears to differ from that in the rabbit only in its spontaneity.
Furthermore; estrogen-induced ovulation in pregnant rats is
blocked by anti-adrenergie and anti-cholinergie drugs (pento-
barbital and atropine respectively) if these are administered soon
after the estrogen injection.

At present nothing definite is known concerning possible neural
involvement in the reproductive processes of hibernating bats,
but the characteristic reproductive periodicity and the asyneh-
ronous release of pituitary gonadotrophins implied in Herlant’s
(1956) work suggest strongly that neural mechanisms may in
fact be involved. Taking his cue from the work of Sawyer ef al.
(1949) and Everett (1952), Herlant (1954) recently attempted
to determine whether raising the estrogen levels in hibernating
bats (Myotis myotis), which are in a state of ‘‘sub-estrum’’ dur:
ing the winter sleep (Guthrie and Jeffers, 1958; Wimsatt, 1944b ),
would stimulate the ovulation reflex. Eight adult females were
procured in February and maintained at 15°C in the laboratory.
Each bat received subcutaneously 0.1 mg estradiol proprionate at
48 hour intervals, and were autopsied 2 to 10 days after the first
injection. Seven of the eight injected animals ovulated, as did
one of the uninjected controls, although Herlant did not indicate

I 64 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

how many control animals were used. The results are highly
suggestive, but unfortunately were carried out too late in the
year to preclude the possibility that spontaneous ovulation had
oceurred in the activated animals. Should similar experiments
earried out earher in hibernation, when spontaneous ovulation
does not oeeur, prove successful, and especially if the response
is blocked by adrenergic drugs, strong presumptive evidence
would be provided that LIT release in the bat, as in the rat and
rabbit, is mediated by a neuro-chemical mechanism. If this can
be established, then the way is open for an experimental analysis
of the environmental and/or intrinsic factors which activate the
hypothalamic response, and the eventual elucidation of the com-
plete physiology of delayed ovulation in hibernating bats.

Summary

The reproductive cyeles of hibernating bats display many
peculiarities which undoubtedly have arisen in consequence of
the evolution of the hibernating habit and the superimposition
of the period of hibernation on the season of reproduction, for
corresponding peculiarities are not observed in tropical, non-
hibernating bats. While the annual sequence of reproductive
events has been well worked out for many species, an integrated
picture of the underlying endocrine and, possibly, neural mechan-
isms Involved has not yet been achieved in any bat, nor have the
immediate effects of hibernation per se on these mechanisms
been adequately studied. This paper presents a review of the
reproductive peculiarities of hibernating bats, and focuses at-
tention on what, in the author’s opinion, are some of the funda-
mental problems needing further experimental analysis. These
include: in the male, determination of the true endocrine status
of the testis during hibernation, and related to this, an investiga-
tion of the apparent asynehronous production of pituitary
vonadotrophins regulating the two aspects of gonadal function
(production of spermatozoa and androgen respectively) ; and in
the female, elucidation of the physiological mechanisms under-
lying the central peculiarity, the delay of ovulation through
hibernation, and the specific influences of hibernation and ex-
ternal stimuli on the operation of these mechanisms. Recent
experiments by the author and others which indicate their prob-
able nature are discussed.

1960 MAMMALIAN HIBERNATION 265

REFERENCES
BAKER, J. R. AND T. F. Birp
1936. The seasons in a tropical rain forest (New Hebrides). — Part 4.
Insectivorous bats (Vespertilionidae and Rhinolophidae). J.
Linn. Soe. London, 40:143-161.

CHRISTIAN, J. J.
1956. The natural history of a summer aggregation of the big brown
bat, Eptesicus fuscus fuscus. Am. Midl. Nat., 55:66-95,

COURRIER, R.
1927. Etude sur le déterminisme des caractéres sexuels secondaires
chez quelques mammiféres a l’aectivité testiculaire périodique,
Arch. Biol., 37:173-334.

EVERETT, J. W.
1952. Presumptive hypothalamic control of spontaneous ovulation.
Ciba Found. Colloq. Endoerinol., 4:167-178.

GUILDAY, J. E.
1948. Little brown bats copulating in winter. J. Mammal., 29:416-417.

GUTHRIE, M. J.
1933. The reproductive cycles of some cave bats. J. Mammal., 14:199-
215.
GUTHRIE, M. J. AND K. R. JEFFERS
1938. The ovaries of the bat Myotis lucifugus lucifugus after injection
of hypophyseal extract. Anat. Rec., 72:11-36.

HERLANT, M.
1953. Etude comparative sur l’activité génitale des cheiroptéres. Ann.
Soc. Roy. Zool. Belge, 84:87-116.
1954. Influence de oestrogénes chez le murin (Myotis myotis) hiber-
nant. Bull. Acad. Belge, Cl. Sci., (5) 40:408-415.
1956. Corrélations hypophyso-génitales chez la femelle de la chauve-
souris, Myotis myotis (Borkhausen). Arch. Biol., 67:89-180.

KrutscH, P.
1956. The reproductive cycle in the male bat of the species Pipistrellus
hesperus. Anat. Rec., 124:321.

LADMAN, A. J. AND R. J. BARRNETT
1955. The localization of glycoprotein hormones in the adenohypophy-
sis by combined use of differential protein solubilities, histo-
chemical staining and bioassay. J. Histochem. Cytochem., 3:591.

MatTTHeEws, L. H.
1937. The female sexual cycle in the British horse-shoe bats, Rhino-
lophus ferrum-equinum insulanis Barrett-Hamilton and &. hip-
posideros minutus Montagu. Trans. Zool. Soc. London, 23:224-
266,

P66 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

MILLER, R. E.
1939. Reproductive ceyele in male bats of the species Myotis lucifugus
lucifugus and Myotis grisescens. J. Morph., 64:267-295.

Pearson, O. P., M. R. Kororp anp A. K. PEARSON
1952. Reproduction of the lamp-nosed bat (Corynorhinus rafinesquet )
in California, J. Mammal., 33:273-320.

Sawyer, C. H., J. W. Evererr anp J. E. MARKEE
1949. A neural faetor in the mechanism by which estrogen induces the
release of luteinizine hormone in the rat. Endoerinol., 44:218-

oe
ye

Siegen, J. HH.
1955.) Cytochemienal and histophysiological observations on the baso-
phils of the anterior pituitary gland of the bat, Myotis lucifugis
lucif/ugus. J. Morph., $6:225-264.

SLUITER, J. W. AND L. BELS
1951. Follicular growth and spontaneous ovulation im captive bats
during the hibernation period. Koninkl. Nederl. Akad. Wetensch.,
Amsterdam, (C) 54:585-593.

Wimsarr, W. A.
1942.) Survival of spermatozoa in the female reproductive tract of the
bat. Anat. Ree., 83:299-307.
1944an. Further studies on the survival of spermatozoa in the female
reproductive tract of the bat. Anat. Ree., 88:193-204.
1l944b. Growth of the ovarian follicle and ovulation im Myotis licifugus
lucifugus. Am. J. Anat., 74:129-173.

WIMsATT, W. A, AND F. C. KALLEN
1957. The unique maturation response of the Graatian follicles of
hibernating vespertilionid bats and the question of its signifi-
cance. Anat. Ree., 129:115-132.

Winsart, W. A. AND IL. TRAPIDO
1952. Reproduction and the female reproductive cycle in the tropical
American vampire bat, Desmodus rotundus murinus. Am, J.
Anat., 91:415-446,

DISCUSSION FOLLOWING WIMSATT’S PAPER

HOCK pointed out that it is not quite true to believe that
vround squirrels show no reproductive or embryological changes
during hibernation, and that breeding takes place only in the
spring. He noted that the earhest appearing hoary marmots in
the spring have been found to have two 2.5 em embryos in place,
which means that breeding must have occurred the previous fall.

1960 MAMMALIAN HIBERNATION 267

WIMSATT replied that he did not know of this, but realized
that some early spermatogenic changes oeeur in the woodehuck
and hedgehog prior to hibernation but that these are still essen
tially spring-breeding species. He emphasized that the main fact
to remember is that except in bats hibernation is a period of
sexual quiescence.

HOCK indicated that spermatogenesis has proceeded to the
secondary spermatoeyte stage before animals enter hibernation.

MENAIKER asked if bat sperm had been stored in vitro at
temperatures close to those encountered in hibernation. WIM-
SATT knew of no such work and remarked that, if required,
collecting sperm from a female reproductive tract would be a
considerable task. LYMAN asked what the distribution of sperm
was in the female bat reproductive tract. WIMSATT repled that
most of the sperm were located in the fundi of the uterine elands
and in the uterine lumen; usually they are oriented perpendie-
ularly with their heads against the epithelium.

BRATTSTROM stated that Wade Fox (unpublished observa-
tion) has shown that female garter snakes (Thamnophis) can
retain and nurture sperm in special saes for at least six months.

SCHONBAUM pointed out that JOHANSSON had mentioned
the presence of a large quantity of androgen in brown fat, but in
rodents. Were androgens in high titer in bat brown fat? WIM-
SATT rephed that it was possible, but again he knew of no
evidence for this.

FOLK pointed out that he and Grindeland had information
which indicated that the estrous evele of the hamster continues
into hibernation. He asked WIMSATT to comment on. this.
WIMSATT rephed that sex organs proved remarkably unre-
sponsive in the torpid animal in experiments using gonadotrophic
hormones, and that if torpid tissues are unresponsive to hormones
he could not see how hormones could initiate arousal or ‘‘ precip-
itate anything’’ in the torpid animal. He stated that his experi-
ments took place during a short term and do not rule out the
possibility of hormonal effects over a long period in the hiber-
nating animal,

PLATE

Kie. 1. Surviving follicle of hibernation in the ovary of the bat ALyotts
lucifugus lucifugus showing the characteristic vacuolation of the cells of the
diseus proligerus. The follicle maintains this appearance throughout the
hibernation period, and only experiences preovulatory growth and rupture
after the bat emerges from hibernation in the spring (Hematox. & eosin).

hig. 2. Surviving follicle of hibernation in the bat Hptesicus fuscus fuscus
illustrating the rich deposits of glycogen responsible for the vacuolation of
the discus cells (Bauer-Feulgen stain).

SV
STRESS AND NEUROSECRETION IN THE
HIBERNATING HEDGEHOG

By Paavo SUOMALAINEN
Department of Physiologic Zoology
University of Helsinki
Helsinki, Finland

In recent vears, Selve’s theories (1950) concerning the adapta-
tion syndrome and the hypophyseo-adrenal system have aroused
considerable controversy, but have also shed new light on many
problems.

Jf an individual is continuously exposed to stress, the resulting
adaptation syndrome evolves, according to Selye (1950), in
three stages: (1) an initial alarm reaction, which is defined
as the sum of all the non-specific systematic phenomena elicited
by sudden exposure to stimuli to which the organism is not
adapted; (2) the stage of resistance, adaptation proper, when
the body’s compensatory reactions to stress develop; and (38) a
phase of exhaustion which occurs if exposure to stress is exces-
sive.

Agents causing merely local damage, which requires no general
adaptive adjustment, are relatively mild alarming stimuli, while
those which evoke intensive adaptation responses produce severe
alarm reaction svmptoms. Examples of the latter are cold and
fastine, both characteristic of hibernation.

The adaptation syndrome is characterized by a number of
morphologic and functional changes. A convenient indicator of
stress is the blood picture. In the south of Finland, hedgehogs
are in their normal summer condition from the middle of June
to the middle or end of August. On account of the preenancy
of the females in late May and in June we have investigated
only males at this season. Between these summer hedgehogs
and hibernating animals there are remarkable differences (Table
I). During hibernation we find all the blood changes typical
of stress — leucopenia, neutrophilia, eosinopenia and lympho-
penia. But the animals investigated in the autumn, before the
onset of hibernation, and in the spring when they have wakened
up are also interesting. In the autumn there is already a clear
neutrophilia, eosinopenia and lymphopenia. In the south of
Finland, hedeehogs emerge from hibernation in the beginning

272 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

of May. After this the figure for neutrophils is almost the same
as in the autumn. The blood count shows a clear neutrophilia and
Ivmphopenia without eosinopenia. Hedgehogs brought into arti-
ficial hypothermia in a refrigerator in July resemble those in
natural hibernation. The marked ‘‘shift to the left’? during
hibernation indicates that the bone marrow is active even in
hibernating animals.

TABLE I

Leucocyte Picture of the Hedgehog

Neutrophils
Jo | Hosino- 3aso- Lympho- Mono-
WBC Band Seg- phils phils cytes cytes
per cu. mm. form mented % % %o %
In spring 18,700 2.6 47.4 6.1 1.6 38.5 3.
In summer 18,100 1.8 35.0 Fal 2.0 55.1 1.0
In autumn 17,700 5.7 42.3 2.1 0.9 48.3 0.7
In hiber- 4,100 AT. 28.3 ie? 0.8 20.9 1.8

nation

To judge from the blood count, the adaptation syndrome is
already very pronounced in the autumn, even before the onset
of hibernation (ef. the low figure for eosinophils). In hiber-
nating animals the adaptation syndrome is at its most imtense.
In the spring when the animals emerge from hibernation, the
stress picture is less.

In response to the various stress stimuli the body shows a
eommon syndrome, which includes discharge of adrenal hor-
mones, hypertrophy of the adrenal cortex, involution of the
lIvmphatie system and disturbances of the gastrointestinal tract,
kidney and other organs (Selye, 1950).

Table Il shows the variations in the weight of the adrenal
olands relative to the body weight at different seasons of the
year. The adrenal glands are distinctly enlarged during hiber-
nation. We have also estimated microscopically, from sections
made as near the median line as possible, the ratio of the area
of the cortex to the area of the medulla and to the whole adrenal
oland. The figures in Table II show that it is the cortex which
is especially enlarged during hibernation. The clearest change
is in the zona reticularis.

Autumn and spring are obviously times of stress for animals
and also for man. The transition from the summer condition to

1960 MAMMALIAN HIBERNATION 2

the winter condition and vice versa is not at all easy from the
physiological point of view. And these seasons are still more
difficult for animals that hibernate; their heat balance is then
radically changed. In response to the stress stimuli the adrenal
cortex has an important part to play. In the histophysiologic
investigation of the adaptation syndrome, attention was chiefly
devoted to the thickness of the adrenal cortex, and to the number
and position of the lipid granules in the cortex.

TABLE II

Relative Size of the Adrenal Gland and of the Adrenal Cortex
in the Hedgehog

Relative
adrenal

weight Area of cortex /
(mgm/100 gm area of the whole Area of cortex /
body-weight) adrenal gland area of medulla
In spring 53
In summer 50
Tn autumn 53 0.86 7.0
In hibernation 62 0.93 14.2
In early spring 52

In the autumn the histologic picture of the cortex is already
changed (Suomalainen, 1954). The cortex is certainly not yet
enlarged and its zones can be clearly distinguished from each
other. But the lipid granules are situated in the zona reticularis
(in summer chiefly in the zona fascicularis), and are not found
in other zones. A decrease in the sudanophilic substance is a
sign of increased activity and a shght alarm reaction.

In adrenals investigated a couple of weeks before the onset
of hibernation, an exceptionally large number of degenerating
cells were found in the cortex (Suomalainen, 1954). The amount
of lipids was increased in all zones of the cortex, but especially
in the zona glomerulosa. The cortex had passed into the resist-
ance stage of the adaptation syndrome.

During hibernation, the resistance stage seemed to continue
(Suomalainen, 1954). The cortex was clearly swollen and there
were no sharp boundaries between the zones. The zona reticularis
was enlarged and the cells of the zona fascicularis shghtly
swollen. The degeneration of the cells was evidently very
marked. The lipids were concentrated in the outer parts of the

274 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

cortex and elsewhere diminished, but they were still present in
large amounts. In deep hibernation, in the cold of midwinter
when the body temperature had fallen as low as +2 to 4°C,
the cortex, with the exception of the zona glomerulosa, was very
disorganized. The amount of lipids had decreased everywhere,
and the lipids seemed to have broken up into very fine droplets.
The activity of the gland was obviously increased.

In the hedgehog awakened from deep hibernation in winter,
the zona reticularis was very cavernous, and even the zona fasei-
cularis was spongy and contained cells that were in the process
of degeneration (Suomalainen, 1954). The pid granules had
almost entirely disappeared, but sometimes they were present
in the zona glomerulosa as very fine droplets. The histologic
picture corresponded either to a strong alarm reaction or to the
exhaustion stage of the adaptation syndrome.

In addition to the decrease in the sudanophilic substance
of the adrenal cortex, a simultaneous decrease in the sudanophilic
substance of the hibernating gland of the hedgehog can be
demonstrated (Suomalainen, 1954). Investigations made in my
department have shown that the sudanophile substance decreases
in the hibernating gland, especially when the animals emerge
from hibernation, At the same time the granules are greatly
reduced in size. This, too, shows that emergence from hiberna-

tion iS a severe physiologic stress.

TABLE III

Cholesterol Content (mem-; ) of the Adrenals,
Ilibernating Gland and Serum of the Hedgehog

Total cholesterol

Cholesterol esters

Hiber- Hiber-
nating nating
Adrenals gland Serum Adrenals gland Serum
In spring 772 173 193 478 29 137
In summer 1103 218 236 698 50 155
In autumn 1580 223 Ok 1042 36 Ls
In early winter 778 198 L99 318 32 196
In midwinter S95 209 233 DOS 23 170

It is interesting to compare cholesterol determinations made
from the adrenals and hibernating gland with the histophysio-
logic investigations. According to Selye (1950) and others, the
cholesterol content of the adrenal cortex is reduced during the

1960 MAMMALIAN HIBERNATION 275

alarm reaction and the exhaustion stage of the adaptation syn-
drome, and is normal or increased during the stage of resistance.
Table [1] shows the means of our determinations. In the autumn
the Instologic picture of the adrenals revealed a slight alarm
reaction. At the same time their cholesterol content was slightly
reduced. In midwinter the hibernating hedgehogs, according to
the histologic investigations, were in the resistance stage. The
cholesterol content was then shghtly increased. The cholesterol
content of the hibernating gland parallels the changes in the
cholesterol content of the adrenal e@lands.

TABLE IV

Kk * Content of the Blood and Nat, Me*t* and
Ca** Content of the Serum in the Hedgehog (mem/100 ml)

Kt Nat Mgtt Catt

In spring 160 393 4.5 10.1
In summer 161 389 ae 10.3
Lu autumn 127 393 4.0 10.0
5.8 10.4

[In hibernation 104 418

In an intact animal the administration of mineralocorticoids
influences the level of potassium and sodium in the plasma; the
plasma is depleted of potassium, and the sodium concentration
increases. As is evident from Table IV, the K* content of
hedgehog blood is already reduced in autumn before hiberna-
tion and reaches a minimum during hibernation (Suomalainen,
1956). The Nat content of the serum, on the other hand, is
shehtly raised. Hibernation does not appear to have any effect
on the serum Cat* content. A feature typical of hibernation
in the hedgehog, and also of hypothermia in many poikilothermic
animals, 1S an increase in the Me** content of the blood.

TABLE V

The Relative Size of the Lymph Nodules (or Follicles) in the
Cortex of the Lymph Node in the Hedgehog

In the In the

neck froin
In autwnn 179 205

In early winter 99 144

276 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The general adaptation syndrome with adrenocortical en-
largement is usually accompanied by an involution of the lym-
phatic organs (Selye, 1950). Table V shows the means of the
relative size of the lymph nodules in the cortex of the lymph
nodes in the neck and in the groin of active hedgehogs and of
hibernating ones.

The control of the adaptation syndrome is complicated. It
is obvious that the hypophysis plays a central part through
secretion of the adrenocorticotropic hormone (ACTH) following
stress. Again, many experiments of recent years (Scharrer,
19560; Bargmann ef al., 1958) make it appear likely that nerve
fibers from the anterior part of the hypothalamus terminating
around the multitude of capillary loops in the median eminence
of the tuber cinereum liberate some chemical mediator into the
blood by which it is conveyed to the adenohypophysis to regulate
its secretory activity. Under conditions of stress the liberation
of ACTH appears to be excited by nervous influences acting on
the hypothalamus both directly and indirectly through neural
pathways from the cortex and the anterior nuclei of the thala-
mus. Stress may also effect ACTH secretion through variations
of the blood supply to the hypophysis. It is also likely that
adrenaline released from the adrenal medulla in stressful states
may have a subsidiary effect in exciting secretion of ACTII.
But there is some evidence that insulin, by facilitating the pas-
sage of glucose into the cell, plays a part in the reaction to
stress.

The investigations of my department (Suomalainen and
Uuspaa, 1958) make it evident that the adrenaline content of the
adrenal glands of the hedgehog usually increases just at the time
when the cortex, too, is activated on account of physiologic
stress (Fig. 1). This is especially the case during periods of
intense cold in midwinter. But even in autumn, before the onset
of hibernation, the ratio of adrenaline to noradrenaline has
already increased. From the investigations made in my depart-
ment it seems likely that the production of insulin, too, is in-
creased in hibernating hedgehogs (Suomalainen, 1956).

The nature of the humoral mediator (or mediators) liberated
in the median eminence of the tuber cinereum and involved in
the release of ACTH. is at present unknown. It is probable that
it is a polypeptide component of neurosecretory material, lib-
erated by the nerve endings in the median eminence and infun-
dibular stem (Suomalainen and Uuspaa, 1958).

1960 MAMMALIAN HIBERNATION 2

—~!
—~!

Neuroseecretory cells are nerve cells which show eytologie evi-
dence of secretory activity. They elaborate microscopically vis-
ible granules and droplets, which, in most cases, pass along the
axons toward the nerve terminals. In vertebrates, the nerve
terminals in which the neurosecretory material is stored make
up a great part of the posterior lobe of the hypophysis (Scharrer,
1956; Bargmann et al., 1958).

500

vem./gm,

400

Jn. Jy. A. 8. Oo. N. D. JI. FO M. A. M.

Hibernation

Fig. 1. Adrenaline and noradrenaline content of the adrenal glands of the
hedgehog in different seasons. , Adrenaline, @ (3), O (2); ---, nor-

adrenaline, w (2), YV(@). From Suomalainen and Uuspiaé (1958).

The neurosecretory substance stains characteristically with
chrome-alum-hematoxylin-phloxin and aldehyde-fuchsin. It is a
complex protein to which active polypeptides, such as vasopressin
and oxytocin, may be attached (Scharrer, 1956; Bargmann et al.,
1958).

Neurosecretory cells occur in various parts of the nervous
system. In mammals the neurosecretory cells of the hypothala-
mus form two conspicuous groups, the nucleus supraopticus and

278 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

the nucleus paraventricularis. In all vertebrates studied so far,
the axons of these two nuclei form a conspicuous fiber tract, the
tractus hypothalamo-hypophyseus, which descends toward the
hypophysis. In mammals, some of the fibers terminate in the
pars intermedia. The great majority of the axons arising from
neurosecretory cells in the hypothalamus end around blood
vessels in the neurohypophysis. The neurosecretory material con-
tains the hormones, which were formerly thought to be produced
by the posterior pituitary (Scharrer, 1956; Bargmann et ai.,
1958).

Because objective quantitative estimation of the neurosecretory
substance in microscopic slides is almost impossible, we have
used the volume of the cell nucleus as the index of its activity
(Suomalainen and Nyholm, 1956). Using the camera lucida, 100
cell nuclei, magnified 1500 times, were drawn from the nucleus
supraopticus of each animal. The sizes of the nuclei were
measured with a planimeter. The mean values are given in Table
VI.

The Table shows that the cell nuclei are at their smallest in
June. In July-August and in the pre-lethareic period of Sep-
tember-October the nuclei steadily increase in size. In hiber-
nating animals they are always bigger than in active ones. They
are largest in Mareh and then grow smaller during the post-
lethargic period in April when the animals are still hibernating.

The picture of the nucleus supraopticus-hypophyseal system
parallels these conclusions. In summer hedgehogs there is hardly
any secretory substance in most of the cells. The axons of the
hy pothalamo-hypophyseal tract stain poorly. At the distal end of
the tract are the Herring bodies, that is, axons with bulbous
swellings, containing considerable amounts of secretory sub-
stance. There is often a moderate quantity of this substance in
the neurohypophysis, also. The distal end of the latter, however,
frequently contains no secretory substance. We may conclude
that neurosecretion is rather sheht in summer.

In the pre-lethargie period in autumn the nuclei of the
nucleus cells have already increased in size. A large amount of
the secretory substance may even be present, in the form of
large droplets and coarse granules (Plate, fig. 1). There are also
cells containing less of the secretion, in finer granules. In the
intercellular substance there are often copious accumulations of
secretory granules. The axons of the tract may be quite thick
and intensely stained. Furthermore, there are dark Herring

1960 MAMMALIAN HIBERNATION 279

bodies, both large and small (Plate, fig. 2). Conelusion: Neuro-
secretion is more active, but the secretory substance is still stored
in the hypophysis.

TasLue VI
The Relative Size of the Cell Nuclei in the

Nucleus Supraopticus at Different Seasons

Awake Hibernating
In June 2.97 In November
2.60 December 3.80
3.00 3.02 1.85
4.26
Tn July 3.46 449 4.08
3.52
2.93 3.30 In January 4.80
= = 5.3
In August 3.58 498 48]
3.63
3.68 3.65 In February 4.62
——_ -_——- 5.35
In September 453 4.83
October 3.44
3.82 In March 5.05
3.63 4.75
4.35 5.A0 5.07
3.73 3.79 ;
_ In April 4.61
4.30

4.30 4.40

eLwake
In May v.41
3.90
2.89 3.27

From Suomalainen and Nyholm (1956).

In the hibernation period, the nuelei of the nucleus cells and
the cells themselves, too, are large (Plate, fig. 3). The neuro-
secretory substance is present as very fine granules, if it is visible
in the cells. The axons of the tract are thin but stain well.
They are most evident at the distal end of the tract. The eapil-
laries at the proximal end of the neurohypophysis are greatly
enlarged. Conclusion: Neurosecretion is intense during hiberna-
tion. The secretory substance is no longer stored in the neuro-
hypophysis except to some extent in midwinter.

280 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

In general, we can say that there is a clear correlation between
neurosecretion and stress in hedgehogs all the year round. Among
the recent surveys which have emphasized that the neurosecre-
tory substance contains the hormone stimulating the adeno-
hypophysis, I would especially mention the contribution of
Martini (1958) and Saffran et al. (1958) to the second interna-
tional symposium on neurosecretion at Lund in 1957, and the
investigations of Guillemin (such as 1956).

From investigations performed during the present decade it
is obvious that secretory activity also occurs in the subeommis-
sural organ (Seharrer, 1956; Bargmann ef al., 1958). This organ
constitutes a specialized area of the ependyma located beneath
the posterior commissure of the midbrain in the roof of the third
ventricle, in the region where the ventricle suddenly narrows
to become the cerebral aqueduct. Such an organ has been re-
ported from all the vertebrates investigated in this respect, with
the exception of a few mammals. In some cases, among the mam-
mals, subecommissural cell groups have withdrawn from the
ventricular position and have formed a glandular island beneath
the ventricular epithelium. They may also abandon the ancient
mode of secretory release into the ventricle and probably give
off their material into blood vessels. The subeommissural organ
of the hedgehog is apparently of this type. In Figure 4 (Plate)
you see a cross-section through this great group of cells. We have
demonstrated that they contain a substance which stains like the
neurosecretory substance (Plate, fig. 5), and which even forms
Herring bodies (Plate, fig. 6). We have not yet investigated
whether there is any seasonal rhythm in this secretion or in the
changes in the size of the nuclei which secrete it. But even the
discovery of a new center of secretion in the brain is interesting.

REFERENCES

BARGMANN, W., B. HAnstTrROM, B. SCHARRER AND E. SCHARRER
1958. Zweites Internationales Symposium tiber Neurosekretion. Berlin,
126 pp.

GUILLEMIN, R.

1956. Hypothalamic-hypophysial interrelationships in the production
of pituitary hormones in vitro. In: Fields, Guillemin and Carton,
Hypothalamic-hypophysial interrelationships. Springfield, 156
pp. (Pp. 46-57.)

MARTINI, L.

1958. Neurosecretion and stimulation of the adenohypophysis. Jn:

Bargmann et al., Pp. 52-54.

1960 MAMMALIAN HIBERNATION IS]

SAFPRAN, M., A. V. ScHALLY, M. SEGAL AND B. ZIMMERMANN
1958. Characterization of the corticotrophin releasing factor of the

neurohypophysis. Jn: Bargmann ef al., Pp. 55-59.

SCHARRER, E.
1956. Neuroseeretion. Jn: Fifth annual report on stress. Montreal,
Pp. 185-192.

SELYE, H.
1950. Stress. Montreal, 822 pp.

SUOMALAINEN, P.
1954. Further investigations on the physiology of hibernation, Proce.
Finnish Aead. Sei., 19538: 131-144.
1956. Hibernation, the natural hypothermia of mammals. Triangle,

2 227-233
or “avo.

SUOMALAINEN, P. AND P. NYHOLM
1956. Neurosecretion in the hibernating hedgehog. Jn: Brrvin
HanstrrROM. Zoological papers in honour of his sixty-fifth birth
day Noy. 20, 1956. Lund, Pp. 269-277.

SUOMALAINEN, P. AND V. J. UUSPAA
1958. Advenaline/noradrenaline ratio in the adrenal glands of the
hedgehog during summer activity and hibernation, Nature,
182:1500-1501.

DISCUSSION FOLLOWING SUOMALAINEN’S PAPER

BULLARD inquired as to relative versus absolute increase in
size of glandular tissue. SUOMALAINEN indicated that the
elands discussed (adrenal and cells of nucleus supraopticus )
increase in size both relatively and absolutely during hibernation.
BULLARD asked if a concomitant decrease in fat occurs.
SUOMALAINEN replied that it does. LYMAN asked if this
effect and the neurosecretory activity in hibernation were con-
fined to hedgehogs, and the reply was that it had been investi-
gated also in the golden hamster and found to be true there also.
LYMAN asked what time of year these effects were present.
SUOMALAINEN rephed that it was during hibernation.

DAWE observed that the nucleus supraopticus lay under the
thalamus, which could give credence to the existence of an
active ‘‘thermostat’’ in hibernation. SUOMALAINEN said the
supraoptic cells are the most probable source of neurosecretory
substance produced in the hibernating state. KEHIL asked if
the appearance of stainable material in the stalk of the hypo-
physis necessarily indicated active secretion, or rather might

282 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

indicate storage with a decrease in secretory rate. SUOMALAT-
NEN said it left this area and passed along the axons of the
tractus hypothalamo-hypophyseus into the neurohypophysis for
seeretion,

KISHER remarked on the impressiveness of this evidence for
specific tissue changes during hibernation. He asked if SUO-
MAULAINEN would be willing to omit the word ‘‘stress’’ in
discussing such changes. He pointed out that in working with
hibernating animals one must act very quickly, since changes
occur very quickly. From the microscopic pomt of view, one
can say that a stress occurs almost instantaneously. Conversely,
preparation for hibernation takes a very lone period of time
before the tissues are ready. This occurs slowly. As a function
of time, conditions are thus distorted when one refers to stress
changes occurring in hibernation. SUOMALAINEN replied that
winter and hibernation, at least in Finland on the north border
of the distribution area of the hedgehog, are physiological

stressors.

DENYES said that using Bush’s technique of analyzing for
freely circulating sterols it was found that the amount was
halved in hamster blood at cold exposures of 45 hours, and
that only trace quantities were present in hibernating hamsters.
She thought this was interesting since SUOMALAINEN had
found an accumulation of secretory material in the pituitary
during hibernation, whereas she had found practically no cir-
culating sterols. These two facts, she believed, indicated that
during hibernation secretory material is stored rather than cir-
culated in the blood.

ee Sees hae

PLATE

Fig. 1. Nucleus supraopticus cells in the hedgehog in autumn, 1125 x.
Fig, 2. Herring bodies and intensely stained axons in the hypophyseal tract
of the hedgehog in autumn. 1125 x. Fig. 3. Large nucleus supraopticus
cells in the hedgehog in early spring. 1125 x. Fig. 4. Cross-section through
the subcommissural cell group of the midbrain of the hedgehog, 52 x.
Fig. 5. Substance which stains like the neurosecretory substance in the
subeommissural cell group of the hedgehog, 1300 x. Fig. 6. Herring bodies
in the subcommissural cell group of the hedgehog. 1500 x.

XV
SOME PHYSIOLOGICAL PRINCIPLES
GOVERNING HIBERNATION IN
CITELLUS BEEKCHEY!

By FELIx STRUMWASSER
National Institute of Mental Health
National Institutes of Health

Bethesda, Maryland

A list of some eight principles governing hibernation in Citel-
lus beecheyt is presented and eiaborated upon in this paper.
In addition I will assert that hibernators did not evolve qualita-
tively new mechanisms for the manipulation of body temperature
and the maintenance of mammalian life systems at temperatures
just above 0°C. Instead, hibernation will be considered to involve
an extension of already available mammalian regulatory mech-
anisms for operation at low temperatures.

I. Hibernation in Citellus beecheyi
Takes Place in Successive Stages with

Complete Arousal Between These Stages

Figure 1 is a six-day plot of brain temperature in a partly
deafened summer squirrel after it had been in a 7°C environ-
ment for twenty days. As ean be seen, there are six major dips
in the temperature record. Three of them represent the normal
temperatures for sleep in the cold in this squirrel, the lows
falline between 33.8° and 34.6°C. Three other dips, imitiated
forty-eight hours apart, consecutively reached lows of 30.8",
27.9° and 22.9°C. Brain temperature had been continuously
recorded during the entire period in the cold and this was the
first sign of a phenomenon different in degree and pattern from
the usual nightly temperature drops. This pattern of decreasing
body temperature to successively lower levels with a complete
arousal each time is absolutely typical of all the squirrels
studied. Reasons will be developed shortly for calling each
of the hibernation drops, as long as they are followed by a
lower drop, a test drop and each of the minimum temperatures
reached a critical point. Eventually a final plateau of brain

IS6 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

temperature is reached and hibernation persists. The longest
duration of hibernation that I have so far measured in this
species, with continuous recording of brain temperature as the
criterion, was three days at 6.1° in an environment of 2°C.
Time course of the critical brain temperatures. Figure 2 illus-
trates the time course of the critical brain temperatures in both
winter and summer squirrels. The critical temperatures are
plotted at the time of the day at which they were first reached.
As a general rule, whether the animal was a winter or a summer
animal, hibernation took place with the same pattern. However,

~
J}
(
ma
—
>
—_
)
j
J
i

w
ou
SZ
al
J

BRAIN TEMPERA TURE IN °C
3
: ea

nN
on
Sonn ne

——

LIGHT SCHEDULE :

12M I12N 12M 1I2N I2M 12N 12M 12N 12M 12N 12M 12N 12M
TIME OF DAY

Mig. 1. A 6-day segment of the brain-temperature history of a summer
ground squirrel during which time it took its first 3 test drops. Brain tem-
perature plotted every hour. On the time axis midnight and noon are
indicated by M and N, respectively.

during the summer there was a long delay, as long as twenty-six
days in one animal, before the first test drop occurred. As can
be seen, the relationship between critical brain temperature
and time in days after placement into the cold is not linear
and the curves of the two winter animals initially parallel each
other. The curves of the winter animals are separated in time
because one entered hibernation twenty-four hours later than
the other; note also that one animal operates on a twenty-four
hour hibernation schedule initially (closed circle), and the other
on a forty-eight hour schedule (open circle). Because there is

1960 MAMMALIAN HIBERNATION 287

some orderliness in this process I think of these curves as
perhaps paralleling the integrated rate of general metabolic
preparations necessary for the journey into hypothermia. This
is not the only possible process occurring during the preparatory
phase. It is tempting to suggest some alteration in brain circuitry
or adaptation of synaptic transmission for effective operation at
low temperatures as alternative or additional possibilities.

WINTER

~@ SUMMER

a ee Ad

“ EVOKED AROUSAL

CRITICAL BRAIN TEMPERATURE IN °C

——

5 10 15
TIME IN DAYS AFTER PLACEMENT INTO COLD

)

Fig. 2.
squirrels. Critical temperatures are plotted at time of day : yhic Ly
i Is. Critical tem] t plotted at time of day at which they

Time course of critical brain temperatures in winter and summer
were first reached.

II. Body Temperature Can Be Set and
Regulated in This Hibernator Over a Wide Range

Regulation of brain temperature at the critical point. Figure
3 illustrates the kind of regulation of brain temperature ob-
served at three critical points all taken from one squirrel. Note
that the strips read from right to left. In A, as 31.5°C is reached
from a slowly falling temperature, a sharp inerease in tempera-
ture is initiated rising to 32.1°C; from this peak, temperature
again falls slowly, repeating the same pattern as 31.5°C is

Vol. 124

MUSEUM OF COMPARATIVE ZOOLOGY

BULLETIN :

ISS

‘LO.LUDs

VUES Oy UL ‘XOALpoodsoul ‘syutod [VoTLty Judo sep E pv WOTPLING IL ot Jo Suotptod of: 4) pul — a CG pues OD -g pure’ o-: Vf] OT Pye re
“SO Avotpur sour pr oyy 4-p sduqs up ‘yurod [wont of} Je omyriodutoy, uri JO worrpnsoy

WO. Spvo.l

OUT OJON

eS L

Se ees a a

ae a

“BIT

1960 MAMMALIAN HIBERNATION 289

reached, as in Bb. Strips C and D depict the same phenomenon
at the lowest temperature arrived at during another test drop
in this animal. The regulation in strip / is of interest because it
consists, in the right half, of oscillations varying between 0.06°C
and 0.1°C (see G@ also). Such inereases in temperature at the
critical point are brought about by shivering. This behavior
of brain temperature at the lowest level reached for each test-
drop into hibernation — that is, making several passes toward
the critical point — is typical in all the squirrels studied. Ap-
parently, the final lower limits of the temperature of each
hibernation test drop are regulated and are not allowed to fall
below a certain level of the critical point, apparently within
0.1°C and probably less.

Although there are, as can be seen, gaps of the critical brain
temperatures in each of the individual cases of Figure 2, when
all the animals as a group are examined we find no preferred
temperatures and no large gaps in the physiological temperature
range. We are forced to conelude that this squirrel can probably
maintain and regulate its temperature anywhere between 6° and
39°C.

Ill. The Timing of Hibernation Can Be Coordinated with
the Normal Activity Rhythm

The long wait of the summer animals allowed us to study the
timing of the daily temperature rhythm and its relation to
hibernation. For example, we followed one individual’s brain
temperature for twenty-seven days, noting for each day the times
that the evening drop and the morning arousal were initiated.
Excluding the first seven days in the cold when there was a
systematic shift, not to be discussed here, the range of time for
the evening drop extended from 4:26 p.m. to 8:06 p.m. It is
noteworthy that on the three occasions of hibernation test-drops,
the initiation occurred within this range. All arousals were
initiated before the light period but perhaps it is more interesting
that the arousal from each of the three hibernations was initiated
within the normal range of variation, 2:08 a.m. to 7:41 a.m.,
despite the fall of brain temperature being as large as 15°C
in the third hibernation. Figure 4 shows a plot of arousal at
what we call preferred hourly intervals. The groups are ar-
ranged in order of consecutive time of day; members of a group
are arranged in order of consecutive days. As can be seen,

29() BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

there are several groups separated by approximately one hour
intervals.

Where the number of arousals is sufficient to test for signifi-
cance between the means of adjacent groups (groups 2, 3, 4 and
5), the differences are found to be highly significant (¢ test for

" aren

e
° ]
° 12 GROUP 2, 7:12:56

—— GROUP 3, 6:10°5)

TIME OF AROUSAL (A-M.)
a

4 GROUP 5, 4:0023:°8
e
3h
t
e
2r
SSS 22S ses

1 aes cas Cee
2 5 9 15 18231016 20266 7 8 13 142712 I7 19 21 2224254 II
DAY

Fig. 4. Arousals at preferred hourly intervals. Groups arranged in order
of consecutive time of day; members of a group arranged in order of
consecutive days. Limits of groups 2, 3, 4 and 5 are shown by brackets;
mean of each of these groups is given as time of day, with the standard

error in minutes.

small samples, P < 0.001 in all cases). Note that the first hiberna-
tion arousal (day 22) occurred at 4:00 a.m. and the second hiber-
nation arousal (day 24) occurred at 4:06 a.m. amid a group of
seven arousals averaging 4:00 a.m. with a standard deviation
of 10.1 minutes. The third arousal occurred at 6:06 a.m. which
was amid a group of four arousals averaging 6:10 a.m. with a

1960 MAMMALIAN HIBERNATION 29]

standard deviation of 10.1) minutes. Apparently arousals,
whether they are from test-drops or from normal sleep occur
at preferred time intervals, roughly one hour apart while initia
tion of hibernation test-drops, like the normal nightly tempera
ture drops, occurs randomly within a limited portion of the day.

A time sense which is temperature independent. This animal
must then have a time sense most probably due to an internal
clock which must be temperature-independent or temperature-
compensated since the initiation of arousal most usually antici-
pates the heht period and apparently occurs at preferred
hourly time intervals with variation in each class of only a few
minutes despite the large decline in brain temperature. How-
ever, all arousals are not clock-initiated, at least not all from
hibernation. Arousals from test drops are most likely to be since
the test drops are short-lasting phenomena. We have seen arousals
from deep hibernation (where brain temperature is close to
6°C) that seem to be clearly clock-initiated, because they fall into
these preferred hourly intervals, but there are some that are
obviously not. Some other internal mechanism is presumably
responsible for these arousals.

by 1e Triggering of Entrance into Hibernation is
IV. The Trigg
Dependent on the Brain Integrating Three Factors

Three classes of hibernators within rodents. An examination
of the information available justifies the recognition within the
rodents of at least three classes of hibernators: I, those that wait
a variable but relatively lone time in the cold—one to three
months according to Lyman (1948) for the hamster, Mesocricetus
auratus —and then enter deep hibernation in one decline in
temperature ; II, those that do not wait more than a few days and
then enter deep hibernation in one decline in temperature, for
example, pocket mice, Perognathus longimembris (Bartholomew
and Cade, 1957) ; III, those that wait only a few days but never
enter deep hibernation in one decline, going through a series of
consecutively lower temperatures and arousing completely in
between hibernation drops. Besides Cifellus beechey!, this group
probably includes Citellus tridecemlineatus (Johnson, 1951;
Foster, 1934; Zalesky, 1934), apparently the dormouse Myorus
glis (Wyss, 1932), probably the marmot JMarmota marmota
(Dubois, 1896), the woodehuck Marmota monar (Rasmussen,
1916; Benedict and Lee, 1938), and possibly Crtel/us parry:
(Musaeehia and Wilber, 1952; Svihla and Bowman, 1952).

292 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Critical points, test drops and preparations. The difference
between these groups may lie only in the fact that the first waits
until it is fully prepared before entering hibernation, the second
may be virtually prepared at all times or needs very little
preparation, while the third group begins test drops while prep-
arations are in progress and goes only as far as the preparations
have gone. Certainly the fine regulation of the low temperature
reached at each entrance is suggestive that it is rather critical
to the squirrel not to yo below it for that day. This is the reason
these are called critical points. It is proposed that at each

PERMISSIVE
ENVIRONMENTAL
FACTORS

Se ee NEST) Fe ot A BODY _ Tempematuge = 4
cae a DROP THERMOSTAT COMPENSATION

|
THE RMO-
EFFECTORS

Fie. 5. Three-factor theory of hibernation.

drop there is some testing of how well preparations have gone,
so for the time beine these drops before the final plateau are
labeled test drops.

A three-factor theory of hibernation. Figure 5 summarizes
our findings, so far, in a diagram which explains, or attempts
to, how the triggering of hibernation might be accounted for.
There are three main factors which are necessary to account for
the time when the thermostat is turned down.

a. Permissive environmental factors. When environmental
factors are ‘‘permissive,’’ biochemical preparations for hypo-
thermia proceed. Lack of adequate environmental factors may
result in the preparations not proceeding at all (7, Fig. 5).
Both internal and external environmental factors are important

1960 MAMMALIAN HIBERNATION 293

in this concept. One would guess that a serious competitor for
hibernation would be reproductive drive, even in the presence of
permissive external factors; the latter requirement may be dif-
ferent from species to species and would consist of factors such
as noise, terrain, level of environmental temperature, food supply
and so on.

hb. State of preparations. The preparations are considered
to be biochemical and necessary for survival of the total animal
under conditions of prolonged hypothermia. The body thermo-
stat, by being kept informed of the state of preparations, can
pull the trigger for hibernation if these are sufficiently under
way by the correct time of day.

ce. Temperature independent internal clock. The clock allows
the animal to synchronize the various stages of hibernation with
its normal species-specific activity schedule in the field and for
this reason is of considerable survival value. If the preparations
have reached a certain stage and have done so by a certain critical
portion of the day and there are no interfering environmental
Factors (3, Fig. 5), the thermostat activates the adequate thermo-
effectors and the animal at this time is said to be entering
hibernation,

Some requirements for central (brain) integration of the three
factors. Whatever biochemical processes preparatory to the
Journey into, hypothermia are proceeding in the cold environ-
ment, they must exert an influence on the temperature regulating
mechanisms, since this mechanism seems to be able to compute
just how cold it can permit the animal to become. It is also
conceivable that through some feedback mechanism the state of
preparations is informed of just how well the body machinery
is performing at each of the critical temperatures and can make
the appropriate adjustments, if needed, for the next drop (left
dotted line, Fig. 5). Evidence for this may possibly come from
the observation that each succeeding entrance into hibernation
occurs at a faster over-all rate (Fig. 6). Additional flexibility
of the system may be gained if the preparations may be speeded
up (or slowed down) depending on the time of day, (2, Fig. 5).

Another feedback mechanism allows the internal clock to be
informed of the temperature/time characteristics of the drop;
this would give the clock, by a mechanism of computing the neces-
sary compensation for temperature effects, its temperature inde-
pendent appearance (right dotted line, Fig. 5).

To sum up, the squirrel is rather particular as to when it drops
its body temperature; preparations must be at some critical

2O4 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

level before it will do this and when it takes its initial drops it
will go only as far, apparently, as these preparations safely allow
it to 20.

VY. The Curves of Cooling/Time During
Kutrance into Hibernation are Under Close Control
and Represent Something More than Passive Cooline

Form of temperature curves of consecutive entrances inte

hibernation. Figure 6 is a plot of four consecutive entrances
into hibernation from the same squirrel, terminating at four

ENTRANCE INTO HIBERNATION
2nd
Spal © 3rd
z @ 4th
= > 51h
WwW
a
=
Re
act
x
uJ
a
=
WW
"20
z
a
a
fea)

TIME IN HOURS

Fig. 6. Form of temperature drop during 4 consecutive entrances into
90°”

hibernation. Zero time is time at which brain temperature reached 32°C in
all 4 curves. Brain temperatures plotted at least every 15 min.

successively lower critical points. Zero time for the four en-
trances was taken as the time when the squirrel’s brain tempera-
ture had reached 32°C, since, as a general rule, once this tempera-
ture is reached there never occurs a spontaneous turning back.
It can be seen that each time the squirrel enters hibernation
it drops its temperature a little faster than the previous time so
that by the fifth entrance (series began at entrance 2) at 24°C
the squirrel was 20 minutes ahead of the fourth entrance, 45
minutes ahead of the third entrance, and 65 minutes ahead of
the second entrance. It will be noticed that by 28°C there is

1960 MAMMALIAN HIBERNATION 295

already an obvious separation of the four eurves. All four curves,
as is the general rule, have a higher virtually linear rate of
decline in the initial limb of the curve, followed by a decrease
in rate until a plateau is reached.

There is a very significant feature in these curves that should
be noted. Although the decline in the second limb of each eurve
is exponential-like, the animal is not bound to this relationship.
After two rapid temperature inereases in the fifth entrance
eurve (as the critical point was being approached), temperature
each time declined with a steep slope very close to that of the
imitial limb. This immediately suggests that the gradient between
core and air temperature is not the only factor in determining
the shape of the cooling curve. Shivering as deteeted electrically
is responsible for the shape of the second limb of the curve.

Significant details of expanded temperature curve. Figure 7
shows actual records of portions of the four consecutive entrances
into hibernation referred to in Figure 6. First, it can be seen
throughout the records that there are plateaus and gentle steps
of declining temperature. The steps are not of uniform ampli-
tude or rate, nor are the plateaus shown of uniform length. These
three aspects of the plateaus and steps depend on the portion of
the temperature curve studied and which of the series of en-
trances is being examined. For example, the two steps (A
and £6) in entrance 2 are small and have very gentle slopes.
Compare this to the steps in entrance 5 (particularly L and JW)
which have considerably steeper slopes. In general it has been
found that the later entrances of a series have on the average
shorter plateaus, and greater amplitude steps or faster rates of
decline of temperature within the step or a combination of these
last two factors. It is these factors apparently which explain
the faster over-all rate of temperature decline in consecutive
entrances. As the critical point is approached, plateaus become
longer. Small oscillations of temperature — a rising and falling
of about 0.1°C — become apparent (strip #’) and the amplitude
of the steps is quite small (strip A’), approaching the dead band
of the potentiometer recorder. Maximum slopes of some of the
steps are in the neighborhood of 0.3°C/min. (for example,
strip G).

Heat-production and heat-loss mechanisms. During entrance
into hibernation, the squirrel intermittently shivers as detected
by the chronic muscle leads (Fig. 11). Figure 8 is a plot of brain
temperature at 1-minute intervals as related to the actual record

296 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

A ENTRANCE 2

G ENTRA NCE 4

a -
eee oe , ae io
]
eae Sesion Saeares uname Sages ae
4 niieoninicdaal besiiihioniemeateoes ees
: ee =a ons

i ENTRANCE 5

ssscneiititita cstiiummssisaiasiniaitcsisuanaptieeh seieisihameaibitee

+ '
i ;
} i UM.
ot SH Raw Manes vee Se inoue } Ln
; i - }
N ; 4 ; } - — —
imine Tn Ee { +2 6 -
i ! i
| | ;

Fig. 7. Steps and plateaus in declining brain temperature curve during
entrances into hibernation. Strips selected come from the 4 entrances into

TIME

hibernation, completely portrayed on a compressed time scale in Figure 6.
Temperature graduations are 0.2°C. Time reads from right to left.

1960 MAMMALIAN HIBERNATION 297

of surface skin temperature obtained by a permanently im-
planted thermistor. The initiation and length of shivering is
illustrated by the solid bar while on the line below the hatched
area represents the presence of continuous small amplitude
muscle activity — muscle tone — and the clear area its absence.

If one follows the graph through, it will be seen that the
following phenomena occur: 1) shivering during the step down

(A, B, C); 2) shivering during the plateau after the step down
ie oy sm] sper a
he F a amet ig Maeve’ oe —
eae, net pees ON ia ek meihas ce Ss
Rote
BS @\9 028) 010! -8 8
a c
34

NO SHIVERING

MUSCLE TONE
DIMINISHED

———_ Time: 1 ov = 30 Sec(]JJJIIII])

36°C 27
BRAIN SKIN :
a eee Oe
35 | oe
E ore? a

O° fy)
NN, posi! D e oo ove
000 34 oo ®°%o0 00000

000°? 22
335
“pM

of?
Fig. 8. Heat-loss, production and conservation mechanisms during en

trance into hibernation. Record of skin temperature (thermistor) is con-
tinuous but brain temperature (thermocouple) is plotted every minute (open
circles). Below, the solid bars indicate shivering while the horizontally
hatched bars indicate the maintenance of muscle tone. Time reads from
right to left.

(D); 3) a fall in temperature of the skin concomitant with a
rise of temperature in the brain when there is no shivering (F) ;
4) when brain temperature falls, there is a rise of skin tempera-
ture preceding the drop in the brain with a return to original
temperature, the major peak of this double wave lasting about
5 minutes and having an amplitude of 0.8°C (Ff); 5) where
only somewhat late low amplitude increases in skin temperature
occur during a fall in brain temperature (#7), muscle tone and
shivering are absent.

298 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

We cannot be at all precise about the time relations since the
error in producing a graph on such a slow time scale from dif-
ferent recorders may be as large as 15 seconds (about the size
of the open eireles). However, about 244 minutes after the
start of a shivering burst lasting 1 minute a plateau in brain
temperature is obtained (J). The rise in skin temperature occurs
!,-1 minute before a step down in the temperature of the brain.

Shivering as a brake during entrance into hibernation. The
fact that the fine detail of the decline in brain temperature con-
sists of long plateaus and shorter steps means that some time
is being spent without any temperature change. Three-tenths
degree centigrade per minute represents a normal rate of decline
of temperature during a step down in the later entrances. If it
could be continued at this rate, the squirrel would be able to
drop 80°C in 100 minutes. Dropping this much takes a squirre]
entering hibernation about 18-22 hours. The production of
plateaus as initiated and maintained by shivering is responsible
for this slowing of the temperature decline. Shivering then acts
as a brake slowing down the rate of fall of temperature as the
animal enters hibernation.

Mechanism of the **step down.” The mechanism of the deelin-
ing steps in brain temperature appears to be a vasodilation on
the skin of the back of the animal, which is exposed out of the
nesting material to the cold air, and a reduction of muscle tone
during steep drops, but with the maintenance of muscle tone
and actual shivering in drops which are less steep. Apparently
a rise in brain temperature can occur without shivering by
vasoconstriction on the skin of the back. All in all, there appears
to be a coordinated interplay between the cooling powers of the
skin and its ability to reduce heat loss and the heat production
capacity of shivering and maintenance of muscle tone. By econ-
trolling these processes, the animal entering hibernation can
control the decline of temperature rather precisely. The physio-
logical significance of avoiding a rapid cooling is not known
but apparently the squirrels studied never do otherwise. We
feel, for reasons discussed elsewhere (Strumwasser, 1959), that
Lyman and Chatfield’s (1955) suggestion, ‘‘that the decline
in metabolic rate is the cause of the decline in body temperature, ’’
is an oversimplification. An active inhibition of general metabolic
rate during entrance into hibernation seems quite unlikely from
the fact that rapid alterations of deep body temperature are
possible and furthermore appear to be under fine control (Figs.
Oy Os Tare)

1960 MAMMALIAN HIBERNATION 299

VI. The Heart-Rates During Entrance and Arousal
Follow Different Paths with Respect to Temperature

When heart rate is plotted for several hours preceeding and
during entrance into hibernation (Fig. 9), it is obvious that there
is a striking decline, as the hibernation drop becomes continuous
(occurring between 34.2° and 33.6°C), from the rate of 153
beats to 68 beats/min.; that is, a decline of more than one-half
the rate in 30 minutes with a lowering of temperature during

ST i aki
33
RATE —— TEMPERATURE 4220,,
oO a
° =)
z
229 Z
WwW
5
e i Qa
é 2
ud a
ao| WwW
=e foal
lJ
Kb be
z <
alle ; Li
ioe x
@

10 13

>
TIME IN HOURS

Fig. 9. Heart rate during entrance into hibernation. Heart rate and
temperature plotted every 10 min,

this time of only 0.6°C. The decline in heart rate is rather more
undramatie though, if one considers that rates as low as 108
beats/min. may occur at a time when brain temperature is
fluctuating between 35°-37°C several hours prior to the act of
entrance.

When the heart-rate/temperature relation is analyzed in the
same squirrel during a consecutive entrance and arousal (Fig.
10), it can be seen that the curves are fair mirror images of
each other, together having the appearance of a hysteresis loop.
For arousal, heart rates as high as 246 beats/min. may occur

300 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

at brain temperatures as low as 16°C, while during entrance,
rates as low as 84 beats/min. occur at temperatures as high as
34°C,

The mirror-image-lke relation of the heart-rate/temperature
curves for the squirrel arousing from and entering hibernation
sugeests that the heart is being driven to either side of the
Arrhenius constant which would be obtained from the de-
nervated or isolated heart, with strong excitation during arousal
and strong inhibition during entrance. Dawe and Morrison
(1955) who used hedgehogs and two species of ground squirrels

=
374

b e e wm %“e 80 6

8 e ©

ie é

7 e ei?
O337 ° or
°o e e

x © e e
~ t ENTRANCE. AROUSAL
29
> -F
a =
B<6 es ie
weor es
a ee e}
> 8
WW L ae
ee
= [ &
= cn
oc Bo
@i7/ i

| ° O° e a

[ me

13 oc wl wee *°

“to = so a | eer Ee

HEART BEATS PER MINUTE

Fig. 10. Heart rate/temperature relation during a consecutive entrance
into and arousal from hibernation.

for their study reported somewhat similar results. This suggests
a dominant sympathetic influence on the heart during arousals,
and during entrance a predominantly parasympathetic one.
One should note however, that the Q,9 between 23.5° and
17°C (eritical point of this drop was 12.5°C) in the animal
entering hibernation falls between 3 and 2 which would indicate
relative temperature dependence for the heart in this region.
Prior to this range the active inhibition of heart-rate might be
associated with the diminished oxygen needs of a very relaxed
musculature (probably beyond that present with normal sleep).

1960 MAMMALIAN HIBERNATION 301

Vil. The Spontaneous Electrical Activity of the Brain is
Maintained During Entrance Into and Maintenance of
Hibernation Despite Declining and Low Temperatures

Some neural correlates during entrance. So far brain-wave
records taken from squirrels hours before entrance into hiberna-
tion have revealed no signs of specific activity by which the
entrance could be predicted. The appearance of excessive spind-
ling in most areas of the cortex occurs at a time when the
temperature is already dropping. However, these are intermit-
tently broken up by desynehronizations lasting as lone as 3-5
minutes during which time temperature is stable and may be
associated with movements of the squirrel in its nest and/or
periods of shivering. As soon as brain temperature declines below
approximately 34°C, (the normal nightly sleeping temperature )
several interesting electrical patterns appear with rather specific
interrelations.

Figure 11 illustrates localized patterns of activity within the
lateral motor cortex, the basolateral amygdala and the dorsal
hippocampus. The specific discharges are centered around shiy-
ering which is recorded on the fourth channel together with the
activity of the right sensory cortex.

Motor cortex. Note in strip A of Figure 11 (occurring durine
a plateau of brain temperature at 23.4°C) the grouping of a
specific 9-10 eps rhythm in the motor cortex growing in ampli-
tude and continuity until apparently as the peak amplitude is
reached shivering takes place. Compare the activity to that in
the sensory cortex which shows only a nonspecific spindle burst
close to shivering (strips A and (). Note the continuation of
the 10 eps rhythm in the motor cortex throughout shivering and
for several seconds after shivering. These 1-2 second bursts of
10 eps aetivity start to build up some 4-9 seconds before shiver-
ing but low amplitude 10 eps activity in the form of waxing and
waning bursts seems to be ever-present during entrance into
hibernation and is not as apparent at other phases of the
hibernation or sleep cycle.

Strips C and D (occurring during a plateau at 21.4°C) repeat
an illustration of the points made but are presented to show the
long 10 eps after-shivering discharge present in the motor cortex
concomitant with the maintenance of muscle tone (compare the
thickened line in channel 4 strip D with the initial part of
and the terminal part of B).

302 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

A
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R-L HPC si cnaseaif eaRcachistael Wie ener tad eee |z00
R SC-EMG SMidemas serpents tenant lll |200

B
TF THELU outfanic ss epi dalaagade ainda nies cenae toned amask a
ert Hy ena A real tat ne ANN Ml ip IN ANAM ey Hayes

rane tA try eli V/A AAA senate A
Kichler RANA nelle neta Astana rvhepeyitiet

c
Po SAc ET, ayy at a ‘ati ui

ws AA val aly att i A oh Oy

paasror natn —_ Laat ate rte inten hae AMA attend

se enenaieea ARIANA id. parce nanan ill es

D
ts i mag inl ene lin Ilo thi Pa
Urea Ah Nn et sail fy i" ‘ | AACE Nit Aa y\\" sihesut \ ANY

se eaten adapmbeaatie d derheoeinadee es ts alco tn et tt iene l aaa att homoncens Woemmnadaa iad
se alATiA AIH 4 ieee amend ali senaaimae\ Lah bad celdabuilamadeneniepiiea dabaatida

— 2 SECS.
E

F
NW nny Were i Yd Whew fat we Nome Mi
| i \\\ R-L MCP FRAN INR 600
AM Ne Nadie h
vw ANAC A ANA
VATA ieorecane os

proneram near AAAS AA AAN AeA)
| aig
Paiiabinvilat nt A Nemo natA, EKG | 100
SES: —— 5 SECs.

Fig. 11. Activity in the brain correlated with shivering during entrance
into hibernation. A and B, C and D are continuous strips. FE is a faster
record showing the degree of synchronization between the hippocampus and
amygdala during the 5 eps burst. F is from a different squirrel which had
a positive motor cortical lead but shows the difference in the activity of the
motor cortex during arousal shivering. Calibration is in uv. AMYG-
amygdaloid nucleus; HPC — hippocampus; L and & — left and right sides
of brain, respectively; M/C — motor cortex; OB — olfactory bulb; SC —

sensory cortex,

1960 MAMMALIAN HIBERNATION 303

Amygdaloid-hippocampal interrelations. A rather constant
finding after shivering as the motor cortex activity dies is the
appearance of high amplitude 5 eps rhythm in the amyedala
during which an identical rhythm in the hippocampus breaks
out. Strip # is a faster record to show the fairly close synchrony
between these two areas; note that the hippocampal rhythm
appears in the midst of the amygdaloid burst which outlasts the
hippocampal activity.

Absence of these specific correlates during arousal shivering.
It is estimated that around 500 shiverings were recorded in all
the squirrels with some or all of the three positive brain leads ;
the interrelated pattern of the rhythms described was always
present with each shivering, and at no other time. These patterns
are not seen with the shivering present at other stages of the
hibernation cyele.

Interpretation of the specific EEG patterns. The motor corti-
cal discharge may well be the sign of a facilitative downstream
discharge which initiates or aids in the development of shiver-
ing; at any rate this is a suggestion based on the long latency
before shivering begins. The amyegdaloid-hippocampal rhythm
is mostly apparent when the motor cortex 10 eps discharge is at
its low or absent or beginning to disintegrate (strips A, B and C
of Fig. 7). In D, where the motor cortical discharge takes
quite long to, dwindle down, the amygdaloid pattern is present
but is not as large as in B. It is interesting to note that when
this occurs the hippocampal rhythm is weak and the synchrony
not as good. For these reasons it is felt that the amyedala and
hippocampus are involved in a mutually inhibitory mechanism
with the motor cortex or something preceding it but are not
involved at the level of the fundamental shiverine mechanism.
Both excitatory and inhibitory influences from the hippocampus
and amygdala on the autonomic aspects of mammalian activity
have been demonstrated by numerous investigators, In particular
IKaada (1951). It is well known that the motor cortex is not
necessary for shivering in non-hibernating mammals during a
temperature stress (Isenschmid and Schnitzler, 1914) and it is
presumably not involved in shivering in the ground squirrel
at other times, but it is clearly correlated with shivering during
entrance into hibernation. This suggests that a hierarchy of brain
control systems is probably exerting its influence on the funda-
mental system responsible for shivering; that is, these ‘* higher”?
systems are determining the initiation and termination of shiver-
ing and its integration into the coordinated pattern responsible

304 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

for the precise form of the curve of the temperature drop.
This may indeed be a general phenomenon in animal behavior ;
similar activities in the same animal may be brought about by
different hierarchical systems, each system complete within it-
self and organized to accomplish a particular goal.

VIIL. During Deep Hibernation Certain Compiex Regulations
and Behavior are Compatible with a Striking Decline in
Cerebral Electrical Activity

Behavior during deep hibernation. Postural adjustments.

Durine maintained hibernation at brain temperatures as low
as 6.1°C the deatened hibernator is not completely motionless.

Fig. 12. Heart rate during a series of postural adjustments. Brain tem-

perature remained constant throughout at 10.7°C. Darkened burst of muscle
activity is synchronous with a postural adjustment.

At this temperature there are about two or three somewhat peri-
odie postural adjustments per hour. This in its simplest form
consists of a slow raising of the head and forepart of the body
with a gentle return to the original position. However, there can
be more complex coordinations in this behavior. After raising its
head and the forepart of its body, the squirrel may slowly shift
its position in the nest several degrees by properly orienting the

1960 MAMMALIAN HIBERNATION 305

forepart of its body before gently returning to its nest. During
these postural adjustments, cardiovascular compensations occur.
As can be seen in Figure 12 heart rate increases. Not infre-
quently there is a clear-cut increase in heart rate anticipating the
postural adjustment (strip #). Note the striking rebound
inhibitions present after a particularly long duration of accele-
rated heart-beat in G.

Response to sound stimuli. The ability of three undeafened,
unimplanted deeply hibernating squirrels to respond to various
sounds in their environment has been studied. Oral temperatures
have been measured from a few minutes to an hour after ter-
mination of the particular experiment. Upon tapping sharply
two or three times on the metal top of the aquarium or on one of
the glass panes, one first observes slow uneurling of the pinna if
it is relaxed against the side of the head. If the tapping is re-
peated (sometimes it takes 1 or 2 more presentations), the
squirrel invariably cocks its head, directing one ear toward the
source of the sound by slow rotation of the neek with or without
raising the forepart of its body. Even if the tapping is not
presented for the next 20-30 minutes, the pinna remains erect
and the head remains in the same position or may be slowly
turned in the opposite direction after a variable lapse of time.
iventually, the head is rotated to its original position and the
animal continues to hibernate as judged by respiratory rate
and behavior for at least 2 hours, a time during which most of
the arousal is normally accomplished. An oral temperature of
».8°C in an environment of 2.1°C was the lowest temperature
recorded within 20-30 minutes of this sequence. If vigorous
tapping is maintaind for a few minutes an arousal is invariably
initiated.

Vocalization in deep hibernation. When a squirrel in deep
hibernation is touched, one of the first responses is a sustained
loud shriek lasting about 0.55 second (Fig. 13) after which,
of course, an arousal is initiated. There are differences between
the hibernation vocalization and the normal squirrel chatter in
pattern and in leneth although it is interesting, as high speed
analyses have shown, that there are no conspicuous differences
in the frequeney range of the notes. Peak frequencies for the
vocalization of the hibernating squirrel are in the neighborhood
of 3200 eps at 10.7°C while for the normal squirrel chatter at
37.2°C they are 3750 eps.

Focusing of attention, discrimination and localization of the
stimulus source, The fact that the squirrel has an auditory-pinna

306 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

reflex and adjusts the position of its head toward the source of
a sound stimulus is evidence for the ability of the animal to focus
its attention and attempt to locate in space the source of the
sound, The fact that squirrels do not awake to this sound unless
it is maintained tor at least a few minutes is evidence for dis-
erimination.

1O.7°C.

Fig. 13. Voealization pattern of a hibernating (upper) and non-hiber
nating (lower) disturbed squirrel. Vocalizations were tape recorded and
later played back into a cathode-ray oscilloscope and photographed, Tem-
peratures indicated are cerebral; time line displays a 10 eps sinusoidal

oscillation.

[f this discrimination is not sufficiently sophisticated, con-
sider Mullally’s (1953) observation that squirrels (Citellus lat-
eralis) often hibernated in the presence of active members of
the same species: ‘‘At times, with four in a box, two were
hibernating and two were not. Although scuffed by the active
ones, the hibernating individuals did not awaken until they were

1960 MAMMALIAN HIBERNATION 307

handled’? (by the experimenter). This observation suggests
species-discrimination in an animal whose core temperature was
probably below 10°C (see his Table Il for hibernating rectal
temperatures)! All these behavioral phenomena during deep
hibernation are of great interest, in particular when the electrical
activity of the brain at these low temperatures is examined.
Regulatory mechanisms and brain waves in deep hibernation.

Brain temperatures and heart-rate during deep hibernation.
After the final critical poimt is reached (see Fig. 2) brain tem-
peratures remain constant for prolonged periods of time (10 hrs

ae
\

TART BEATS PER MINUTE
BRAIN TEMPERATURE IN °C.

oe)
8

O73: 40AM 5 10 20 25 30

15
TIME IN HOURS

Fig. 14. Heart rate and brain temperature during maintained hiberna-
tion. Heart rate and brain temperature plotted every 10 min. Brain tem-
perature plotted at shorter intervals during changes.

to 68 hrs, with later entrances having longer maintained
plateaus) with no change in temperature within =0.05°C. Figure
14 is a plot of heart rate and temperature over 32 hours as a
squirrel approached, maintained and aroused from its final
critical point for the first time. During this time environmental
temperature fluctuated between 7-8°C. It can be seen that during
the maintained low brain temperature of 10.7°C, both the aver-
age heart rate and amplitude of oscillations of heart rate are

308 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

greater than the period during which brain temperature ap-
proaches the critical point. This is a remarkably constant feature
of such plots. It suggests that the relative level of sympathetic
tone is increased at this lower level of temperature.

An increase in temperature from the critical point to a higher
plateau before an arousal (defined as the point from which a
continuous rise without any decline in temperature oceurs, such
as at 29.75 hours after 0 in Fig. 14) is often seen, but there
are cases when it is absent. Note the continuous increase in aver-
age heart rate during the higher plateau and before the next

1\sec. |
T T i ||

Kig. 15. Intermittent initiation of motor unit discharge during deep
hibernation. Strips 7 to 7 and S to 74 are continuous, The smal] unit in
strip 7 stopped its discharge 12 see after the end of the strip; 38% min
clapsed between strips 7 and S. Brain temperature maintained at 8.8°C.

rise. The increase in heart rate prior to a spontaneous arousal
is In agreement with the observations of Dawe and Morrison
(1955) and comparable to the increase seen in the heart rate
before the temperature rise of an evoked arousal in the hamster
(Chatfield and Lyman, 1950).

Muscle tonus during deep hibernation. During these prolonged
maintained low temperatures, tonus is present in muscle. Kigure
15 is a typical continuous record of two muscle units with a

1960 MAMMALIAN HIBERNATION 309

pause of 51, minutes between strips 7 and 8 recorded while
brain temperature was maintained at 8.8°C. The dark bursts
of activity are respirations while the larger, longer duration
deflections are heart beats. It will be noted that the large unit
begins firing at relatively high frequency (4/sec) near the middle
of strip 7. Not until near the start of strip 4 (some 45 sees later)
did the second smaller unit start firing, quite obviously out of
phase with the first and outlasting it. The small unit did not stop
firing for another 12 seconds after the end of strip 7. As strips
8 to 14 show, the cycle may be initiated and completed the very
next time with only the large unit firing.

We have had cases where five or six units were identifiable in
the record. These have been analyzed to determine which unit
starts the cycle, the order of appearance of the units, the length
of the eyele, and the silent interim, over continuous periods as
long as 12 hours. No simple formula or pattern is apparent and,
to date, it has not been possible to predict which of the units
will initiate the cycle or how many will come in, or the order of
their appearance. We also find that there is no obvious relation
to any of the oscillations of temperature measured on the surface
of the skin. The phenomenon included long periods of silence,
as long as 5 minutes. More often than not, there is seen a clear
cut specific discharge in the motor cortex before the muscle
units appear in the record, with maintained but lower amplitude
fast activity lasting a variable period of time (strip A, Fie. 16,
brain temperature at 6.1°C). The maintenance of muscle tone
in deep hibernation is extremely interesting in view of the low
cerebral temperatures during this state. The phenomenon of
periodie initiations of unit discharge with units appearing in
apparently random order and phase relations each time may be
considered the resultant of a central switching mechanism. The
‘‘central switching mechanism’’ appears to be operating quite
normally despite cerebral temperatures as low as 6°C.

Brain waves in deep hibernation. Brain wave activity is far
from absent even at temperatures as low as 6.1°C. Figure 16,
strip A, demonstrates activity in the motor cortex, medial pre-
optic area, septum, and the ventromedial nucleus of the hypo-
thalamus, and relative silence in a subcortical sensory area, the
lateral geniculate body. Note the discharge in the septum. It is
quite coincidental to the activity of the cortex and presence of
muscle units but it is a rather typical example of a pattern, in
this instance seizure-like, unique to each particular electrode
location, which in deep hibernation periodically repeats itself.

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312 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

We have never seen persistent silence in any brain area during
maintained deep hibernation. If one examines long term EEG
records, there is always, in the most inactive cases, some pat-
terned activity which repeats itself.

igure 17 displays this principle in a purely sensory area, at a
brain temperature of 10.7°C. Strip A shows a 4-6/see. rhythm
building up in the right and left olfactory bulb with the appear-
ance of a similar rhythm in the dorsolateral preoptic area no
more than 0.5 mm ventral to the anterior commissure. Respira-
tions (occurring at every shortened cardiae eyele) tend to break
up the rhythm momentarily but do not initiate the typical slow
wave and 50 eps activity seen in non-hibernating squirrels.

When simultaneous records of each bulb relative to an indif-
ferent grounded poit are analyzed (strips B-F’) it is seen that
several phenomena oceur: a) The right bulb may start this
discharge first and as it builds up a similar rhythm can be seen
starting in the left bulb at a time when activity begins to appear
under the electrode close to the anterior commissure (strip £).
b) The left bulb may start the discharge first and the right then
follows at a time when activity in the commissure appears (strip
I’). Note that some of the waves in this latter channel are pre-
dominantly up and some predominantly down, indicating per-
haps communication through the commissure first in one direc-
tion and then the other. ¢) The left bulb may start firing without
a discharge in the right, or vice versa, even though similar
rhythmie activity appears in the commissure (strip C shows the
left, strip B the right). d) Sometimes the right or left bulb
may show a small isolated burst without any apparent activity
in the commissure (strip D).

Kifect of pentobarbital on brain wave activity in deep hiberna-
fion. Such bursts are wiped out within 10 minutes after an
intrathoracic injection of pentobarbital, 30 mg/kg (84 the dose
necessary to anesthetize a non-hibernating squirrel for 1 hr.).

Strip B in Figure 16 is taken from a squirrel with a brain
temperature of 10.7°C. Note the relative silenee of points
in the right and left mesencephalic reticular formation. Strip C
is the same squirrel; the strip begins 114 minutes after touching
the squirrel with a glass rod. Activity immediately appeared in
the relatively quiet areas, the heart speeded up and continuous
muscle activity was initiated.

Strip D occurs 5 minutes after 30 mg/kg of pentobarbital was
injected intrathoracically. Heart beat, muscle activity and brain
activity are obviously declining. Strip / occurs 15 minutes after

MAMMALIAN HIBERNATION

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314 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

D. Whatever fluctuations were present from now on in the brain
channels remained even after the heart eventually stopped some
10 hours later and were indistinguishable from the noise level of
this particular instrument. The animal stopped its respiration
35 minutes after the anesthetic was given. In one more squirrel
injected with 40 mg/ke of pentobarbital, the same sequence of
events occurred, After a preliminary activation due to picking
up the animal, all activity present declined to a level indis-
tinguishable from the instrument noise level even though a good
deal of maintained spontaneous activity was present prior to
picking up the squirrel. The heart beat, however, lasted 13
hours before total failure.

Spontaneous activity in the brain of the hibernator. Acecord-
ing to Lyman and Chatfield (1955) in a review on hibernation,
“... im deep hibernation, the cerebral cortex of the hibernating
animal shows no spontaneous electrical activity, but the species
vary in the temperature at which electrical activity ceases.’? At
the lowest critical points, 6.0° and 6.1°C in two. squirrels,
spontaneous activity was consistently seen (as these 2 animals
together re-entered hibernation a total of 6 times) in the motor
and sensory areas of the cerebral cortex. Lyman and Chatfield’s
statement is based partly on their own research on acutely meas-
ured electrocorticograms of arousing hamsters (Chatfield et al.,
1951) and measurements made on one woodechuck (Lyman and
Chatfield, 1953) in deep hibernation with two implanted cortical
electrodes. Apparently they used low and constant amplification
in all published records, even though temperatures fluctuated
between 7° and 18°C for the woodchuek and 11.4° and 29.4°C
for the arousing hamsters.

It should be emphasized once more that there is a good deal
of spontaneous activity present in a variety of cortical and
subcortical sites during deep hibernation in Citellus beecheyr
(Fig. 16) but sufficient amplification must be used. Even when
‘“*silent’’ areas of the brain are observed they are not persistently
silent. Long term records with or without the presence of the
investigator in the room have shown periodic bursts of activity,
sometimes generalized, however mostly independent and unique
and repeatable in pattern for each area without any change in
cerebral temperature. It is interesting to note that Lyman and
Chatfield (1953), despite their already-stated conclusion, ob-
served for the one marmot studied, ‘‘slow nondescript, spon-
taneous cortical activity’? at 7°C, ‘‘although it was sporadic.’’

ws
—
|

1960 MAMMALIAN HIBERNATION

In the records from Kayser’s group (Kayser et al., 1951; Roh-
mer et al., 1951) on cortical aetivity during deep hibernation
in the Kuropean squirrel, Citellus citellus, the same phenomenon
is apparent but it is not clearly stated in their two papers
whether the attachment of leads to the squirrel in their system
always initiated an arousal or not; apparently they never re-
corded in the cortex from animals entering hibernation.

The analysis of discharges during deep hibernation in the two
olfactory bulbs and its interconnection suggests some of the
factors involved in these intermittent discharges; it is assumed
that the activity recorded in the third channel is volume con-
ducted from the ventral border of the anterior commissure at
which the electrode was aimed. An anatomical factor involved
in these discharges is the symmetrical interconnecting arrange-
ment of the brain allowing for activation of one side if the
other is discharged by some means. Of the physiological factors,
apparently a certain level of activity is necessary before trans-
mission out of a particular bulb ean oceur (D, Fig. 17) and
even when transmission to the other bulb occurs there may be
no activation of the discharge (B, C, Fig. 17), a faet probably
indicating that the elements responsible for the discharge in the
contralateral bulb are fluctuating in their excitability due to
intrinsic or extrinsic causes.

Significance of the brain’s spontaneous activity in deep hiber-
nation. Apparently the activity of the brain in deep hibernation
may be thought of as related to two states. a) There is activity
related to homeostatic mechanisms at low temperatures, which
seem to be functioning in a remarkably effective manner. The
maintenance of brain temperature within =0.05°C at a few
degrees above environmental temperature during deep hiberna-
tion should be emphasized. The brain activity includes, for ex-
ample, the motor cortical discharge as related to muscle tone
(Fig. 16 A) and the maintained activity of the septal and
hypothalamie areas (as opposed to the relative silence of the
lateral geniculate and mesencephalic reticular formation), prob-
ably related to maintained autonomic control (Figs. 12 and
14). Note that these records at a lower brain temperature and in
sustained deep hibernation appear more lke desynehronized
records typical of an alert state in a non-hibernating squirrel but
merely at a lower amplitude (close to 10 per cent of normal)
than records from animals entering hibernation (Fig. 11). 5)
There is activity not obviously related to homeostatic mechan-
isms, for example the repetitive patterns already mentioned even

316 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

in relatively ‘‘silent’’ areas and purely sensory areas (olfactory
bulb) quite unrelated to known environmental inputs.

This latter activity may be the activity needed to keep the
particular area functioning; perhaps if stopped for too long
some dynamic patterning may be lost (perhaps something learned
or innate). Or, it may be related to the nervous system being
poised at all times for an arousal.

It should be pomted out that the relative quiescence in some
areas — the mesencephahe reticular formation, for example
(Fie. 16 B) —does not imply a temperature depressed excita-
bility, for as soon as the squirrel was touched and the stimulus
withdrawn, activity immediately appeared, remained and grew
(Fig. 16 C).

The pentobarbital experiments should be interesting ones to
continue. It makes one wonder whether it is possible in the
deeply hibernating mammal to wipe out the brain’s spontaneous
activity, even transiently, without producing an animal incapable
of arousal.

Special properties of the central nervous system of the hiber-
nator. The ability of the squirrel in deep hibernation with brain
temperatures close to 6°C, to focus its attention, to attempt to
localize stimuli in space, discriminate, vocalize and adjust its
posture beside maintaining autonomie regulations is remarkable,
particularly when it is realized that there is a 90 per cent redue-
tion in the amplitude of the general electrical activity of the
brain. If only 10 per cent of the total neuronal population is
active, on the average, then I suspect it is a ‘‘carefully selected”’
10 per cent rather than a random distribution.

The maintained excitability of the nervous system despite low
cerebral temperatures is not unexpected since, of course, it is
known that autogenous complete arousals are possible. However,
the immediacy, degree and growth of the central response to a
simple stimulus despite the low temperature (Fig. 16) may call
for temperature-compensated synaptic transmitter mechanisms.

One of the more specialized properties of the hibernating
central nervous system is its ability to maintain homeostatic
mechanisms so well at low cerebral temperatures. However, it is
felt that the most specialized cerebral property les in the hiber-
nator’s ability to turn down neuronal activity to a level, at
6°C brain temperature, which does not interfere with complex
behavior, with arousability, with instincts and probably a myriad
of learning.

at |

1960 MAMMALIAN HIBERNATION

REFERENCES

BARTHOLOMEW, G. A, AND 'T. J. Cape
1957. Temperature regulation, hibernation, and aestivation in the little

pocket mouse, Perognathus longimembris. J. Mammal., 38:60-72

Brenepicr, F. G@. AND R. C. Lre
1938. Hibernation and marmot physiology. Carnegie Inst. Washington

Publ., 497 :1-239,

CHATFIELD, P. O. aND C. P. LYMAN
1950. Cireulatory changes during process of arousal in the hibernating
hamster. Am. J. Physiol., 163 :566-574.

CHATFIELD, P. O., C. P. LYMAN AND D. P. PurRPURA
1951. The effects of temperature on the spontaneous and induced
electrical activity in the cerebral cortex of the golden hamster.
Electroencephalog. and Clin. Neurophysiol., 3:225-230.
Dawe, A. R. AND P. R. Morrison
1955. Characteristics of the hibernating heart. Am. Heart J., 49:367-
384.

Dupots, R.
1896. Physiologie comparée de la marmotte. Ann. Univ. Lyon, Paris,

268 pp.

Foster, M. A.
1954. The reproductive cycle in the female ground squirrel Citellics
tridecemlineatus (Mitehill). Am. J. Anat., 54:487-511,.

ISENSCHMID, R. AND W. SCHNITZLER
1914. Beitrag sur Lokalisation des der Wirmeregulation vorstehenden
Zentralapparates im Zwisehenhirn, Arch. exp. Pathol. Pharma-

kol., 76:202-223.

JOHNSON, G. E.
1931. Hibernation in mammals. Quart. Rev. Biol., 6:439-461,

ICAADA, B. R.
1951. Somato-motor, autonomic and electrocorticographic responses to

’? and other struetures

electrical stimulation of ‘‘rhinencephalic
in primates, cat and dog. Acta physiol. scand., 24, suppl. 83,
285 pp.
\Yser, C., F. ROHMER AND G. HIEBEL
1951. L’EEG de Vhibernant; léthargie et réveil spontané du spermo
phile. Essai de reproduction de I?7EEG chez le spermophile
réveillé et le rat blane. Rev. Neurol., 84:570-578.

LYMAN, C. P.
1948. The oxygen conswuption and temperature regulation of hiber

nating hamsters. J. Exper. Zool., 109:55-78.

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LYMAN, C. P. AnD P. O. CHATFIELD
1958. Hibernation and cortical electrical activity in the woodchuck
(Marmota monax). Science, 117:533-534.
1955. Physiology of hibernation in mammals. Physiol. Revy., 35:403-

425.

MULLALLY, D. P.
19538. Hibernation in the golden-mantled ground squirrel, Citellus
lateralis bernardinus. J. Mammal., 34:65-73.

MusaccuHia, X. J. AND C. G. WILBER
1952. Studies on the biochemistry of the Arctie ground squirrel.
J. Mammal., 33:356-362.

RASMUSSEN, A. T.
1916. Theories of hibernation. Am. Nat., 50:609-625.

RouMer, F., G. HIEBEL AND C. KAYSER
1951. Recherches sur le fonetionnement du systeme nerveux des hiber-
nants. Les ondes cérébrales pendant le sommeil hibernal et le
réveil. Etude sur le spermophile, C. R. Soe. Biol. (Paris), 145:

747-752.

STRUMWASSER, F.
1959. Thermoregulatory, brain and behavioral mechanisms during en-
trance into hibernation in the squirrel, Citellus beecheyi. Am. J.
Physiol., 196 :15-22.

Svinta, A. AND H. C. BowMAN
1952. Oxygen carrying capacity of the blood of dormant ground squir-
rels. Am, J. Physiol., 171:479-481.

Wyss, O. A. M.
1932. Winterschlaf und Wirmehaushalt, untersucht am Siebenschlife:
(Myorus glis). Pfligers Arch. ges. Physiol., 229:599-635,

ZALESKY, M.
1934. <A study of the seasonal changes in the adrenal gland of the
thirteen lined ground squirrel (Citellus tridecemlineatus) with
particular reference to its sexual cycle. Anat. Rec., 60:291-321.

DISCUSSION FOLLOWING STRUMWASSER’S PAPER

FOLK congratulated the speaker but noted that a correction
is essential: the ‘‘test drops,’’ he believes, are improbable in the
case of the thirteen-lined ground squirrel. In experiments over a
2-year period using a controlled constant environment, it
was noted that animals would drop into hibernation immediately
without ‘‘test drops.’? On one occasion, six animals entered

1960 MAMMALIAN HIBERNATION 319

hibernation within less than 24 hours after leaving the warm
room. STRUMWASSER rephed that test drops are not phe-
nomena that can be ascertained by mere visual observation of
the hibernating animal; continuous temperature recording of
the undisturbed animal in a properly controlled environment is
necessary to ascertain whether test drops oceur or not. If such
experiments are performed with the thirteen-lined ground squir-
rel and entrance into deep hibernation (core temperature down
to 6°C and lower) is observed to occur in one continuous tem-
perature decline, then these animals belong in Group [ or If of
the scheme outlined in his paper.

LYMAN then substantiated STRUMWASSER’S observations
by remarking that he was sure such ‘‘test drops’’ occur with
Citellus beecheyi. He thought, on the other hand, that it some-
times occurred with the thirteen-lned ground squirrel and the
woodchuck, and sometimes not. He stated that sometimes these
animals when put in the cold would go into hibernation im-
mediately without a test drop, but at other times they would go
‘‘up and down with several test drops.”’

FOLK commented on the preparatory state for hibernation,
pointing out that it, too, must be an internal rhythm. He
raised the question of photo-periodicity as the preparatory stim-
ulus for hibernation. STRUMWASSER noted that only pre-
cisely controlled environments would give such answers.

GRIFFIN asked whether the brain temperatures given always
referred to temperatures taken at the same spot in the brains
of experimental animals. STRUMWASSER said the thermo-
couple was within 3 mm of the same area. GRIFFIN noted that
Hammel working on dogs saw gradients in brain temperatures.
He asked if gradients appeared in areas of little circulation
during hibernation. STRUMWASSER said he would not expect
such gradients in brain temperature. GRIFFIN asked what the
lowest temperature he recorded was. STRUMWASSER said he
never saw a brain temperature below 6°C, and believed this was
in some measure related to the California species he was working
with. He also noted that it is quite possible that brain tempera-
ture is regulated at a considerably higher level in deep hiberna-
tion than the rectal temperatures so far reported in various
species.

GRIFFIN said he assumed the electrodes used were fairly
big. STRUMWASSER replied that small stainless steel wires.

320 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

S7y and 8Ov in diameter, were chosen to minimize damage for
subcortical recordings.

BISILTOP asked what would happen to the ‘‘test drops’” if
the environmental temperature was lowered very gradually to
ceive adaptation of sense organs. STRUMWASSER said that one
would have to do this over about a 15-day period very slowly,
which would be a pretty tricky affair, but in his belief, ‘‘ test
drops’’ would still occur. BISHOP doubted this.

XVI
THE MECHANISMS OF HYPOXIC
TOLERANCE IN HIBERNATING AND
NON-HIBERNATING MAMMALS’

By Roperr W. BuLuarp, GEORGE Davip and
C. Thomas NICHOLS
Department of Physiology
Indiana University School of Medicine
Indianapolis, Indiana

Different mammalian species possess varying degrees of toler-
ance to hypoxia. Much of the work on comparative altitude
physiology is reviewed by Denzer (1950). In general, the
poikilotherms are extremely tolerant to almost complete anoxia
or altitudes well above 80,000 feet. Likewise the bats, perhaps
because of their poikilothermic nature, are extremely tolerant to
altitude. Diving homeotherms are also quite tolerant to brief
hvpoxic periods (Prosser, 1950). Spallanzani first noted in 1803
that bats and marmots had a higher degree of tolerance to oxy-
een deficiency. Many experiments were done with animals in
the hibernation state throughout the nineteenth century. Some
were relatively crude such as those of Carlisle (see Bidrek et al.,
1956), who in 1805 submerged the hibernating hedgehog in
water for 30 minutes with no untoward effeets. Hiestand ef al.
(1950) have shown that hibernating adult homeotherms are more
tolerant than non-hibernating adult homeotherms.

There are many possible ways of explaining the nature of the
increased tolerance of the hibernators. For example: (1) the
hibernators have been shown to utilize unusual vasomotor control
during the dehibernation process (Adolph and Richmond, 1955;
Lyman and Chatfield, 1955). Can they eall forth this mechanism
of restriction of blood flow to only vital regions during a
hypoxic exposure as divine mammals can? (2) Hibernators
have hkewise been shown to be capable of ‘‘volitional’’ body
cooling. heart rate decrease and metabolism decrease associated
with entrance into hibernation (Lyman, 1958). Does the hiber-
nator upon hypoxie exposure utilize such mechanisms as pro-
tective responses? (3) It has been suggested that in hibernation

LThis project was supported in part by the Research and Development Division,
Office of the Surgeon General, Department of the Army under Contract No
DA-49-007-MD-947.

322 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

anaerobic metabolism is an important share of the total metabo-
lism (IXlar, 1951). Can hibernators in the non-hibernating state
make greater use of these metabolic pathways than other species?
(4) The limited data on oxygen-hemoglobin dissociation curves
indicate that the hibernators possess hemoglobins of high oxygen
affinities (Prosser, 1950; Barker, 1957). Is this of survival value
during hypoxie exposure ?

bd oe

RECTAL TEMPERATURE DEPRESSION,

EX POSURE TIME IN MINUTES

23.0 °C. Ta ---- 14.0 °C. Ta
= =

O RATS @ GROUND SQUIRRELS

Mig. 1. Changes in reetal temperature of rats and ground squirrels taken
(oO various altitudes at the rate of 4000 feet/min at chamber temperatures of
¥3°C and 14°C. Standard error of the mean is indicated when six or more

animals were used for the determination of a curve.

In this paper some physiological responses to hypoxia are re-
ported for several mammalian species in an attempt to account
for differences in hypoxic tolerance.

Body temperature changes upon hypoxic exposure. Figure
1 shows the changes in colonic temperatures of rats (Wistar
strain) and thirteen-lined ground squirrels (Citellus tridecem-
lineatus) of similar body weight upon exposures to various de-
erees of hypoxia in an altitude chamber. The rates of cooling
increased as barometric pressure decreased and at all barometric

1960 MAMMALIAN HIBERNATION 323

pressures the ground squirrels always cooled faster than did the
rats. The curves tended to level off at a lower body temperature
as the exposure progressed. The ground squirrels at 200 mm Ie
and 150 mm He cooled at approximately the same rate, although
there is a marked difference in oxygen deficiency. The e@round
squirrels at 150 mm He and at a chamber temperature of 23°C
cooled almost as fast as those at 14°C. If the cooling constant

THORACIC
TEMP.

SOCENTIGRADE

TEMPERATURE

RECOVERY|

ALTITUDE EXPOSURE PERIOD 4 PERIOD

20 80

40 60
MINUTES

0
TIME

Fig. 2. The changes in body temperature of one squirrel taken to a 150

mm Hg barometric pressure at a chamber temperature of 14°C.

is estimated from Newton’s law of cooling, which takes into
aceount the difference between body temperature and ambient
temperature, the constant is higher in the squirrel at 28°C. This
may be indicative that the cooling process is of a regulated nature
and that the squirrel can utilize a maximum cooling procedure
if necessary. In another series of experiments, the living ground
squirrels in the first 30 minutes of exposure to 150 mm He
cooled faster than dead rats, and at approximately the same rate
as dead ground squirrels.

Localized temperature changes upon hypoxrie exposure.
ieure 2 shows the curves for various body temperatures ob-

tained for one ground squirrel. Typically, the colonic and

324 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

thoracic temperatures fell in a parallel fashion. Only very sheht
temperature gradients between abdomen and thorax were seen
during the hypoxie exposure. At the end of the exposure the
typical arousal response is seen with immediate and rapid warm-
ing of the thorax followed by a more gradual increase in rectal
temperature. The skin temperature shown was obtained from
the lower back. In all cases the skin temperature never fell as
rapidly as the rectal or thoracic temperatures at the beginning
of hypoxie exposure. In some cases it tended to stay constant
and in others it rose shehtly. After approximately 10 minutes
the skin temperature started to fall and the three temperatures
approached the same value. A delayed fall in surface tempera-
ture coimeident to a rapidly falling core temperature can only
be explained by vasodilation of peripheral vessels. Thus, in
hypoxia, the ground squirrel was not making an attempt to con-
serve body heat by vasoconstriction. Similar results were ob-
tained with the hamster and rat. Even though the hibernator
possesses the machinery of the diving mammal circulatory reflex,
it does not utilize this in hypoxia, as has also been reported by
Adolph and Riehmond (1955). However, at the end of the
hvpoxie exposure the hibernator immediately utilizes a selective
vasoconstriction for rapid rewarming. A lowered body tempera-
ture is of little detriment to a hibernator beeause of this ereat
rewarming ability.

TABLE [
Time of Death at 150 mm He in min.

Rat Squirrel
Chamber —— = - — ——
temperature Time of Time of
*¢ death tange death Range

38°C 2.8 0-6 14.2 9 - 25
33°C 5.0 0-7 16.4 6-33
28°C 8.0 5-10 35.3 Li 5
23°C 12:7 10-19 Survived 2 Hours
18°C 18.0 13 - 23 ‘6 SL
14°C 19.7 Ls-48 66 ee ce
14°C (shaved ) 2TEL

8°C 48 O's G8 oe oe oe

Survived 2 Hours!

1 Rats survived if precooled by exposure to S°C for 1 hour before the hypoxic
period.

1960 MAMMALIAN HIBERNATION 325

Temperature depression and survival. Table I indieates the
importance of body cooling in hypoxie survival. The rate of
body cooling could be altered by simply changing the chamber
temperature. The time of ‘‘death’’ was taken as the time respira-
tions ceased. Klectrocardiograms were taken but showed great
variability with arrhythmic, low voltage waves persisting for
some time in many animals. At 38°C the survival times of both
the rats and the ground squirrels were severely limited. As
chamber temperature decreased, survival times increased. At
25°C the ground squirrels survived the 2-hour exposure period.
At a chamber temperature of 8°C, the rats were capable of sur-
viving the 2-hour exposure period. However, in these experi-
ments a 60-minute control period prior to hypoxia was main-
tained, and during this time, at an ambient temperature of 8°C,
considerable cooling of the rat took place which may have en-
hanced survival. In both species, if the animal could lower its
body temperature to below 30°C in the first 30 minutes of ex-
posure to a barometric pressure of 150 mm He, it could survive
the 2-hour period. Four shaved rats were exposed to hypoxia at
14°C, and showed an increase in survival of 8 minutes when com-
pared to controls. However, the shaved rats did not cool as
rapidly as the ground squirrels at this temperature. An im-
portant finding here is that at all ambient temperatures, inelud-
ing those at which no body cooling was possible, the hibernator
outlived the non-hibernator.

Figure 3 represents a plot of survival times of several species
at 150 mm He versus various environmental temperatures. It
can be seen that the cooler the chamber, the greater is the
survival. However, the hamster (Jesocricetus auratus) has
ereater survival time as does the ground squirrel but did not cool
as rapidly as the squirrels at a barometric pressure of 150 mm
He. At a chamber temperature of 23°C the cooling undergone by
the hamster was only about half of that undergone by the
squirrel. Although body temperature decrease is important in
hypoxic survival if is not the only factor involved. Wiestand
et al. (1950) reported that the ground squirrel was more resistant
to hypoxia than the hamster. Our data indicate that the hamster
has the greater tolerance. However, different experimental ap-
proaches as to the rate of ascent and to the final degree of
hypoxia were used.

Survival benefits of body temperature depression. It ean
be demonstrated that body cooling enhances survival in hypoxia.
Body cooling in aceordance with Van’t Hoff’s law will decrease

326 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

the metabohe oxygen requirement of the animal thus forestalling
anoxic damage. Another less obvious benefit is that oxygen vol-
ume and tension will be greater in the cooler alveolus due to
the diminished vapor tension of water. For example, in the
lune of the euthermie animal exposed to a barometric pressure
of 150 mm He, 47 mm He or approximately one-third of the
pressure in the lung will be due to water vapor pressure. In

T TT 2 ae
140

n
3)
i=
>} 120-- HAMSTER GROUND =
Zz SQUIRREL
=
100 + |
Zz
4
< 80 + 4
> |
>
(oo
=)
n

CHAMBER TE VMsPoE CR AIT URES OG?

Fig. 3. Survival times at 150 mm Hg barometric pressure and at various
chamber temperatures for hamsters, ground squirrels, rats and mice.

the hypothermic animal at 30°C, 31 mm Hg or approximately
one-fifth of the lung pressure is due to water vapor pressure.
As water vapor pressure is decreased the oxygen tension will be
increased. This increase may mean the difference between death
and survival.

Studies (Barker, 1957) have shown that a major factor in the
tolerance to hypoxia of several mammalian species is the affinity

1960 MAMMALIAN HIBERNATION 327

of hemoglobin for oxygen. Cooling of the blood will inerease
the affinity of hemoglobin for oxygen or shift the hemoglobin
dissociation curve to the left much like that oeeurring with alti-
tude adaptation. This likewise may account for survival of the
cooler mammal.

The mechanism of body temperature depression. What is the
nature of this cooling response upon hypoxic exposure? The
cooling curves indicate that as exposure continued the tempera-
tures tended toward a new lower level. This may indicate one of
the followme: (1) that an equilibrium has been established
between lowered metabolism and reduced heat loss due to de-
creased temperature gradients between animal and environment ;
(2) that regulatory mechanisms in hypoxia are now in action;
or (5) that this new temperature level represents a new regu-
lated level permitting survival during the stress of hypoxia.

The peripheral vasodilation which is seen may be interpreted
as an attempt at regulation of body temperature at a preferred
lower level or hypoxic interference on blood vessels directly or
through the vasomotor centers.

These experiments indicate that the cooling is of survival
value but they do not indicate whether or not the cooling is
simple hypoxic interference upon temperature regulation or an
actual regulated response. There is evidence from two Hungarian
laboratories that this response is regulated in the rat. Don-
hoffer et al. (1957) have shown that appropriate bilateral epi-
thalamie lesions will abolish the hypoxic cooling response and
the hypoxie decrease of oxygen consumption, thus implicating
the involvement of the central nervous system. Jarai and Lend-
vay (1958) have exposed rats to 400 mm He barometric pressure
and followed the oxygen consumption and cooling curves. In
rats given injections of dinitrophenol, oxygen consumption and
heat production were increased, vet the degree of rectal tempera-
ture decrease was the same as that seen in the controls. They
interpreted this as an indication that the hypoxic temperature de-
pression is of a regulated nature.

The hibernating mechanism as a factor in hypoxic toler-
ance. It appears that there are mechanisms other than simple
body cooling that protect the hibernator in hypoxia. It was seen
that the ground squirrel would outlive the non-hibernating rat
even though the ambient temperature was such that no body
cooling could occur. The hamsters did not cool rapidly at
higher temperatures and showed the greatest survival ability.
When the tolerances of individual animals were studied, it was

328 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

seen that those animals surviving longest at any set of pressure
and temperature conditions did not always show the greatest
rate of temperature depression. Perhaps hypoxic survival is
dependent upon a marked reduction of the metabohe rate. Popo-
vie (personal communication) has demonstrated that the metab-
olism of a hibernator in natural hibernation is lower than that
of the hibernator artificially cooled to the same body temperature.
Is it possible that the hibernator not in the hibernating state

—
GROUND SQUIRREL |
TT T Li T T —T | So T a Saaean | T
200+ 500 |
; Mas
i=)
fc 456 RECTAL and THORACIC TEMP.]
S} 175+ a
- 35
2 0 THORACIC TEMP.| ~~ iT
S | 400+ “
=) 150+ & 6
Zz 3] 350+
S| | z ;
1254 B
te ‘| 300+ S
3 2 +
& < i
~| 100+ &| 250+ >
a = 4
o a
(2)
es &| 200+ fs
Wl 5+ < :
ind T15] 0
bs &| 150+ METABOLISM
4. m
wy 50 + = 5 5
a =| 100+ @ 10
<
Be) 25+ - i
+ 5
: oN A
o+ o+4+—_++ + t—— t +——+ +—}+———+—_++
0 10 20«30—S 40 50 60 7089 90 100 110
T IoM.E IN M UN UTES

Fig. 4. Changes in metabolism (Og consumption), body temperatures and
heart rate oceurring when ground squirrels breathed a 5-6 per cent mixture
of oxygen in nitrogen at a chamber temperature of 12°C. Each curve repre-
sents a mean of six experiments. Standard error of the mean is indicated

at selected times.

when confronted with hypoxic stress can simulate the same
mechanisms utilized when entering hibernation?

The recent data (Lyman, 1958) obtained with chronic prepara-
tions on entrance into hibernation have given us criteria which
can be used to determine whether or not responses to hypoxia
are similar to those of hibernation. In the entrance into hiber-
nation, declines in heart rate and metabolism occur. These
decreases are not temperature-dependent as body temperature
falls at a slower rate than does either heart rate or metabolism.

1960 MAMMALIAN TIIBERNATION 329

Likewise, during the entrance into hibernation peripheral vaso-
dilation may be occurring. As an experimental approach these
criteria were tested on animals exposed to cold and hypoxia.

In the first series of experiments, hamsters, rats and ground
squirrels were restrained in small eylinders and placed in a 12°C
water bath. Electrocardiograms, skin, chest, and rectal tempera-
tures were continuously recorded. Oxygen consumption was
measured according to a method deseribed by Adolph (1950).
Carbon dioxide production was similarly measured.

H A M S T E R
L
T T ie a T > > T T 1
5 mee oe |
4 +35
< |
RECTAL TEMP.
> rm [RECTAL TEMP) |
4 ~
a| 150+ & ee
eq &
a 5| 350+
Z iS 4
0) 125+ ile SI
: | <} 300 +25}2
i n ia?)
° 3 nm
a
&| 1007 py] 250 7- HEART RATE | 20|>
Zz re) a
w a a
© a 200+ 2
foe] 75 Bb {oo}
a) < +15
Ae fm 180 METABOLISM BS
& e
z sor & ©
le ay ° o +10
A f/ 100+ ©
° m
ma
<| 25
2 OT (5% Op ON AIR 1s
=
0 0
0 10 20 30 40 50 60 70 80 90
TIME IN MINUTES

Fig. 5. Changes in metabolism (Os consumption), body temperatures and
heart rate occurring when hamsters breathed a 5-6 per cent mixture of
oxygen in nitrogen at a chamber temperature of 12°C. Each curve repre-
sents the mean of six experiments. Standard error is shown at selected

times.

Figures 4 and 5 represent the data obtained on six hamsters
and six ground squirrels when the air going through the cylinder
was changed to a mixture of 5 to 6 per cent oxygen in nitrogen.
Upon hypoxie exposure a decrease in heart rate not dependent
upon body temperature decrease occurred. For individual
animals, the heart rate decrease was not consistent but varied
considerably, dropping rapidly at times, rising slightly and

5330 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

plateauing at other times. After the first large decrease in heart
rate, the mean heart rates showed a generally slower decline
which was perhaps dependent upon decreasing heart tempera-
ture. The rise in heart rate upon the end of the hypoxie exposure
appeared to be temperature dependent but with a higher tem-
perature coefficient than that of the latter part of the hypoxic
exposure.

The data indicate that there was a decrease in metabolism.
as expressed in per cent of the prehypoxie exposure value, not
dependent on body temperature decrease. Considerable fluctua-
tion of metabolism was seen in individual animals. Upon the
end of the exposure, metabolism rose sharply and did not appear
to be body temperature dependent. The metabolism data of
these experiments must be considered with extreme caution.
The prehypoxic exposure metabolism is not basal, but represents
that of a restrained animal exposed to cold. Metabolism was
above basal for two reasons: (1) increased thermogenesis due to
cold exposure, and (2) occasional struggling of the animal. These
experiments must be repeated using unrestrained animals traimed
to rest quietly in a eylinder.

The responses obtained appear to meet the criteria of hiberna-
tion entrance mechanisms, in that there are non-temperature-
dependent decreases in heart rate and metabolism and, as has
already been shown, peripheral vasodilation. It may be argued
quite successfully that all of these responses are hypoxic re-
sponses and only similar to hibernation responses by coincidence.
The chief objection is that rats, non-hibernators, exposed to 6 per
cent oxygen in similar experiments also showed very sharp non-
temperature dependent decreases in metabolism and heart rate
which were followed by immediate death. At higher oxygen ten-
sions, 75 mm He or 10 per cent, which allowed survival, the rat
showed more gradual and steady declines in heart rate and
metabolism which again were not strictly temperature dependent.
In these experiments the rats could not rewarm themselves fol-
lowing the hypoxie exposure, and body temperature, heart rate
and metabolism continued to fall at the same rate.

In a series of experiments which is being performed in this
laboratory at the present time, heart rates are measured while
the animals are taken to altitude at varying rates in the chamber.
The results so far may be described briefly as follows. If the
chamber temperature were high and decompression slow, an
increase in heart rate of rats and ground squirrels would be seen

1960 MAMMALIAN HIBERNATION 301

followed by a rapid decline not dependent on thoracic tempera-
ture decline. Hamsters did not show this increase at any tempera-
ture but always showed a very rapid decrease in heart rate. Per-
haps this is related to the increased tolerance of the hamster.
If the chamber were colder and decompression rapid, the squir-
rels likewise would show a very rapid decline in heart rate which
was not temperature dependent. Often the heart rates would
climb to higher levels again and then decline in a temperature
dependent fashion.

It appears from these recent experiments that cold and rapid
onset of hypoxia enhance the heart rate depression in the squirrel.
It may be speculated that the cold ground squirrel is partially
‘*set’’ to enter hibernation, and the hypoxia serves as a trigger.
Ilowever, we cannot conclude that these hypoxie responses are
the same as hibernating responses in the hibernating: species.
The conclusions of Donhoffer et al. (1957) and Jérai and Lendvay
(1958) are that mammals possess mechanisms for depression of
metabolism and temperature in hypoxia. Is it these mechanisms
that the hibernators use for entering natural hibernation? With
the widespread occurrence of hibernating animals in the dif-
ferent mammalian orders it hardly seems likely that parallel
evolution of hibernation mechanisms could have occurred. It is
more likely that the hibernators make use of equipment already
present in these orders. The evidence for neural regulation
of metabolic reduction in rodents has been presented (Donhoffer
et al., 1957; Jarai and Lendvay, 1958). Perhaps the hypoxia
brings about a forced metabolic reduction similar to the ‘‘active
metabohe reduction’? of hibernation as deseribed by Lyman
(1958). It may be that hypoxia can initiate the neural activity
which is involved in hibernation but not the lone-term endo-
erine regulation.

Hypoxia and artificial hibernation. Differences between hi-
bernation and artificial hypothermia exist. Before we conclude
that they are completely different we must critically examine
all methods of inducing hypothermia. The responses obtained
in cold and hypoxia are similar if not the same as the responses
of entrance into hibernation. Is the end result the same? Ground
squirrels were exposed to 150 mm He barometric pressure in
a 7°C chamber for 5-6 hours. During this period all body temper-
atures fell to about 2 degrees above the chamber temperatures.
Heart rates ranged from 7 to 27 beats per minute. When atmos-
pherie pressure was restored to normal, temperatures remained
the same. Heart rates remained in the above range and were

Og
i

332 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

extremely arrhythmic as has been reported for true hibernation
(Lyman, 1959). After four hours the chamber temperature was
increased to 15°C. When the chamber temperature reached
approximately 15°C, rapid rewarming of the squirrels began
with chest temperatures and heart rates rising rapidly as has
been reported for arousal from natural hibernation (Lyman
and Chatfield, 1955). Oxygen consumption data have not been
obtained, and it is not known whether or not these squirrels
will rewarm spontaneously from extremely low body tempera-
tures.

These squirrels may be in a state of *‘neural’’ hibernation but
not ‘‘endoerine’’ hibernation. We hope that this symposium will
establish definitive eriteria for hibernation so that such ques-
tions may be answered.

Conditioning of responses to hypoxia. It has been noted that
after ground squirrels were used for several hypoxic exposures,
for example, three exposures in eight days, responses to hypoxia
changed. During the control prehypoxie period these squirrels
maintained a constant thoracic temperature while rectal tem-
peratures fell rapidly. Immediately upon the onset of decom-
pression of the chamber, before a real hypoxie environment had
been established, thoracic temperature fell rapidly toward the
level of the rectal temperature. When the thoracic temperature
decreased to less than a degree above the rectal temperature, both
temperatures fell in a parallel fashion. Heart rate in these
animals declined rapidly and did not show the fluctuations found
in squirrels upon the first exposure. Metabolism also fell rapidly
without fluctuation. At the end of the hypoxic exposures re-
warmine was faster, heart rate increased faster and metabolism
increased faster and to a much higher level than those of first-
run squirrels. In a limited series of rat experiments the re-
sponses to hypoxia did not show any statistically significant
changes with repeated runs.

The change in response of the squirrel with repeated exposures
may represent adaptations at cellular levels or a conditioning
at the regulative levels. If much central nervous system involve-
ment is necessary for these responses then the development of a
conditioned reflex is a strong possibility. The fact that the
thoracic temperature began to decrease before a real hypoxic
environment had been established indicates that it may be a
central nervous system response. More experiments are needed
to truly define the nature of these changes.

1960 MAMMALIAN HIBERNATION 333

Diseussion

It has been shown that rapid body cooling enhances survival
in hypoxie exposures. It has likewise been pointed out that cool-
ine in itself will not account for species differences in hypoxic
resistance. None of the mammals studied utilized the vascular
responses similar to those utilized by diving mammals. There is
evidence that the hypoxie metabolie depression and temperature
depression are regulated by the central nervous system and are
not entirely direct effects of hypoxia on tissues. It can be stated
that hypoxic responses and hibernating responses appear quite
similar; whether or not they are the same responses remains to
be conclusively proven. Perhaps when more data are obtained
for both hibernators and non-hibernators for exactly the same
type of hypoxie exposure the answer will be found. It may be
speculated that the forced metabolic depression in hypoxia is of
henefit to the animal.

Other possibilities must still be considered. One factor which
may give the hibernator enhanced survival in altitude is the
possession of hemoglobin of high oxygen affinity. The very
limited published data indicated that this may be the case. For
example, the marmot hemoglobin has a one-half saturation
oxygen tension of 23.8 mm He (Prosser, 1950), the hamster, ap-
proximately 20 mm He (Barker, 1957), the rabbit 32 mm He,
mouse 72 mm-He, and rat 40 mm He (Prosser, 1950). These
values should be considered with caution as the ‘‘physiological’’
eurve may be different from the laboratory derived curve. The
hemoglobins of the hibernators seem to have high oxygen affinity.
lt has been demonstrated that the higher the oxvgen affinity
the greater the hypoxie tolerance (Barker, 1957). More data is
needed for the rest of the hibernating species before this factor
can be evaluated.

It has been pointed out that anaerobic metabolism may be the
predominant metabolism of hibernation (Klar, 1951; Bidrek
ct al., 1956). However, further biochemical studies are needed,
especially during the hypoxie exposure. Whether or not the
hibernator may utilize anaerobic pathways more extensively in
hypoxia is another possibility that must be investigated.

REFERENCES
ADOLPH, E. F.
1950. Oxygen consumption of hypothermic rats and acclimatization to
cold. Am. J. Physiol., 161:359-373.

334 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

ApoupnH, E. F. AND J. RICHMOND
1955. Rewarming from natural hibernation and from artificial cooling.
J. Appl. Physiol., 8:48-58.

BARKER, J. N.
1957. Role of hemoglobin affinity in determining hypoxia tolerance of
mammals during infancy, hypoxia, hyperexia and irradiation.
Am. J. Physiol., 189:281-289.

BiorcK, G., JOHANSSON AND H. SCHMIDT
1956. Reactions of hedgehogs, hibernating and non-hibernating, to the
inhalation of oxygen, carbon dioxide, and nitrogen. Acta physiol.
seand., 37:71-838.

DENZER, H. W.
1950. Comparative altitude physiology of animals. Chapter IV-K. Jn:
German Aviation Medicine. World War II. Vol. I, U. S. Gov.
Print. Off., Washington.

DONHOFFER, Sz., Gy. MestTyAn, L. NAGY AND Gy. SZEGVARI
1957. Uber den Mechinismus der hyperthermischen Steigerung und der
hypoxischen Senkung des Energiewechsels. Acta Neuroveget.,
16 :390-399.

Hirstanp, W. A., W. T. RocHoup, F. W. STEMLER, D. E. STULLKEN AND
J. E. WIEBERS
1950. Comparative hypoxie resistance of hibernators and non-hiber-
nators. Physiol. Zool., 23:264-269.

JARAI, 1. AND B. LENDVAY
1958. The action of gdinitrophenol on heat production and body
temperature in hypoxic hypoxia. Acta physiol. Hung., 13:147-
151.

Kiar, E.
1951. Beitriige zur Biologie des Winterschlafes. Zschr, ges. exp. Med.,
109 :505-516.

LYMAN, C. P.
1958. Metabolie adaptations of hibernators. Fed. Proe., 17:1057-1080.
1959. Blood pressure and other measurements on the ground squirrel
during the hibernating cycle. Fed. Proe., 18(378) :96.

LYMAN, C. P. AND P. O. CHATFIELD
1955. Physiology of hibernation in mammals. Physiol. Rev., 35:403-
425.

Prosser, C. L., Editor

1950. Comparative animal physiology. Philadelphia, 888 pp.
SPALLANZANI, L.

1803. Mémoires sur la respiration. Geneva, 373 pp.

1960 MAMMALIAN HIBERNATION 335

DISCUSSION FOLLOWING BULLARD’S PAPER

DAWSON inquired as to the change in RQ during the meta-
holie changes of hypoxia. BULLARD rephed that he measured
not RQ but respiratory exchange ratio which is changed by
ventilation, but that the values are not as low as reported for
hibernation since the RQ for hibernation is a reflection essentially
of a prolonged hypothermia in which body fat is utilized.

JOMHANSSON pointed out that BULLARD used stoppage of
respiration as the termination point of biological activity follow-
ing hypoxia, but he wondered about the heart activity which con-
tinued. BULLARD said they had not seen ventricular fibrilla-
tion, but only low voltage waves which continued for some time.
This time was highly variable; respiratory cessation was also
variable but gave a better end-point.

JOHANSSON stated that many believe that the basic differ-
ence between hibernators and non-hibernators is the ability of
the autonomie nervous system to be functional at a low tempera-
ture. Of even greater importance, JOHMANSSON felt, is a basic
difference in metabole patterns between the two kinds of ani-
mals. One way of showing this is to remove the heart and perfuse
it with Tyrode solution. In this way nervous and humoral influ-
enees would be removed more or less completely. In such an
experiment, the heart of a hibernator will stop beating at a much
lower temperature than a non-hibernator’s heart.

ADOLPH volunteered a method for clarifying the distinctions
between hypoxia and hypothermia. In order to avoid hypo-
thermia, if one wishes to study hypoxia alone, thermistors are
placed in the animals, which operate a heating device. In such
a situation, the hypoxic animal continuously activates the mstru-
ment to keep itself warm, because the animal will inevitably
tend to cool in hypoxia. ADOLPH also pointed out that in
hypoxia there are diminutions in oxygen consumption, and often
in heart rate, although the hypoxie animal continues to breathe
excessively in the presence of low oxygen.

BULLARD stated that Hungarian papers are available which
deseribe an abolition of the depression in body temperature and
in metabolism following hypoxia by appropriately placed brain

lesions.

XVII
ON THE CARDIAC RESPONSE IN
HIBERNATION AND INDUCED
HYPOTHERMIA

FUNCTIONAL, PATHOLOGIC AND METABOLIC
ASPECTS’

By H. 8. 8. Saragas
Institute of Physiology, University of Helsinki
Helsinki, Finland
and Wenner-Gren Cardiovascular Research Laboratory
Stockholm, Sweden

In the course of the last few years it has become increasingly
evident that natural hibernation and indueed hypothermia in
non-hibernating mammals represent fundamentally different
physiologic states. In natural hibernation the main vital fune-
tions continue, even if profoundly retarded, down to body tem-
peratures near zero, a considerable degree of homeostasis being
maintained. That the neuroendocrine system, for example, re-
tains a relative integrity in deep hibernation is suggested by the
mere fact that the hibernating animals are capable of arousing
themselves by various external stimuli, even including intense
cold. Non-hibernating mammals rendered hypothermic by the
customary technic, consisting of a suppression of the thermo-
regulatory defense by anesthetics followed by intense cooling of
the body, behave in a different fashion. With decreasing body
temperature their vital functions are also retarded. However, at
body temperature levels of about 10° to 20°C fatal cardiae crises
usually supervene even when artificial respiration is employed to
maintain sufficient blood oxygenation. Moreover, mammals in
deep tolerable hypothermia are generally unable to arouse
themselves spontaneously no matter what the external stimulus.
The only means of reviving them is by the external or internal
appheation of heat. Then, when body temperatures above the
levels of the so-called **cold narcosis’’ are attained, and pro-
vided there is no residual anesthesia, the hypothermic mammal
may eventually rewarm itself by means of its thermoregulatory
mechanisms.

1 Support for the writing of this survey and for some of the experiments sum-

marized here was provided by the Emil Aaltonen Foundation and the Sigrid
Jusélius Foundation.

BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The main physiologic differences between hibernation and
induced hypothermia have been authoritatively discussed by
Popovie (1952), Kayser (1955), and Lyman and Chatfield
(1956), among others. The present paper is an attempt to
analyze the cardiac response in hibernation and induced hypo-
thermia with reference to some differences in the cardiae fune-
tion, cardiac pathology and myocardial metabolism. The cardiac
reactions in hibernation will be contrasted with those in hypo-
thermia, induced in anesthetized mammals under artificial respir-
ation by surface cooling. As is known, hypothermia of this type
has been most widely studied by physiologists and most widely
practiced in surgical therapy. Moreover, such a restriction seems
justified in view of the facet that, depending on experimental
variables, a wide variety of physiologic states may occur in hypo-
thermia, just as they may in normothermia.

Cardiac function. Generalizing, it may be said that with de-
creasing body temperature the heart function is progressively
retarded in both hibernation and induced hypothermia. How-
ever, closer observations on the heart rate, cardiae rhythm and
configuration of the electrocardiographic records in association
with hibernation and hypothermia reveal remarkable differences.

At first, the definitely different critical body temperatures for
the heart function may be recalled. In hibernating animals a
coordinated heart function is maintained down to body tempera-
tures near zero (Suomalainen and Sarajas, 1951; Sarajas, 1954;
Biorek and Johansson, 1955; Dawe and Morrison, 1955; Lyman
and Chatfield, 1956). According to Adolph (1951), infant non-
hibernatine mammals with insufficient thermoregulation can
tolerate body temperatures of the same general order. Adult
non-hibernating mammals, on the contrary, generally succumh
with ventricular fibrillation or eardiae standstill when body tem-
perature levels ranging from about 10° to 20°C are attained.
This terminal cardiae crisis is usually preceded by ventricular
ectopic beats and nodal or idioventricular rhythms (Crismon,
1944: Bigelow cf al., 1950; Biérek and Johansson, 1955). Finally,
it may be mentioned that isolated heart preparations from
hibernating and adult non-hibernating mammals stop funetion-
ine at temperature levels closely corresponding to the critical
hody temperatures for heart function in these two groups of
mammals (Hirvonen, 1956).

Until recently, information on the earliest cardiac events i
animals entering hibernation has been seanty. However, Lyman
(1958) has shown that in woodchucks the heart rate tends to

1960 MAMMALIAN HIBERNATION 339

decline prior to the fall in body temperature, and judging from
the subsequent report of Strumwasser (1959a) the same holds
for the California ground squirrel. These observations are also
compatible with our observations on hedgehogs (Suomalainen
and Sarajas, 1951; Sarajas, 1954). All the investigators agree
that during entrance into hibernation there is initially a steady
decline in the heart rate fairly proportionate to the fall in body
temperature (Suomalainen and Sarajas, 1951; Sarajas, 1954;
Dawe and Morrison, 1955; Lyman, 1958; Strumwasser, 1959a).
In deep Inbernation, however, the heart rate appears largely
independent of the body temperature levels; prolonged periods
of extreme bradycardia are interrupted by short periods of rela-
tive tachyeardia. This phenomenon has been shown in different
hibernators including the hamster (Chatfield and Lyman, 1950;
Lyman, 1951), the hedgehog (Suomalainen and Sarajas, 1951;
Sarajas, 1954; Bidrek and Johansson, 1955; Dawe and Morris-
son, 1955), the four species of ground squirrel (Lyman, 1951;
Dawe and Morrison, 1955; Nardone, 1955; Kayser, 1957 ; Landau
and Dawe, 1958; Strumwasser, 1959b), and the woodehuck (Ly-
man, 1958). In the framework of the present diseussion this
phenomenon is of significance in that it clearly demonstrates
that even in deep hibernation the heart funetion is influenced by
neural and/or endocrine mechanisms.

The cardiac reactions in hibernating mammals roughly charae-
terized above differ essentially from those in non-hibernating
mammals subjected to induced hypothermia. The changes in the
heart rate in the early stages of hypothermia appear to be
largely determined by the depth of anesthesia. In lightly
anesthetized animals there occurs an increase in the heart rate
in the early cooling period, followime which, at about 35°C, the
heart rate begins to fall in a fairly linear fashion (Grogsse-
Brockhoff and Schoedel, 1943a; Pree et al., 1949; Bigelow et al.,
1950; Biorek and Johansson, 1955). This initial increase in the
heart rate is obviously effected by direct cutaneo-cardiac reflexes,
increased ‘“*‘venous return’’ on shivering, and generalized syin-
pathetic activation elicited by cold stimulus, and is even rein-
forced by the increased excitability of the medullary (Grosse-
Brockhoff and Schoedel, 1948b) and hypothalamic (IKoella e¢ al.,
1954) centers reported to occur in the early stages of hypother-
mia. When deeper anesthesia is used, this initial activation of
the heart function on cooling is largely abolished ; then the heart
rate tends to decline linearly from the very beginning of cooling
(Hook and Stormont, 1941; Bigelow ef al., 1950; Bidrek and

340 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Johansson, 1955). Nevertheless, it may be poimted out that the
progressive bradycardia by induced hypothermia is somewhat
relative, contrary to that in hibernation. In our experience with
dogs, and judging from the data given by others (Hook and
Stormont, 1941; Grosse-Broekhoff and Schoedel, 1943a; Bigelow
et al., 1950; Bidrek and Johansson, 1955), the heart rates charac-
teristic of normal, resting, unapprehensive dogs (Murphy, 1942)
are not attained before body temperature levels of about 25°C.
This might be largely due to the fact that, for example, pento-
barbital and ether, both commonly employed to assist the indue-
tion of hypothermia, do considerably accelerate the heart rate,
the former apparently by its vagal blocking action (Nash ef al.,
1956) and the latter by generalized sympathetic activation
(Brewster ef al., 1953). With these statements in mind, it seems
evident that in the early stages of hypothermia there is, at all
events, some trend to sympathetic stimulation with a resultant
acceleration of the heart rate. Remarkably enough, just the
reverse apparently holds for animals entering hibernation, for
Strumwasser (1959a) has recently presented evidence that dur-
ine entrance into hibernation there is a shift of balance toward
the parasympathetie nervous system. This would offer a reliable
explanation of why, in the hibernating animals, the heart rate
heeims to decline even before the fall in body temperature.

Several investigators (Grosse-Broekhoff and Schoedel, 1948a;
ITaterius and Maison, 1948) have found that neither sectioning
of the vagi nor atropinization have any appreciable influence
on the bradycardia of induced hypothermia. Moreover, the re-
activity of the autonomic centers has been reported to be de-
creased in deep hypothermia (Koella ef al., 1954). It appears,
then, that the hypothermic bradyeardia is mainly effected by the
direct depressant action of cold on the cardiae pacemaker activ-
ity, and that, on the other hand, deep hypothermia, in contrast
to hibernation, results in a breakdown of the homeostatic mech-
anisms normally controlling the heart function.

The general configuration of the electrocardiograms taken dur-
ine hibernation and induced hypothermia also reveals definite
differences. According to our own observations (Sarajas, 1954),
and to those of Biorek and Johansson (1955), and of Dawe and
Morrison (1955), the electroeardiographie intervals are fairly
uniformly prolonged in hibernating animals, while the con-
figuration of the deflections does not otherwise show any sienifi-
eant deviation from normal. This is in strikine contrast to the

1960 MAMMALIAN HIBERNATION 34]

clectrocardiographic changes accompanying induced hypother
mia. In hypothermic animals, the Q-T interval is characteristic-
ally more prolonged than the other intervals. Moreover, there
are associated changes in the configuration of the deflections.
The bizarre deformation of the whole ventricular complex con-
stitutes a particularly prominent feature (Grosse-Brockhoff and
Schoedel, 1943a; Lange ef al., 1949; Bigelow et al., 1950; Hee-
nauer et al., 1950; Bidrek and Johansson, 1955).

Judging from the electrocardiographie records, and as may
well be expected, the funetional cardiae changes in hibernation
are completely reversible (Sarajas, 1954). According to Chat-
field and Lyman (1950), and Dawe and Morrison (1955), the
heart rate begins to increase before any increase in the body
temperature becomes apparent. It has also been found that with
increasing body temperature the heart rate increases, at first
slowly and then more rapidly (Chatfield and Lyman, 1950; Suo-
malainen and Sarajas, 1951; Sarajas, 1954; Bidrek and Johan-
sson, 1955; Dawe and Morrison, 1955; Landau and Dawe,
1958). From their pharmacologic observations, Chatfield and
Lyman (1950) concluded that the rapid increase in the heart
rate during arousal is effected by a mass activation of the sym-
pathetico-adrenal system, as previously suggested by Britton
(1928) and Barcroft (1934). Our own observation that in hedee-
hogs the heart beats at supernormal rates in the later stages of
arousal, and that normal heart rates are attained several hours
after the body temperature has become normalized (Sarajas,
1954), also seems consistent with this concept. Finally, it may
be stated that, at least in hedgehogs, the electrocardiogram fol-
lowing arousal appears completely normal (Sarajas, 1954).

There is somewhat controversial evidence as to the degree of
restitution of the cardiac function on rewarming from tolerable
levels of induced hypothermia. Fatalities, indeed, are known
to have occurred during and following rewarmine (Haterius and
Maison, 1948; Pree et al., 1949; Fedor e¢ al., 1958). Moreover,
Swan (1956) has recently presented evidence of acute circulatory
collapse with associated tachycardia in dogs following rewarming
from moderate hypothermia. He was emphatie in stating that
an animal cooled and rewarmed is not returned to physiologic
normality. On the other hand, some investigators maintain
that the electrocardiogram is normalized upon rewarming (Took
and Stormont, 1941; Pree et al., 1949; Hegnauer et al., 1951;
Santos and Kittle, 1958), while others have recorded residual
changes especially in the ventricular complexes (Bigelow ef al.,

342 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

1950; Gunton et al., 1956; Fedor et al., 1958; Helbig and Hein-
rich, 1958).

Cardiac pathology. In the previous section the evident type
difference between the electrocardiographie changes accompany-
ine hibernation and those in induced hypothermia was stressed.
Toward the end of the last decade Lange ef al. (1949) found that
artificial respiration even with pure oxygen, as commonly em-
ployed in actual experimentation with hypothermia, does not
prevent or alter the electrocardiographie abnormalities, inelud-
ine the bizarre deformation of the ventricular complex associated
with tolerable hypothermia. Remarkably enough, however, they
noted that acidification of the blood, which tends to counteract
the shift of the hemoglobin dissociation curve to the left by cold,
resulted in a normalization of the electrocardiogram. The same
could also be effected by increasing the amount of oxygen
physically dissolved in the plasma. As a general result these
investigators coneluded that in induced hypothermia there may
exist cardiae hypoxia, without concurrent hypoxemia. They fur-
ther suggested that the deaths from acute exposures to cold may
be due to hypoxie damage to the heart, of too early a stage to be
detected by the routine histopathologic technics.

The theory of cardiac hypoxia in conditions of induced hypo-
thermia was initially advocated by some workers (Bigelow et al.,
1950; Hegnauer ef al., 1950), but subsequently it has been
mainly abandoned (Penrod, 1951; Hegnauer, 1954; Berne, 1954;
Jude et al., 1957). Moreover, until recently it has been generally
accepted that hypothermia is a safe and non-injurious procedure.
There follows some data on our recent studies intended to eluci-
date the pathology of the heart in association with hypothermia
in dows. As will be seen, the results furnish evidence that in
hypothermia the heart may actually suffer from hypoxia, with
resultant injury to the myocardium. The dogs, totalling 100
individuals, were subjected to graded hypothermia with or
without subsequent rewarming, by use of routine technies. The
results, some of which have been reported elsewhere (Sarajas
and Nilsson, 1954; Sarajas, 1956), may be outlined as follows:
in dogs cooled until fatal termination or held from one to four
hours at body temperature levels of about 22° or 27°C without
subsequent rewarming, the myocardial wall exhibited shght but
detectable fatty degeneration and early necrobiotic changes in the
form of coagulative necrosis. In addition, some histochemical
evidence of glycogen and potassium loss from the muscle fibers
was observed. In dogs subjected to hypothermia of the same

1960 MAMMALIAN HIBERNATION 343

degree and equal duration and then rewarmed and killed 3 days
to 3 years following rewarming, definite, even grossly detectable
inyoeardial necroses were found which showed different stages
of development and healing aceording to the length of survival.
In both the acute and long-term experiments the lesions were
found in the great majority of cases. The dynamics of the lesions
corresponded to that of experimental myocardial infarction in
dogs which indicates, among other things, that they were patho-
genetically related to the hypothermic period. On the other hand,
the lesions were mainly restricted to the subendocardial muscle
layers of the left ventricle. This furnishes evidence for the
hypoxic origin of the lesions, for due to certain peculiarities of
the coronary circulatory dynamies the subendocardial muscle
layers of the left ventricle are the first to suffer from any type of
hypoxia (Raab, 1956; Schiitz, 1958). The histochemically de-
monstrable depletion of myocardial glycogen associated with loss
of the intracellular potassium gives further support to the
hypoxic origin of the lesions, for hypoxic myocardium is known
to lose both glycogen and potassium (Dennis and Moore, 1938;
Merrick and Meyer, 1954; Raab, 1956).

For the present not much is known about the mechanisms
which would render the heart muscle hypoxie in induced hypo-
thermia. Lowered arterial pressure (Bigelow et al., 1950) in
the presence of inereased blood viscosity (Heenauer et al., 1950)
with eventual intravascular aggregation of the red cells (Bigelow
et al., 1950; Konrad and Zindler, 1958), all known to oceur in
hypothermia, may be proposed as possible factors. On the other
hand, when the tachycardia inherent to the early stages of cooling
was discussed, it was concluded that in the early stages of cooling
there is at all events some trend to sympathetic activation. This
is in harmony with our recent studies (Sarajas eft al., 1958),
suggesting that the induction of hypothermia generally evokes
a stress response, even if some stress manifestations may be
obscured by certain specific effects of hypothermia. It may there-
fore be of interest that, according to Raab (1956), epinephrine,
the release of which is inherent to any stress situations, is an
hypoxiatine agent and is capable of causing cardiac lesions
similar to those encountered in the present dog cases. Moreover,
Selye (1957, 1958) has recently produced similar cardiac lesions
in specially conditioned animals by various stressors including
administration of epinephrine, neuromuscular exertion, and cold
and hot baths. Also, the shock-like state of rewarming, which was
previously referred to, may seriously impair the myocardial

344 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

nutrition and thus contribute to the genesis of the eardiac
lesions under consideration. Even if, accordingly, the causation
of these hypothermic cardiae lesions can only be speculated upon,
the fact remains that similar cardiae lesions have recently been
found also by other investigators (Cecconi and Parentela, 1957;
Fisher et al., 1957; Heinrich and Schautz, 1958). Furthermore,
quite recently Hannon and Covino (1958) concluded from meta
bolie studies of heart shees and homogenates that a mild cardiac
hypoxia may exist in hypothermia. The possible functional sig-
nificance of these cardiac lesions which appear to result from
hypothermia has been discussed elsewhere (Sarajas ef al., 1956).
Suffice it to say that they constitute a plausible explanation for
the erave, and even fatal, disturbances in cardiae function as-
sociated with induced hypothermia. To our knowledge, there are
no reports that pathologic cardiac changes would result from
natural hibernation. Neither can such a possibility be seriously
considered.

Myocardial metabolism. Generalizing, it may be said that any
morphologically demonstrable vital reactions are preceded by a
metabolic interference in the tissue involved. It may therefore
be of interest that the hypothermic heart, contrary to the hiber-
nating one, appears to suffer from metabolic disturbances. The
histochemical evidence for loss of glycogen and potassium from
the heart in conditions of hypothermia, as previously mentioned,
has been confirmed by chemical methods (Szekeres et al., 1954;
Covino and Hegnauer, 1955; Gollan et al., 1957). According to
Gollan ef al. (1957), hypothermia gives rise to a marked potas-
situ loss from the myocardium, which is only shehtly aggravated
when the hypothermic animal is subjected to acute respiratory
hypoxia. Szekeres et al. (1954), in turn, have found that hypo-
thermia not only results in a depletion of the cardiae glycogen
but also in a loss of the high energy phosphates, both com-
mensurate with those evoked by respiratory hypoxia in normo-
thermic animals. These changes may be of significance at least
in two respects. First, they give further support to the concept
of eardiae hypoxia in conditions of hypothermia. Secondly, they
suggest that vital functions of the cell membrane such as selec-
tive permeability and active ion transport, which also are pre-
requisites for normal cardiae function, are disturbed in the hypo-
thermie heart apparently as a result of failure in the resynthesis
of high energy phosphates. In principle, just the reverse cardiac
metabole alterations are encountered in the hibernating animals.
According to the chemical and histochemical studies of Lyman

1960 MAMMALIAN HIBERNATION 345

and Ledue (1953) and Zimny and associates (1957, 1958), the
elyeogen content of the heart is increased in hibernation. Jude-
ing from our own determinations of the specific activity of the
heart glyeogen in awake and hibernating hedgehogs given
C1l4tlJabeled elucose, this glycogen accumulation in the heart is
rather tremendous (Forssbere and Sarajas, 1955). On the other
hand, Suomalainen (personal communication) has presented his-
tochemical evidence that the glycogen accumulation in the heart
is accompanied by an increase in the intracellular potassium,
and the recent studies of Zimny and Gregory (1958) indicate
that the cardiac high energy phosphates are maintained during
hibernation,

Several investigators including Lyman and Chatfield (1956)
and Zimny and Gregory (1958) have proposed that in hiberna-
tion the heart is storing glycogen for energy in awakening. As
Was mentioned, the process of arousal is essentially a mass activa-
tion of the svmpathetico-adrenal system, which then accelerates
heart function and metabolie activity. On the other hand,
Landau and Dawe (1958) have coneluded from the dark color
of the blood and of the mucous membranes of the mouth during
arousal that the metabolic activity increases more rapidly than
oxygen Is supphed. From these statements, and considering the
previously mentioned hypoxiating effect of epinephrine, it be-
comes apparent that during arousal the heart is beating under
impending hypoxia. Nevertheless, in this critical period ely-
colysis apparently safeguards the maintenance of the cardiac
energy metabolhsm, for Zimny and Gregory (1958) have pre-
sented evidence that the elvcolytie activity in the heart during
early arousal is essential in resynthesizing the high energy
phosphates. This, in turn, agrees with the generally accepted
concept that glycogen in the heart muscle is a reserve fuel to be
utilized for the resynthesis of the high energy phosphates under
anaerobic conditions only (Raab, 1956). Thus the known tol-
erance of experimental anoxia by the hibernating animals
(Biorck et al., 1956) may also be related to the high levels of
cardiac glycogen.

From the foregoing it may be coneluded that while the hiber-
nating heart is metabolically prepared to sustain the work load
imposed by the process of arousal, the hypothermic heart, when
faced with the hazardous stage of rewarming, is obviously sut-
fering from metabolic failure.

346 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

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LYMAN, C. P. AnD P. O. CHATFIELD
1956. Physiology of hibernation in mammals. Jn: The physiology of
induced hypothermia. Publ. 451. Nat. Acad. Sei.——- Nat. Res.
Council. Washington, D.C. Pp. 80-124.

LYMAN, C. P. Aanp E. H. Lepuc
1953. Changes in blood sugar and tissue glycogen in the hamster
during arousal from hibernation. J. Cell. Comp. Physiol., 41:
471-491.

Merrick, A. V. AND D, K. MEYER
1954. Glycogen fractions of cardiae muscle in the normal and anoxic
heart. Am, J. Plrysiol., 177:441-443.

MuRPHY, Q.
1942. The influence of the accelerator nerves on the basal heart rete
of the dog. Am. J. Physiol., 187:727-730.

NARDONE, R. M.
1955. Klectrocardiogram of the aretie ground squirrel during hiber-
nation and hypothermia. Am. J. Physiol., 182:364-368.

Nasu, C. B., F. Davis Aanb R. A. Woopsury
1956. Cardiovascular effects of anesthetic doses of pentobarbital
sodium, Am. J. Physiol., 185:107-112.

Prnrop, K. E.
1951. Cardiac oxygenation during severe hypothermia in dog. Am, J.
Physiol., 164:79-85.

Popovic, V.
1952. Contribution a l’étude de la thermogenése de l’homéotherme
refroidi. Mon. Acad. Serbe Sci., Belgrade, 199(7), 156 pp.

PREe. O:, 5
1949. The cardiovascular effects of acutely induced hypothermia. J,
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. ROSENMAN, K. Braun, S. RopBARD AND L. N. Katz

Raab, W.
1956. The adrenergic-cholinergic coutrol of cardine metabolism snd
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350 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Santos, E. M. anp C. F. Kirrie
1958. Electrocardiographie changes in the dog during hypothermia.

Am. Heart J., 55:415-420.

SaraJaAs, H. S.S8.
1954. Observations on the electrocardiographic alterations in the
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SARAJAS, H.S. 8. AnD T. E. NILSSON
1954. Beobachtungen ueber die Pathologie der experimentellen
Hypothermie beim Hunde. Langenbecks Arch, dtsch. Z. Chir.,
279:750-756; Verh. dtsch. Ges. inn. Med. 60. Kongress, 118-124.

SarRaJAS, H. 8. 8., P. NYHOLM AND P, SUOMALAINEN
1958. Stress in hypothermia. Nature, 181:612-613.

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Scut1z, E.
1958. Physiologie des Herzens. Berlin-Gottingen-Heidelberg, 570 pp.

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STRUMWASSER, F.
1959a. Thermoregulatory, brain and behavioral mechanisms during
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1956. The cireulation during hypothermia. Jn: The physiology of
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99-100.

1960 MAMMALIAN HIBERNATION aol

ZIMNY, M, L. aNd R. GREGORY
1958. High energy phosphates during hibernation and arousal in the

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1957, Carbohydrate metabolism during fasting and hibernation in
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DISCUSSION FOLLOWING SARAJAS’ PAPER

MENAKER asked if young, non-hibernating mammals which
have not yet acquired the ability to thermoregulate showed heart
damage on cold exposure. SARAJAS said he could not give any
definite answer to the question, yet he believed that young, non-
hibernating mammals may tolerate low body temperatures better
than adults because they have, e.g., a certain reserve for
anaerobic metabolic activity and because their neuroendocrine
apparatus only weakly responds to cold.

WIMSATT cited work of C. F. Bond and P. W. Gilbert (Anat.
Ree., 125:646, 1956) which demonstrated a profound slowing of
cardiac activity in diving birds when they submerge and during
the period of submergence, in contrast to non-diving birds in
which the opposite effect is seen. He generalized further that as
far as he knew, there were no metabolic rates of specific organs
which correlated ideally with a general reduction in metabolism.

RV TT
CIRCULATORY CHANGES IN THE
THIRTEEN-LINED GROUND SQUIRREL
DURING THE HIBERNATING CYCLE’

By CHARLES P. LYMAN and Regains C. O’BRIEN
Department of Anatomy, Harvard Medical School, Boston
and Museum of Comparative Zoology, Harvard University

Cambridge, Massachusetts

In the past few years the function of the heart in hibernation
has received considerable attention. For example, it has been
shown that there is a marked decline in heart rate before a
decline in core temperature when the animal starts to enter the
hibernating state (Lyman, 1958). In hibernation, the heart rate
may vary greatly with no apparent changes in body temperature
(Dawe and Morrison, 1955; Lyman, 1958). On arousal from the
hibernating state the heart rate increases prior to a change in
body temperature (Lyman and Chatfield, 1950, and references
above). These observations suggest that there must be important
changes in the circulation during the hibernating cycle and that
measurements of these changes might give further insight into
the phenomenon of hibernation.

Although some measurements have been made on the blood
pressure of mammals waking from hibernation (Dubois, 1896;
Chatfield and Lyman, 1950; Chao and Yeh, 1951) nothing has
been reported on the blood pressure of mammals either entering
the hibernating state or in natural, deep hibernation. The teeh-
nique developed by Still and Whitcomb (1956) for chronically
intubating the aorta of small mammals gave the opportunity of
measuring the blood pressure of hibernators over lone periods
of time. Measurements could be made as the animal passed from
the active condition into hibernation, as it remained in hiberna-
tion, and as it aroused from the hibernating state. The tube also
offered a means of introducing drugs of known pharmacological
effect into the circulation at any point during the hibernating
evele without disturbing the animal. Using in-dwelling thermo-
couples and electrodes, the body temperature and the electro-
cardiogram (KKG) could be monitored coneurrently with the
blood pressure,

!This research was supported in part by the U. S. Air Force under contract
no, AF41(657)-190 and in part by U. S. P. H. grants nos. RG-5197 and RG-5611.

354 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

This paper is a description of the changes which take place in
the blood pressure, body temperature and EKG during the
hibernating cycle of the thirteen-lned ground squirrel (Citellus
tridecemlineatus), and the effect of drugs during various parts
of this evele.

Materials and Methods

A total of 47 ground squirrels were intubated for this study.
Of these, five yielded satisfactory records of the various phases
of the normal hibernating cycle and seven others were used
successfully in the study of the effect of drugs on the circulation
during hibernation. Of the former, continuous records of 2 to
8 days were obtained from single animals. The animals were
kept in individual cages in a cold room which was maintained
at 6 =4°C. They were given shavings for bedding and Purina
laboratory chow and water ad libitum. Animals which had hiber-
nated over protracted periods of time were used preferentially.
Prior to intubation, the animals were aroused in a warm room,
and then anesthetized with an intraperitoneal injection of pento-
barbital sodium (80 me/kg¢). The aorta was exposed by an
abdominal incision. A small slit was made in the vessel about
1 em posterior to the renal arteries and a thin polyethylene tube
(PE 10, ID 28 mm, OD 0.6 mm), bevelled at the end, was
inserted into the slit and pushed rostrally 1.5 em. No modifica-
tions were made in the technique deseribed by Still and Whit-
comb (1956) except that the instrument used to make the slit in
the aorta was a curved Bard Parker blade (size 12), ground
to 0.5 mm width and sharpened to a needle-like point. Once
the tube was fastened in place with a tie to the muscles of the
back it was filled with heparin-saline (25 me heparin/10 ml
physiological saline) and closed at the distal end with a knot.
The abdominal incision was closed and the tube anchored again
with a tie at the caudal edge of the incision. The tube was then
led subcutaneously to an exit between the scapulae, and fastened
to the skin of the baek. The animal was given 40,000 units of
procaine penicillin and returned to its cage in the cold room.

If intubated animals were observed to re-enter hibernation,
they were removed in the hibernating state and fitted with one
or two thermocouples made of 34 ga. iron and constantan wire
individually protected with PE 10 polyethylene tubing. Usually
one thermocouple was fastened subcutaneously in the region near
the heart and the other fastened intraperitoneally at the mid-
abdomen. The thermocouple wires were led subcutaneously to

1960 MAMMALIAN HIBERNATION 355

the exit between the scapulae. Three silver wire electrodes were
sewed into the skin of the back. The tube from the aorta was
spheed using a section of 427 hypodermic needle and a long
piece of PE 10 tubing. All wires and the tube were passed
through a helical spring for protection, and the spring was sewed
to the back where the tube and thermocouple wires made their
exit from the animal.

The ground squirrel was placed in a round battery jar meas-
uring 23 em in diameter with food and water and the bedding
from its cage. The helical spring was led through a wire screen
which closed the top of the jar and was suspended with an
elastic band so that the animal could move freely in the cage
without being bothered by the cable.

Throughout the chrome experiments, temperatures from heart
and abdomen were each recorded every thirty-two seconds on a
Leeds and Northrup Speedomax thermoelectric recorder with an
accuracy of £0.25°C. The EKG and blood pressure were ob-
tained every four minutes, for a period of one minute.

Blood pressure was measured directly from the polyethylene
tube using a Statham P23D pressure transducer.” This was
amplified with a Grass low-level DC preamplifier, model 5P1A,
and Polygraph DC driver amplifier, model 5.°. Various sensitivity
settings were used during the experiments and a drift of as much
as 25 mm He. could take place in a twenty-four hour period.
However, the machine was calibrated at least twice a day, and
more often when exact measurements were required. Thus the
accuracy did not vary more than = 5 mm He which is a sheht
change compared to those which actually took place in the blood
pressure. In order to prevent clotting in the tube, a flow of
heparin-saline (0.6 mg/ml) of approximately 0.5 ml per day was
perfused through the tube by means of a slowly driven screw-
drive syringe. Because it was possible that the length of the
polyethylene tube might seriously affect the recorded pulse pres-
sure, various lengths of tubine were tried under known econdi-
tions of blood pressure. It was found that, within the conditions
of the experiment, neither the varying lengths nor temperatures
of the tubes made any appreciable differences in the blood pres-
sure measurements.

The apparatus was kept running day and night during the
measurements. At various times, records were obtained of ani-
mals in the active condition in the cold, during the process of

”

2 Statham Instruments Ine., Los Angeles, Calif.

° Grass Instrument Co., Quiney, Mass.

356 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

entering hibernation, in the hibernating state, and arousing from
hibernation. In the experiments using drugs, the agent was in-
troduced into the animal via the polyethylene tube. In all eases,
the approximate dose was determined by giving graded doses
of the drug in question to intubated, nembutalized rats. Awake,
intubated ground squirrels were given doses below those which
affected the rats, and the doses were increased until some effect
was noted. Subsequently, a comparable dose was used initially
in each experiment and increased gradually if no effect was
noted. Periodically, tests were made to be sure that the same
amount of heparin-saline solution did not produce a similar
result. In order to produce vasodilation individual doses of
acetylcholine chloride (Merek*) varying from 0.15 to 1.2 mg/ke
were given while Benodaine hydrochloride Merck’ was given at
8 to 41 me/ke. To produce vasoconstriction l-Norepinephrine
(Levophed bitartrate, 0.20¢, Winthrop) was used in coneentra-
tions of 6 to 44 ue/ke.

Results

The normal non-hibernating ground squirrel in the cold main-
tained a fairly steady heart temperature of 37 £1°C. The ab-
dominal temperature averaged 0.5 to 11°C below the heart
temperature. Blood pressure and heart rate varied considerably,
depending chiefly on the activity of the animal. Often the heart
rate was reduced as much as one-half in a few seconds, accom-
panied by a reduced blood pressure and an increase in pulse
pressure. Though this oecurred invariably if the animal were
alarmed, it also took place for no apparent reason. Over periods
when two annnals failed to hibernate for several davs the mean
blood pressures averaged 119 mm He, but the highest mean
pressure was 158 and the lowest was 76. Tlighest svstolic and
lowest diastole pressures were about 20 mm He above and below
these figures. Tleart rates from the same observations averaged
299 beats per minute, with a high of 468 and a low of 184.

Entrance into hibernation was usually preceded by some sort
of aetivity, for the blood pressure and heart rate rose transiently.
After this period of activity there was a sudden drop in heart
rate, accompanied by a decrease in systolie and diastolic pressure
(Fig. 1). Although the heart rate might deerease to one-third

'We are extremely obliged to Merck & Co. of Rahway, New Jersey, for giving
us the acetylcholine.

9 2-(1-Piperidylmethyl)-1, 4-benzodioxan hydrochlovide.

Qa
|
~

1960 MAMMALIAN HIBERNATION

of its original value in fifteen minutes and the blood pressure
drop precipitously, still the latter remained in the lower part of
the range found in the resting, awake animal.

After heart rate and blood pressure declined, the body temper-
ature started to deerease. Often after a few minutes the heart
rate again speeded and the blood pressure rose. This was fol-
lowed by a rise in body temperature. A second decline in heart
rate and blood pressure was again followed by a drop in body
temperature. Although the heart and abdominal temperatures
were not the same at the beginning of the hibernating state, they

“40- ] aa

Q
“io
20

S 300 N
rob. .
errr 200 8
o HEFT KATE x

(00

iSe-eal T T 1 Ls T LE i aT Qa
0 Z. ¥ é 72 7a 74 74
fot eS

Fig. 1. Blood pressure, heart and abdominal temperature, and heart rate
of ground squirrel entering hibernation. Blood pressure in dark area is
highest systole and lowest diastole recorded every four minutes for a one-
minute period. Note declines in heart rate and blood pressure, followed

by body temperature.

soon became identical and remained the same until the animal
was near the temperature of the environment. At this time the
heart temperature was about 0.5°C above the abdominal tempera-
ture and remained so while the animal stayed in hibernation.
During the first part of entrance into hibernation, heart rate
and blood pressure were irregular. Bradyeardia often occurred
for a few seconds followed by tachyeardia, with a concurrent
decline and rise in blood pressure (Figs. 1 and 2a). As the
entrance into hibernation proceeded, the pattern of the heart

358 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

rate became more regular, and the fluctuations in blood pressure
beeame less pronounced. Thus, when the heart temperature
reached 32-21°C, the graph of highest systole and lowest diastole
became much more even (Fig. 1). Slowine of the heart was
accomplished both by quite evenly occurring skipped beats and
by reduction of the even rate of the heart (Fig. 2b). Oceasion-
ally, while the body temperature was still dropping, the heart
rate increased transiently and muscle action potentials appeared
on the EIXG. An increase in heart rate and a rise in blood pres-
sure occurred at the same time (Fig. 3a). Such transient bursts

Fame: Vater. beper rEme FIP C.
[Pare 2P2- fabom TEMA 361°C.

cat Ppl ar a A ii :

Time: T:06FR -LERRT TEMP 767°C

Kore: 6F M8007. 7FETAP 26.7 TC. SIS
AP ANNALARA ie
AACA NOACTANTAN is

i a tt 10 s£COWDS = ise

Fig. 2a. Blood pressure and EKG of same animal starting to enter
hibernation. Note uneven pattern of beats. Time: 4:26 p.m. = 1% hours
on Figure 1.

Fig. 2b. Same animal later. Note even pattern of beats and skipped
beats.

of activity occurred at unpredictable intervals and usually lasted
too short a time to cause any difference in the decline in body
temperature, but occasionally they were of longer duration and
actually resulted in a brief rise in body temperature. Also as
hibernation deepened, the heart rate became slower and the pulse
pressure increased (compare Figs. 2a, 2b and 3a). These changes
were accompanied by a slight lengthening of systole and an
increasingly long diastole (compare Figs. 2 and 3, a and b).

1960 MAMMALIAN HIBERNATION 359

The marked inerease in the length of diastole indicated an
increase in peripheral resistance as hibernation deepened. Be-
eause the systolic pressure varied greatly, if was possible to
compare the rate of diastolic runoff from the same systolic pres-
sure at all stages of the entrance into hibernation. If the increase
of the anele which the diastolic pressure made with the per-
pendieular was plotted against temperature, the result was almost
a straight line.

VME: GO! Fl LUEPIROT > TIEPA/P ae: S

WE: Fé. VB OOPA TEV ANN ANNA M i
}— J? SECONDS 2 rn He
VrmEe: Sst Pl), Plenper TE Eb ce [EO
lere: / /PBDoOM, TEM EFC
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(0 SECONDS 4 WW He
eee

Fig. 3a. Same animal as in Figure 2, showing transient increase of heart

rate at low body temperature. Note muscle action potentials on EKG.

Fig. 3b. Same animal, now in deep hibernation. Blood pressure tube
slightly plugged. Blurring of EKG is electrical artifact.

In deep hibernation, two or more heart beats sometimes oc-
curred quite close together followed by a long diastole. In such
cases the second beat occurred before diastolic pressure had had
time to drop markedly, and the next systolic pressure was higher
thar: the first (Fig. 9¢). This implies that there was considerable
blocd in the heart after the first beat. At other times the heart
rate was fairly regular, though it was never absolutely even.
In this case systolic pressure rose to about the same height with
each beat, and the drop in pressure during the latter part of
diastole was so slow, as the blood pressure approached zero, that
diastolic pressure remained extremely even. In the whole series

360 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

of reeords the systole pressure varied between 90 and 40 mm He
and the diastole between 40 and below 10 mm He, with heart
temperatures between 5 and 8.5°C. The lowest precise record
of diastolic pressure was 7 mm He, Very long term records of
animals in hibernation were not made, but heart rates as low as
three beats per minute were recorded.

Kare: HEART vem (FO? H10
Nx 100. AK tA NK Athi
a mote

et ae SECOWDS

JO SECONDS |

aah ens aed aide

Fig. 4a. Another animal entering hibernation. Evenly occurring elec-
trical depolarizations with little or no change in pulse pressure,

Fig. 4b. Extra systole with no change in pulse pressure. Rates measured
w ENG,

Occasionally, as the animal entered hibernation, or when in
deep ‘ibernation, complete sequences of myocardial depolariza-
tions were recorded with little or no change in pulse pressure.
These sometimes occurred at fairly evenly spaced intervals (Fig.
da) with sheht changes in the configuration of the EKG, and
at other times took the form of extra systoles (Fig. 4b) with no
change in pulse pressure.

Complete records were obtained of animals which were stimu-
lated to arouse from the hibernating state (Fig. 5). In some
cases the animals were stimulated by poking, but in animals
which were fitted only with a heart thermocouple arousal was
initiated by insertion of a reetal thermocouple to a depth of
2.0 cm.

1960 MAMMALIAN HIBERNATION 36]

As soon as the animal was disturbed, the heart rate inereased
and ‘iastole was markedly shortened. The increase of heart rate
was accompanied by the appearance of musele action potentials
(Fig. 6a). These changes were often observed within two or
three minutes after application of the stimulus. Later, systolic
and diastolic pressures rose and the heart began to warm (Fig.
6b).

Gu. Fees.

ke
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ae IED UP
gS > a g
; x
lo
PEPBRT TED f rannaenneet . loa
2 ee a Seseuddcvnastecnctasscecoucetucsosaccessbadees
RECTAL FEF
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ba KATE.
t T T T T Qo

hig. 5. Animal waking from hibernation, graphed as in Figure 1.

As arousal continued the heart rate became more rapid, the
blood pressure rose and violent shivering could be seen in the
anterior part of the animal. Although the plot of the highest
systole and the lowest diastole does not show it clearly (Fig. 5),
the pulse pressure was considerably reduced (Fig. 7a). During
this time the temperature of the heart and the anterior part of
tae body increased rapidly, while the abdominal temperature
remained nearly statie (Fig. 5).

As the heart temperature approached 37°C, the abdominal
temperature started to rise and the blood pressure and heart
rate usually, but not invariably, dropped from the extreme
heights to which they had climbed (Figs. 5, 7a and 7b). During
this time diastolic runoff was more rapid, indicating a decrease
in peripheral resistance. The abdominal temperature rose rapidly

362 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

TIME: 6 OUMUTES. AEwRer Tempe ¥°C
Ware : 17 KECTKL TEP L.2°C

PSISININD SIS ISIS IRPSTSSISES

VO SECONDS ° r

tH
Jp pap pp pert pct ppm tre

Vim: SVimineves. at TEP 7EC aad
WATE: BY NECTRL FETIP 2.2°C

AAAI

10 SECONMOS

ool ei en al ipl

Fig. Ga, Same animal as in Figure 5. Note bursts of muscle action

potentials in EKG. Time: minutes after animal was picked up.

Kig. 6b. Same animal. Note increase in systolic pressure, heart rate and

muscle action potentials.

Time JY OULTES. Be. TEMP T/C
Kore: 388 Keorac TEI? 42°C 95°

a Mi aia i ai i ih th a

70 SECONMOS

Time: 2OV muures, lerer rene FE FC.
Ware: WE - F702 NECTAL FETS? PEITC.

Hl .
J ii i _ ‘ Na Wh ih . VARY iis nnnnd iid wih 75°

70 SECONDS

Fig. 7a. Same animal, before posterior has warmed. EKG discontinued
because of fast rate and blurring by muscle action potentials.
Fig. 7b. Same animal. Posterior now warming. The even variation in

blood pressure is caused by respiration.

1960 MAMMALIAN HIBERNATION 363
and within 245 to 34% hours after the initial stimulus the
animal was completely aroused. For an hour or more after this
the heart and rectal temperatures averaged at least a deeree
above that found in the normal, awake animal.

A sinele record of an animal which started to arouse spon-
taneously at 2:30 a.m. showed the same sequence of events,
with heart rate and blood pressure rising before heart tempera-
ture.

180

Lavon fKews

Leccour ma
P/E

| WERRT TEMP L 7 S
wy 4 eo . a <u

y RECTAL TEMP <7

[(EART RATE i

T T : T = o
0 f 2 Sores. ce %

Fig. 8. Graph of partial waking and re-entranee into hibernation, as in
Figure 1.

Occasionally during the winter months an animal, when stim-
ulated during hibernation, started the arousal process, but did
not complete it and returned to the hibernating state (lig. 8).
In these cases the arousal was precisely as described above, with
a rapid rise in heart rate, blood pressure, temperature, and
trequency of muscle action potentials. Quite suddenly, however,
the heart slowed and the muscle action potentials were reduced.
Peripheral resistance increased, as measured by the slope of the
diastole runoff time as deseribed above. The blood pressure
dropped, but not as rapidly as the decrease in heart rate. As the
animal re-entered the hibernating state, the heart temperature
declined slowly and the abdominal region, which had remained
cold during the transient period of arousal, rose slowly to nearly

364 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

the temperature of the heart and then declined with the heart
temperature.

Reaction to Drues

When the experiments with drues were begun, it was apparent
that the heart of the hibernating animal was extremely sensitive
to liquids introduced via the intubated aorta. As little as .07 ml
physiological saline introduced quickly occasionally caused a
sheht transient increase in heart rate. For this reason the effect
of the drues was repeatedly checked against control injections of
saline solution. Although the same drug was often used several

TE: 11-02 AUP Leper 7EHIE LT
We a ALB OOP, TEV: 7 FIC.
/
|
1] ee a0
YO SECOWDS nite
HG
Vint: [| O84) | 00 UN KETER AOE TVA CH OLS UE
KATE: LO JEW SATE es

b 2

Time: /OSIBD? ewer rere FIO /i0
Kare: € safe

a ee 4
¢ : of
TIME: 10:3GR 10 | PHN PETER. CENODPINE MOSECOUES: mmrie
Ka Te 1S TEP SAE. SdO

of dl SF tf
d ¥0

Fig. 9a. Pulse pressure in deep hibernation.

Fig. 9b. Pulse pressure after acetylcholine. Note faster diastolic runoff
from slightly lower systolic pressure than in Figure 9a.

Fig. 9c. Pulse pressure in deep hibernation.

Fig. 9d. Pulse pressure after Benodaine hydrochloride. Note faster
diastole runoff from same systole pressure.

times on a single animal, every experiment was repeated on at
least two animals.

As might be expected, fairly large doses (0.15 to 0.9 me/ke)
of acetylcholine were necessary to override the presence of
cholinesterase in the awake ground squirrel and cause a clear-cut
effect. Onee the effective dose was reached, there was a drop in

1960 MAMMALIAN HIBERNATION 365

blood pressure and a compensatory increase in heart rate of
55 to 70 per cent. There was no observed bradyeardia caused by
this drue.

Similar doses produced a marked effect on the hibernating
animals. his effect consisted of a rapid decrease in peripheral
resistance coupled with a rise in heart rate. Unlike the situation
in the awake animal, the systohe and diastolic blood pressure
showed little or no change during this time (Figs. 9a and 9b).
If the infusion of acetylcholine was continued, the heart rate
increased further and the animal started the process of arousal.
Although the long-term effect of acetylcholine is typical of a
normal arousal, we were not able to determine whether this drug
is actually the neurohumeral agent which mediates the waking
process. It is possible that vasodilation and speeding of the
heart were in themselves as much of a stimulus to waking as
would be an externally applied physical stimulus. However,
within the dose ranges used, short-term, rapid injections of
acetylcholine did not cause arousal, while one sharp mechanical
stimulus almost invariably produced this result.

Acetylcholine had apparently no effeet on the distribution of
blood once arousal was fully underway, for doses as large as 2.82
me/ke failed to cause a change in the blood pressure or a rise
the abdominal temperature,

Benodaine hydrochloride was chosen as an adrenergic bloeking
agent rather than Dibenamine hydrochloride Merck or other of
the better-known drugs because its effeet is of short duration
(Goodman and Gilman, 1958). In low doses the primary effect
of this drug was a speeding of the heart and a resulting rise in
blood pressure in both the awake and hibernating animals. Larger
doses caused a drop in blood pressure in the awake animal al-
though the heart rate was increased. In the hibernating animal
doses of $8.5 to 25 me/ke caused a marked decrease in diastolic
runoff time along with an increase in heart rate (Figs. 9¢ and
oa).

Norepinephrine infused rapidly into the active ground squir-
rel caused a rise in blood pressure, an increase in pulse pressure
and a slowing of the heart which was probably compensatory.
In contrast, the effect of this drug on the hibernating animal in
doses of 6 to 20 uge/ke was an increase in heart rate and pulse
pressure and a rapid rise in blood pressure. Because of this
rapid rise, sufficient comparative measurements of diastole run-
off time could not be made, but there was no evidence that peri-
pheral resistance was increased by norepinephrine during hiber-
nation. The rise in heart rate and inerease in pulse pressure

366 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

alone were enough to account for the initial rise in blood pres-
sure.

Norepinephrine at doses of 17 to 44 ue/ke was also introduced
into animals during arousal, when the heart temperature had
reached 37°C and the temperature of the posterior part of the
hody had started to rise. At this time the drug caused an im-
mediate rise in blood pressure, and the abdominal temperature
ceased rising and remained level for one minute or more. After
this time the blood pressure resumed its original level and the
abdominal area again started to warm. It was not possible to hold
the blood pressure at the high level or stop the abdominal region
from warming over long periods of time in spite of the infusion
of large amounts of norepinephrine. However, the abdominal
temperature could be made to rise in a step-wise fashion by
periodic introductions of norepinephrine into the bloodstream.

Discussion

Records of blood pressure in any stage of hibernation are
scanty, and none have been reported on mammals entering hiber-
nation or in the undisturbed hibernating state. Dubois (1896)
reported very low blood pressures after the cannulation of the
carotid artery in the hibernating marmot. Blood pressure became
higher as the animal aroused from the hibernating state, but
Dubois did not trace the changes during the arousal process.
Chao and Yeh (1951) measured the blood pressure of hiber-
natine hedgehogs by acute cannulation of one carotid artery.
The conditions of the experiment were very different from those
described here, as the animals were strapped to a board through-
out the experiment. These authors report that the carotid
arteries were completely bloodless during hibernation, which is
certainly not the case in hibernating rodents.

Chatfield and Lyman (1950) measured the blood pressure of
hamsters arousing from hibernation by acutely cannulatine a
carotid artery. The conditions of the experiments were com-
parable to those reported here for the process of arousal, except
that there was a time lag of 25-35 minutes to perform the cannu-
lation. The results differ in that the rise in blood pressure was
much more rapid in the hamster, but did not reach the high
pressures observed in the waking ground squirrels. The obser-
vations on hamsters should be repeated using chronic intubation,
not only to clarify this discrepancy, but also because the varia-
tions in the physioloey of hibernation in these two species should

1960 MAMMALIAN HIBERNATION 367

supply interesting comparisons on the condition of the circula-
tion.

As far as the present results are concerned, it is apparent that
there is a decrease of heart rate and blood pressure as the ground
squirrel enters hibernation, and that this decrease occurs before
a detectable decrease in body temperature. The equal tempera-
tures of heart and abdomen as the animal enters the hibernating
state indicate that blood flow to the anterior and posterior parts
of the body is evenly distributed. As body temperature drops,
peripheral resistance increases. A part of this increase in
peripheral resistance is probably caused by the increased viscosity
of the chilling blood. However, part of the resistance must be
eaused by changes in the vascular bed, for the stimulus of
waking, or a vasodilatory or an adrenergic blocking drug, can
quickly reduce the peripheral resistance before there is any
measurable change in temperature.

The result of the increased peripheral resistance and concur-
rent rise in pulse pressure is that the mean blood pressure re-
mains at remarkably high levels, even with a heart rate of only
three or four beats per minute in the deeply hibernating animal.
We have observed in chilled, nembutalized ground squirrels that
the peripheral resistance does not rise appreciably as the animal
cools, nor does the pulse pressure increase. The net result of a
low systolic pressure and a rapid diastohe runoff time is a very
low mean. blood pressure. This may contribute to the early death
of the hypothermed potential hibernator, while the animal in
natural hibernation may live for many days.

The great increase in peripheral resistance with hibernation
Was unexpected. From our observations on the equal rate of
decline of temperature in various parts of the woodehueck (Tiy-
man, 1958), we had postulated that the animal was vasodilated
as it entered hibernation. It appeared reasonable that any vaso-
constriction would cause marked differences in temperature in
various parts of the body as is observed in the waking hiber-
nator. The possibility of a gradual, evenly distributed, vaso-
constriction over the whole body had not even been considered.
It now appears likely, however, that the hemodynamies of the
ground squrrel and the closely related woodchuck during the
hibernating evele are identical, for in both animals the tempera-
ture distribution is the same on entering and waking from hiber-
nation and in both the heart rate anticipates any change in
temperature.

368 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The hibernating thirteen-lined ground squirrel is therefore
probably evenly vasoconstricted over its whole body. Prior to any
measurements of blood pressure in hibernation we had suggested
that the pink feet of the hibernating hamster might indicate a
condition of vasodilation (Lyman and Chatfield, 1955). Although
hibernation in hamsters and ground squirrels differs im many
ways, an alternative explanation for the pink feet of the hamster
could be the cherry-red condition of the blood during hibernation.

The presence of electrical depolarization of the heart during
hibernation with little or no change in pulse pressure may be
explained, in the case of extra systoles (Fig. 4b), by lack of
filling time before the next beat. When depolarizations occurred
at more even intervals (Fig. 4a) some effect of the deep respira-
tions of hibernation might have reduced or obliterated the
arterial pulse. On the other hand, depolarization without visible
beats in isolated hearts of the ground squirrel (Landau, 1956)
and hamster (lyman and Blinks, 1959) has been reported, and
complete uncoupling of the membrane phenomena from the con-
tractile process is at least theoretically possible (Brooks eft al.,
1955, p. 317). Whatever the explanation, it is interesting that
the effective arterial pulse in hibernation can be even less than the
very slow electrically measured heart rate.

When the hibernating ground squirrel starts to arouse, there
is a rapid rise in heart rate and decrease in peripheral resistance,
Similar results may be produced by vasodilatory drugs. In
neither case is it possible to tell whether the decreased peripheral
resistance causes a compensatory speeding of the heart, or
whether the decrease in peripheral resistance and increase m
heart rate occur at the same time. Shortly thereafter, the heart
starts to warm though the posterior remains cold.

One is forced to conclude that there is a differential vasodila-
tion in the anterior part of the body which is a vital part of
the waking process. That vasodilatory drugs do not cause a
warming of the posterior part of the body suggests that vascular
beds of the anterior and posterior parts have different thresholds
at this stage in the hibernating cycle.

Although peripheral resistance is reduced as arousal starts,
the heart is able to maintain the blood pressure by increasing
its rate. Indeed, as arousal progresses, the blood pressure rises
and the heart rate increases, in spite of an ever-decreasing pert-
pheral resistance. The confinement of the active circulation to
the anterior part of the body results in a high blood pressure and
an efficient and rapid warming of this area. In contrast, if an

1960 MAMMALIAN HIBERNATION 369

active ground squirrel is given acetylcholine, the result is a drop
in blood pressure even though the heart may almost double its
rate. Evidently the heart cannot maimtain a high blood pressure
when the whole capillary bed is vasodilated at the same time.

A similar condition is found in animals during the later stages
of arousal from hibernation. Durine this time the posterior
portion of the animal is warming rapidly, indicating an unre-
stricted blood flow. The blood pressure, which reached its height
when the anterior part of the body was still warming, now
decreases because of the increase in the amount of open vascular
bed. If norepinephrine is injected at this time, the blood pres-
sure increases temporarily and the abdominal temperature re-
mains static for a short time. One can thus produce with a
vasoconstrictor the condition which obtained early in the wakine
process, but this condition cannot be maintained for long.

The fortuitous observations of partial arousals from hiberna-
tion fill out the general picture developed here. The arousal is
normal until the heart begins to slow and the blood pressure
drops. The fact that the blood pressure does not drop as fast
as the heart rate indicates that peripheral resistance must now
be increasing in the anterior part of the body. Since the posterior
part of the body warms very slowly, circulation of blood between
anterior and posterior must be sluggish, which emphasizes that
the peripheral resistance in the latter must be high.

The picture during the hibernating cycle is of a circulation
under remarkably precise control at all times. With our present
knowledge we can only speculate about mechamsms which cause
the observed changes. However, it seems clear that shifts in
temperature alone do not mediate the complex imterrelationships.
Furthermore, whatever is controlling the heart, this organ is
remarkably sensitive to stimuli at all stages of the hibernating
eyele. Further study is in progress with other pharmacological
agents in an attempt to clarify these problems.

REFERENCES

Brooks, C. McC., B. F. Horrman, E. E. SUCKLING AND O. ORIAS
1955. Excitability of the heart. New York, 373 pp.

CHao, I., anp C. J. YEH
1951. Hibernation of the hedgehog. IIT. Cardiovascular changes.
Chin J. Physiol., 18:1-16.

OHATFIELD, P. O., aND C. P. LYMAN
1950. Cireulatory changes during process of arousal in the hibernat-
ing hamster. Am. J. Physiol., 163:566-574.

370 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Dawe, A. R., AND P. R. Morrison
1955. Characteristics of the hibernating heart. Am. Heart J., 49:

367-384.

Dubois, R.
1896. Physiologie comparée de la marmotte. Ann, Univ. Lyon. Paris,
268 pp.

GOODMAN, L.8., AND A. GILMAN
1958.) The pharmacological basis of therapeutics. New York, 1831 pp.

Lanpau, B. R.
1956. Physiology of mammahan hibernation. Dissertation Abstr.,

16:2195.

LYMAN, C. P.
1958.) Oxygen consumption, body temperature and heart rate of wood

chucks entering hibernation. Am. J. Phwsiol., 194:85-91.

LYMAN, C. P., anp D. C. BLINKS
1959. The effect of temperature on the isolated hearts of closely re-
lated hibernators and non-hibernators. J. Cell. Comp. Physiol.,
54:53-64.
LYMAN, C. P., AND P. O. CHATFIELD
1950. Mechanisms of arousal in the hibernating hamster. J. Iexper.
Zool., 114:491-515.
1955. Physiology of hibernation in mammals. Physiol. Rev., 35:403-

425,

StTiLL, J. W., AND KE. R. WHITCOMB
1956. Technique for permanent long-term intubation of rat aorta.
J. Lab. and Clin. Med., 48:152-154.

DISCUSSION FOLLOWING LYMAN’S PAPER

SOUTH inquired as to changes in blood viscosity with respect
to temperature change as a contribution to the blood pressure
picture in hibernation. LYMAN replied that Dr. John Pappen-
heer had worked on the effect of cold on the viscosity of blood,
but the results had not been published.

LANDAU remarked that diastolic pressures rather than slope
of runoff curve may be more critical in estimating peripheral
vascular change.

GRIEEIN inquired about a part of an electrocardiogram

which seemed to be evelic. LYMAN assured him this was an
artifact periodically appearing as a 60 evele hum. GRIFFIN

1960 MAMMALIAN HIBERNATION 3

further expressed his amazement at the high peripheral resist-
ances in hibernating animals; he wondered if the measurements
reflected the difference between arterial and venous pressures
across arterioles and capillaries, or a total resistance of the whole
circulatory system due to high viscosity of the blood, or other
causes. LYMAN rephed that, though blood viscosity certainly
contributed to peripheral resistance, still the rapid changes in
resistance upon injection of drues or arousal must be due to
changes in the vascular bed, since they occur before a change
in temperature.

BARTHOLOMEW observed that the Mohave ground squirrel
has the capacity of warming the whole body at once or the front
end first, depending on the circumstances. Usually the whole ani-
mal warms up at the same rate.

LYMAN suggested that if these animals were vasoconstricted
throughout during arousal one could estimate the degree of vaso-
constriction by giving norepinephrine and noting the change m
blood pressure and peripheral resistance.

DAWE inquired as to the status of the ‘‘pink paw problem.’’
LYMAN rephed that he now believes it is due to the cherry-red
color of the blood of hibernators showing through the paw sur-
face; he no longer believes it is a vasodilation phenomenon.

BISHOP asked if the heart keeps up with body metabolism.
LYMAN rephed that it essentially did so, though an exact cor-
relation has not been made. BISHOP then further remarked
that a discrepancy between the rate of metabolism and the heart
rate may give data usable in this context.

JOHANSSON inquired as to heart rate-peripheral resistance
relationship. LYMAN said that when the peripheral resistance
decreases, there is a compensatory increase in heart rate. In
arousal, dilation occurs first in the anterior end of the body with
a decrease in peripheral resistance there. He stated that he was
not sure which came first, the increase in heart rate or the vaso-
dilation.

MAYER said that he saw a warming of the anterior end of
the Arctic ground squirrel on arousal from hibernation, followed
by a steady wave of warming toward the posterior end of the
hody. LYMAN remarked that he did not believe it was a steady
wave, but rather a sudden dilation and shunting of blood into the
posterior part. This phenomenon is not as striking, he said, in

372 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

the ground squirrel as in the hamster. There is a drop in blood
pressure corresponding to this opening up of the vessels and
the ground squirrel warms very rapidly thereafter. MAYER
stated in rebuttal that if thermocouples are placed alone the
leneth of an arousing hibernating animal they warm in se-
quence, not at once, and the pattern goes back over the animal
as a wave of warming. LYMAN pointed out that heat conduction
could mask changes in blood flow. LANDAU commented that
the anterior temperature could fall when the posterior was
Warming,

LYMAN then asked the group if anyone knew of the possi-
bility of other physiological conditions in which there may be
complete electrical depolarization recorded without change in
blood pressure. BULLARD replied that he thought such a
situation occurred in certain conditions of iome imbalance,
namely calcium lack.

XIX

VASCULAR CHANGES RELATED TO
HIBERNATION IN THE VESPERTILIONID
BAT MYOTIS LUCIFUGUS*’

By FRANK C. CALLEN
Department of Histology and Embryology
Cornell University
Ithaca, New York

In a recent paper, Riedesel (1957) has summarized previous
hematological studies on hibernating mammals and commented
on the contradictory evidence for and against hemoconcentration
associated with hibernation, which appears in the literature.
The study presented here represents an attempt which I have
made to ¢larify our present state of knowledge in this matter.

Plasma volume determinations have been made whieh give
direct evidence for an over-all hemoconcentration in the hiber-
nating little brown bat, Myotis lucifugus. In addition, data have
been obtained relating to plasma specific gravity, hematocrit, and
liver and spleen weight. Collectively, these values allow certain
conclusions to be drawn concerning changes in the distribution
of cellular and plasma components of the blood which are related
to hibernation in this bat, and the probable effect of these changes
on circulating blood volume. In heht of the conflicting evidence
from previous studies, it is perhaps of special interest to note
that some of the results to be presented for the hibernating state
would, taken by themselves, constitute evidence for a general
hemodilution.

The vascular picture during hibernation has been compared
with data obtained from bats under the influence of several states
of activity, including arousal from hibernation and flight. This
preoecupation with the active state was predicated largely by the
fact that, in contrast to other hibernating mammals, resting bats
have been found to lower their body temperature toward that of
the environment whether they are in seasonal hibernation or
not (Hock, 1951). It was therefore deemed necessary to deter-
mine whether observed changes in the circulation are dependent

1This work was supported by National Science Foundation grants (G-2188,
G-7474) to William A. Wimsatt.

2The material presented in this paper is taken from a thesis to be submitted
to the faculty of Cornell University in partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

374 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

on hibernatine temperatures or whether there are conditions
under which the hibernating picture is duplicated over tempera-
ture ranges at which bats are commonly deseribed as being
active.

Materials and Methods

Bats for this study were captured during hibernation from
caves in Pennsylvania and New Jersey. They were stored in a
dark, very humid cold room at 5°C and provided with ample
drinking water, but no food. Determinations were made within
3 months of capture.

‘*Tlibernating’’ values were obtained on bats which had been
transferred from their storage cage to individual specimen jars
in which they were kept, in the dark, at 2°C for 8 to 24 hours.
They were then anesthetized with ether and experiments were
begun in a darkened cold room (2-3°C), using precooled ap-
paratus. After samples had been obtained, all further procedures
were carried out at room temperature.

A state of ‘‘arousal’’ was elicited by transferring the bats
from storage to small, open-topped chambers which were located
in a warm room, but were surrounded with crushed ice to cool
their interiors. All experimental apparatus was chilled, but no
ether was used. These were the only bats used in the study which
were killed before bleeding, two by breaking their necks and the
rest by freezing their heads in a dry ice-acetone mixture. The
animals were bled immediately thereafter.

Bats deseribed as ‘active’? were brought to room temperature
(21-29°C) and provided with water. After they had ceased the
initial intense activity which accompanied their arousal from
hibernation, the resting animals were picked up and held for a
few seconds until they were actively struggling to free them-
selves. They were then etherized and determinations were made.
Bats referred to as ‘‘excited’’ were treated similarly except that
they were brought to a room temperature of 30°C in individual
specimen Jars and were encouraged to struggle for a five minute
period prior to anesthesis. <A final series was made up of
‘active’? bats which had been made to fly for three to ten min-
utes Immediately prior to the administration of ether.

Plasma volumes were determined by a modified Evans blue
dye dilution procedure. Details of this technique, together with
evidence for its validity and further values for active bats will
be presented in a separate report (Kallen, 1960). Briefly,
the method used involved the following: .040 ml of a .5 per cent

{

~
S)

1960 MAMMALIAN HIBERNATION

solution of T-1824 in .9 per cent saline was injected into the
uropatagial vein (Grosser, 1901). After a 5-minute mixing
period, a blood sample of approximately .06 ml was taken by
eardiae puncture (left ventricle) from the opened chest using
a hypodermic needle fitted directly to a graduated capillary
tube in which the sample eould be centrifuged. After (total)
hematocrit was determined, a plasma sample was withdrawn,
diluted and colorimetrically compared to a standard curve.
Plasma volume was calculated by subtracting the volume of dye
injected from the circulating fluid volume indicated by the extent
of dye dilution within the animal.

No sexual or seasonal differences have been observed in any
of the additional determinations which would appear to affect
the plasma volumes or hematocrits to be presented here.

Samples for additional heart hematocrits, and plasma specific
eravities, were obtained by the bleeding method just described.
Splenic hematocrits were obtained by excising the enlarged
spleens of hibernating animals in the cold room, transferring
the organs to small vials and immediately collecting the blood as
it drained from the spleen, with graduated capillary tubes, fitted
in this case with a short length of polyethylene tubing instead
of a needle. The samples were then taken from the cold room
and centrifuged.

Specific gravities were determined by the falling drop method
of Barbour and Hamilton (1926). The small size of the blood
samples made it necessary to use a 104 micropipette for dropping
plasma. Samples for whole blood specific gravities were drawn
from the heart directly into standard Lamotte dropping pipettes
fitted with hypodermic needles,

All equipment used to contain blood was rinsed with an
aqueous .2 per cent heparin solution and dried before use. Ap-
propriate precautions taken to prevent evaporation and hemoly-
sis in the extremely small blood samples included the use of
parafilm as a sealing material wherever feasible.

Mean values to be presented in the text are accompanied by
their standard errors. Although P values (‘‘Student’s’’ ¢ test)
have been ealculated for all data, the values shown in Figure 1]
are presented with twice their standard errors to permit graphic
visualization of significant differences. Differences between means
have been taken to be highly significant when they have a
Be 0 A P > .05 has been assumed to be the result
of random distribution. The degree of variability in individual
determinations has been expressed by the use of the coefficient
of variation (standard deviation x 100 / mean value).

376 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Results and Discussion

Qualitative observations. Several obvious differences were ap-
parent when circulatory phenomena were observed grossly in
hibernating bats. The hibernating animals showed neghegible
patagial circulation and a markedly lowered heartbeat, suggest-
ing a lowered blood pressure. Also, as might be expected, they
were much harder to bleed. Whereas it was often possible to
draw as much as .8 to 4 ml of blood from the heart of an
active animal, the amount obtamable from a bat in hibernation
was rarely as much as .1 ml. The only vessels in the hibernating
animals which were found to bleed at all freely were the
jugular veins.

The enlargement of the spleen in hibernating bats, described
by previous authors (Worth, 1932; Evans, 1938; Lidicker and
Davis, 1955), was also noted when the active bats used for com-
parison had been at room temperature for short periods, but
animals which had spent 4 to 5 hours at room temperature had
spleens as large as those found in hibernation.

Since ether was found not to constrict the spleen in Myotis,
it was considered a satisfactory anesthetic for this study.

In contrast to the spleens, the livers were paler in the hibernat-
ine state, and bled less when removed. Varyine degrees of red-
denine were typically observed as arousal began, accompanied by
an extremely variable amount of spleen drainage.

Autopsy of the bats injected with T-1824 indicated a homo-
veneous distribution of dye in the blood of both aetive and
hibernating bats. In contrast, the arousing animals often
showed a redder color in the mesenteric vessels, or a less in-
tense blueness in the vessels of the arms than of the lees which,
in view of the uropatagial injection site, suggests that arousal in
the bat is aecompanied by the same preferential increase in the
thoracie cireulation which has been found in studies on other
hibernators, notably the hamster (Lyman and Chatfield, 1955).

The blood cells exhibited differences in redness similar to those
deseribed for the blood of ground squirrels by Landau and
Dawe (1958). The bright cherry-red color in blood of hiber-
nating bats under ether was only shehtly darker when hiber-
nating animals were studied without anesthesia. During arousal,
an extreme darkening was a consistent feature, while active
animals showed varying degrees of brightness, but by no means
to a degree comparable to the brilliance observed in the hibernat-
ing state. The volume of leucocytes in the samples was markedly

ca |
ot |

1960 MAMMALIAN HIBERNATION 3

reduced during hibernation. The buffy coats averaged about .2
per cent of the blood samples taken from active bats, but only
half that in hibernating animals, often being absent entirely.

Quantitative observations: liver weights. Representative de-
terminations are those made on April 20, 1959. Four male and 4
female bats in hibernation (etherized) weighed 5.84 ©.19 @, their
livers (gall bladder removed) weighed 4.91 ©.16 @/100¢ body
weight. On the same day, a similar sample of active (etherized )
bats weighed 6.05 £.12 @, and liver weight (gall bladder re-
moved) was 4.70 =.12 ¢/100¢ body weight. The differences are
not significant (P>.3 in both cases).

Blood specific gravities. The number of animals, and body and
spleen weights were comparable to those of the animals shown
in Figure 1, which were run on the same days. Blood of hiber-
nating bats (etherized) showed a specifie gravity of 1.0570
+0015 on Nov. 23-25, 1958, while the higher value for active
bats on Nov. 16-17, 1958, was 1.0598 =.0014, which indicates no
significant difference (P>.2). Hematoerits and plasma specific
eravities suggest that more determinations might have been in
order.

Splenic hematocrit. The blood drained from the spleens of
4 male and 4 female hibernating bats (etherized) on Dee. 13-19,
1958, had a hematoerit of 75 = 2, a value significantly higher
than any heart hematocrit encountered.

Plasma volume, plasina specific gravity, spleen weight and
heart hematocrit. Plasma volume or plasma specific gravity was
taken concurrently with the other values on each bat. The follow-
ing is a discussion of the results, which are presented in Figure
1, and to which the reader is referred.

The data for active bats injected with T-1824 indicates that
hibernating bats had a significantly smaller amount of circulating
plasma than was found in the aetive state. No mechanism which
compensated for the added dve was suggested by the results,
since the determined plasma volumes were the same whether
.020 or .040 ml of dye were injected. In light of the specific
gravities to be discussed, the shghtly greater variability encoun-
tered in the series injected with .020 ml might be attributable to
the disturbance caused in the storage cage by removing bats for
the .040 ml injections on the previous day. By the same reason-
ing, the active animals injected must also be considered as having
heen reeently disturbed during hibernation. Notice that, al-
though the active bats had been at room temperature for periods
ranging from 30 minutes to 8 hours, their plasma volumes were

124

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; es «| VSG ONY ‘LINOOLVW3H = ‘LHOIBM-—-N3BTdS NI S3SNVHD || ONS

9001/9
“LM N331dS

uaajds 40

1960 MAMMALIAN HIBERNATION 319

remarkably consistent, suggesting no great fluctuation in plasma
levels. This finding was confirmed by incorporating additional
determinations in a comparison of values for 10 bats active for
30 minutes to 1 hour (7.2 =.3 ml/100¢ body wt.) with those for
11 bats active for 1 to 3 hours (7.2 © .2 ml/100¢@) and those for
9 bats active for 3 to 8 hours (6.7 =£.2 ml1/100g¢). These means
do not differ significantly (P>.1 in all cases).

The determinations for bats in a state of arousal clearly show
that a rise in plasma volume had been initiated in the 5 to 10
minutes which had elapsed since the animals had been in hiberna-
tion. Indeed, the values shown are probably lower than the actual
volumes, owing to the dye localizations previously mentioned,
although the extent of this localization was not sufficient to sue-
vest that actual plasma volumes were yet equal to those found
in the active state. Spleens ranged from moderately full to
completely empty, and were far too inconsistent in size to sug-
vest any worthwhile quantitative treatment under the conditions
of this experiment. The rising plasma levels and injected dye
wowld both be expected to depress hematocrit, yet hematocrits
during arousal, though extremely variable, were at the highest
level encountered in the study, suggesting a mobilization of cells
in the thoracic circulation at this time.

Comparison of spleens in all hibernating bats studied shows
a significant decrease in weight aecompanyine dye injeetion,
which suggests increased blood pressure as a factor in splenic
evacuation.

Turning now to the determinations made on undyed bats im
November and December, a significant increase in spleen weight
during hibernation can be seen (this active series had been at
room temperature for 30 minutes to 4+ hours). Spleens taken
from a comparable series of active bats, which had not first been
bled, weighed .401 +.047 ¢/100g body weight, a figure compar-
able to that found for the excited animals shown immediately
below which were splenectomized before bleeding in January.
Thus, although loss of blood is aecompanied by a significant
drainage of the spleen in active bats, bleeding alone cannot
account for the size difference between the active and hibernating
states.

Hibernating bats under ether had significantly lower hemato-
crits than (etherized) active ones, which would imply a hemo-
dilution in the heart region. A dilution of the plasma itself was
implied in the significant drop in the corresponding plasma
specific gravity. These specific gravity differences became less

380 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

marked when the spleen was removed before bleeding the
(etherized) hibernating animals (P>.01) and disappeared
(P>.1) when the spleen was removed and blood subsequently
taken from hibernating animals on which no ether was used.
The progressively increasing coefficients of variation reflect the
greater variability in plasma specifie gravities which might be
expected in the hearts of more disturbed animals if plasma
proteins had not been uniformly distributed in the resting state.
Hibernating hematocrits underwent similar changes, although
they never approximated those of the active bats (in no case was
P>.05). In the case of the splenic weights, however, the least
variation was found in the animals which had not been ether-
ized. This suggests a relatively constant amount of blood trapped
in the splenic pulp in a manner which prevented its release by
any mechanism as simple as the squeezing action of the abdominal
skeletal museles which was observed in the absence of ether.

When comparing the first two groups of exeited bats which
were alike except that one series was bled before splenectomy
and the other after, it is seen that spleen sizes are comparable
to those found in active bats in December. Hematoerits have
dropped to slightly lower levels (when compared to those in the
hibernating state), and plasma specific eravities no longer differ
significantly from those found during hibernation. This drop
Was even more consistent in bats which had been flown. Although
bleeding resulted in a significant drainage of the spleen, no sig-
nificant change in heart hematocrit was observed, which may
reflect a temporary trappine of blood from the spleen in the
liver.

The data from the pool of 9 males and 9 females shows no
significant difference in splenie weight or hematoerit between
bats which had been left undisturbed for a matter of weeks
(Jan. 12) and those which had been disturbed in storage the day
before (Jan. 13-14). The plasma specific gravity of the undis-
turbed animals, however, is high enough to differ at a suggestive
level (P<.05) from the lowest hibernating value. This makes
it necessary to call attention to the dates (Nov. 16-17) on which
the active determinations were made; these bats had been left
undisturbed for several weeks. Since the experience in our lab-
oratory has been that bats need ample water and high humidity
to survive hibernation at all, the most reasonable interpretation
of the high specifie gravities which have been encountered ap-
pears to be that bats drink less frequently when hibernation 1s
undisturbed, undergo some dehydration, and have less body

1960 MAMMALIAN HIBERNATION 38]

fluid available to be contributed to the plasma when the animals
increase plasma volume upon entering the active state. This
would imply that the increase in plasma volume may not always
be of the magnitude observed in the dye studies, and that, al-
though significant differences in hematocrit and plasma specific
eravity may be expected after undisturbed hibernating bats have
become active, these differences might lessen or disappear if the
hibernating animals had recently awakened to drink. In all other
cases, reasonably uniform average values for plasma density
were maintained, although they were not uniformly consistent.
Since greater variability appears not to be associated with lower
specific gravity, it cannot be explained adequately by assuming
nothing more than sporadic dilution of heart blood by Lymph
from the thoracic duets; the possibility of a localization of
plasma proteins somewhere in the circulation again suggests
itself.

When excited bats which have been at 30°C for 1 to 2 hours
are compared to those which have presumably come closer to a
resting state after 4 to 5 hours, the larger (P<.05) spleens in
the latter more closely approximate the hibernating condition.
This suggests that more critical studies might uncover a reversion
toward the hibernating picture in bats at rest at room tempera-
tures. The high plasma specific gravity in the active bats, for
example, may prove to be not entirely due to dehydration, but
also to a lower plasma volume than that found in more excited
bats. This tendeney would probably not be detectable by a dye
dilution procedure, since the injection and mixing period prob-
ably serve as excitatory stimuli in themselves.

A final point in regard to specifie gravity is that the decreased
plasma volume during hibernation is not reflected in a higher
density of either blood or plasma in the heart, which suggests
that the system is taking steps to prevent extra work by the
heart at a time when the blood is already more viscous as a
result of cold.

When the animals are segregated on the basis of spleen size
(large vs. small spleens), a significant ebb and rise in hematocrit
is indicated, which has been obscured in most cases by the
rhythmie activity of the spleen. This finding, in conjunction
with the high hematoerit of splenic blood, clearly establishes
the spleen as a source of blood cells for the rest of the eireula-
tion, although the extent of its contribution has vet to be dis-
cussed.

382 BULLETIN: MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Probable changes in circulating blood volume. Sinee inde-
pendent measurements of blood cell volumes were not made,
directly measured blood volumes cannot be given. The rapid
rate of the plasma volume changes, however, together with
Worth’s (1982) observations on the generally quiescent state of
hemopoiesis during hibernation in the bat (only the spleen and
Ivinph nodes were found to be active) make it reasonable to
asstine that a constant volume of blood cells is available within
the animal while these changes take place. Relative comparisons
are therefore possible to a degree.

lor example, if we calculate blood volumes for the hibernating
and active bats injected with .040 ml of T-1824 on the basis of
plasma voltae and observed hematocrit (subtracting the amount
of dye injected and asstunine approximately 4 per cent of the
hematocrit. cell cohunn to be trapped plasma) usine a ratio F
eclls (total body hematocrit /heart hematocrit) of 1 in each case,
hibernating blood and plasma volumes are .590 ml and .300 ml
respectively, while those for active animals are .809 ml and
400 ml. THibernating bats thus appear to have .250 ml of cells
and active bats 409 ml of cells; a difference of .159 ml must be
accounted for.

Observed spleme weight loss was .O38 @ (based on the extremes
in mean splenic weights of hibernating and active undyved bats
of the Nov.-Dee. series), which on the basis of the data presented
would represent only .026 ml of blood cells. Even if we made
the liberal assumption that these spleens actually had contained
04 ml of *textra’’ cells, none of whieh had been accounted for
in the caleulated hibernating cell volume, the splenic contribu-
tion could account for only about one third of the discrepaney.

No other region of red cell concentration remotely comparable
to the spleen has been found in the bat, for Worth’s (1932)
observations would discount bone marrow, and the liver has been
discounted here. We must assume a more general shift of blood
cells away from the heart region during hibernation. Dodgen
and Blood’s (1956) estimate of .5 ml for bat blood volume is
unfortunately based on ‘unpublished data’? with no deseription
of method of determination; however, they were apparently
referring to the volume in Myotis lucifugus. Their value is in
close agreement with the volume of .550 ml calculated here for
the hibernatine bat, which sugeests that the true cell volume
may be as low as .250 ml, implving a bat F cells ratio near 1 in
hibernation and near .6 during: activity. Nevertheless, work has

1960 MAMMALIAN HIBERNATION 383

been initiated in our laboratory to determine cell volumes. di-
rectly, for the I* cells ratio thus impled for the active bat is
extremely low. Reeve ef al (1958) have found F cells of about
1 in normal does, which rose to about 1.1 in normal does
anesthetized with pentobarbital sodium, and fell to about .9 in
splenectomized dogs.’ At present, it seems most reasonable to
conclude that since fewer red cells are needed for respiratory
exchange during hibernation, as evidenced by their color, a large
proportion of them stagnates in the sluggish general circulation,
away from the heart, and that, conversely, ‘‘extra’’ plasma is
present in the capillary beds of the active circulation, but not to
the extent of .6 F cells ratio. The amount of relative change in
these ratios, however, emphasizes the inadequacy of hematocrit
determinations as a source of information on circulatory volume
change during hibernation. Specific gravities also appear to be
affected by varying distribution of blood components, even when
the plasma alone is considered. The contradictory results of
other studies suggest that these phenomena are not peculiar to
bats. An increased emphasis on direct measurements of volumes
appears necessary, therefore, not only for its own sake, but for
proper evaluation of metabohe changes in general which are
reflected in concentration changes of blood constituents during
hibernation.

Summary

A significant decrease in plasma volume during hibernation
was found in the little brown bat, Myotis lucifugus, by using a
modified Evans blue dye dilution procedure. This decrease
was not reflected in higher hematoerits or higher specifie gravi-
ties of blood or plasma taken from the heart in other bats,
which suggests a redistribution of cellular and plasma protein
components of the blood to ease the work of the heart durine
hibernation. A mobilization of blood cells in the thoracic region
during arousal was suggested by the high mean hematocrit
observed; dye studies indicated a preferential thoracic cireula-
tion at this time. A significant rise in hematocrit and plasma
specific gravity after arousal was noted in bats which had been
in relatively undisturbed hibernation, suggesting a dehydration
when compared to hats which had been disturbed recently and,

5'These authors are referring to ratios based on venous hematocrit rather than
heart hematocrit. However, additional data (Kallen, 1960) indicates that mean
venous hematocrit (uropatagial vein) does not differ significantly from mean
heart hematocrit in samplings of active bats injected with T-1824, although
individual variation is masking a slightly higher venous value.

384 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

presumably, had taken water while aroused. Part of this rise,
however, might also be attributed to a reversion toward the
hibernating blood picture whenever bats are at rest, even at
room temperatures, since spleens enlarge to a size previously
described only during hibernation when bats have been at rest
at 380°C for 4 to 5 hours. The spleen was found to contribute
significantly to circulating cell volume and appears to be the only
organ which was doing so. However, the volume of cells in the
spleen is inadequate to account for the extra cell volume implied
by calculating cell volume from plasma volume and heart hema-
toerit when a constant F cells is assumed. A more general con-
centration of cells in the peripheral circulation is suggested dur-
ing hibernation, and the reverse during activity.

REFERENCES

Barpour, H.G. AND W. F. HAMILTON
1926. The falling drop method for determining specifie gravity. J.
Biol. Chem., 69:625-640.

Dopegrn, C. L. Aanp F. R. BLoop
1956. Energy sources in the bat. Am. J. Physiol., 187:151-154.

Evans, C. A.
1938. Observations in hibernating bats with especial reference to
reproduction and splenic adaptation. Am. Nat., 72:480-484.

GROSSER, O.
1901. Zur Anatomie und Entwickelungsgeschichte des Gefissystemes
der Chiropteren. Anat. Hefte, 17:203-424.

Hock, R. J.
1951. The metabohe rates and body temperatures of bats. Biol. Bull.,
101:289-299.

‘

K ALLEN, F. C.
1960. Plasma and blood volumes in the little brown bat. Am. J.
Physiol., 198:999-1005.

LANDAU, B. R. AND A. R. DAWE
1958. Respiration in the hibernation of the 13-lined ground squirrel.
Am. J. Physiol., 194:75-82.

LipickER, W. Z. Jk. AND W. H. Davis
1955. Changes in splenic weight associated with hibernation in bats,
Proc. Soc. Exp. Biol. Med., 89:640-642,
LYMAN, C. P. anp P. O. CHATFIELD

1955. Physiology of hibernation in mammals. Physiol. Rev., 35:403-
425.

1960 MAMMALIAN HIBERNATION 385
Rerve, I. B., M. 1. GREGERSEN, T. TH. ALLEN AND H. SrAR
1958. Distribution of cells and plasma in the normal and splenectom
ized dog and its influence on blood volume estimates with P38
and T-1824. Am. J. Physiol., 17§:195-205,

RIEDESEL, M. L.
1957. Serum magnesium levels ino mammatinn hibernation. ‘Trans,
Kansas Acad. Sei., 60:99-141.

WortrH, R.
1932. Observations on the blood and blood-forming organs of certain

local Chiroptera. Folha Haematolog., 48:387-354,

DISCUSSION FOLLOWING KALLEN’S PAPER

BULLARD asked if the mixing time had been standardized
for each level of animal activity. KALLEN rephed that, using
a d-minute mixing time, he had taken two or more blood samples
from individual active bats, one sample from a wing vein and
one or more from the heart. The similarity of plasma-dye con-
centrations in these samples had appeared indicative of complete
mixing. This procedure had proved impractical in the case of
hibernating animals owing to the difficulty of drawing blood
from the lowered peripheral circulation. However, routine
autopsies consistently showed an apparently homogeneous dis-
tribution of the intensely colored dye in both active and hiber-
nating animals and he (KALLEN) thought it most likely that
mixing had been complete in hibernating bats, especially since
localization of dye was readily observable during states of
arousal. BULLARD asked if mixing time had been changed
with lowered body temperatures, and KALLEN rephed that it
had been kept constant for all states of activity.

ADOLPHE noted that the animals appeared to lose a consid-
erable volume of blood during hibernation — was this due to
dehydration? KALLEN replied that, although some dehydration
appeared to occur during prolonged hibernation, most of the
water lost by the blood appeared to remain within the animal
since higher plasma volumes were observed when hibernating
bats were brought to a state of relatively high activity at room
temperature without having drunk. ADOLPH asked how long
the torpid state had persisted before the animals were used.
K ALLEN said this was difficult to assess, although the data pre-
sented for ‘* disturbed’? and ‘‘undisturbed’’ bats suggested that
it had persisted for some time when their storage cage was left

386 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

unopened. However, in accord with HOCK’S earher evaluations
of time required for the bat to attain a minimum metabolic rate
at low temperatures, all animals used had been kept at 2°C for at
least S hours immediately before determinations were made.
ADOLPH then asked how long the animals had been kept in the
cold. KALLEN replied that the particular animals used had
been hibernating naturally in caves for at least two months, and
kept in the cold room for not longer than three months after
their capture,

XX
PERIPHERAL NERVE FUNCTION AND
HIBERNATION IN THE THIRTEEN-LINED
GROUND SQUIRREL,
SPERMOPHILUS TRIDECEMLINEATUS’

By Tueopore TH. Kenn and PETER MORRISON
Departments of Zoology and Physiology
University of Wisconsin

Madison, Wisconsin

If hibernating mammals are to remain responsive to environ-
mental stimuli throughout hibernation, their nervous pathways
must remain functional at body temperatures so low that condue-
tion would be blocked in most non-hibernating mammals. It has
heen shown by Chatfield ef al. (1948) that the peripheral nerve
in one hibernator, the golden hamster, is functional at somewhat
lower temperatures than in the albino rat, a non-hibernator.
Such an adaptation should have definite survival value, allowing
a hibernating animal to respond to stimulation during hiberna-
tion. One might also expect to see differences in nerve
conduction between hibernating and active phases in the life of a
hibernator, but such were not found in the hamster. However,
the hamster might be termed a ‘‘permissive’”’ hibernator inas-
much as it stores food and does not necessarily hibernate during
the winter; it is, moreover, native to lower latitudes. One might
expect further specialization in more northern species which in-
variably hibernate. This study deseribes peripheral nerve fune-
tion in such an ‘‘obligate’’ hibernator, the thirteen-lined ground
squirrel (Spermophilus tridecemlineatus), in terms of the tem-
perature functions of conduction, excitation and refractory
period.

Materials and Methods

These experiments were carried out durme 1957 and 1958,
The thirteen-lined eround squirrels were trapped in the summer
for use during the followine vear. Although the ages of the
animals were unknown, a ereat number were probably from
spring litters of the vear of capture. Experiments on active
animals were carried out during June and July. During the

1This research was supported by the Wisconsin Alumni Research Foundation,
and U.S.P.H. grant no. H-2095.

388 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

hibernating season (November through March) the animals
were housed in a ‘‘hibernaculum,’’ a special bank of cages pro-
viding isolation from sound and light and in which the tempera-
ture was maintained at 5-10° by the circulation of refrigerated
water through hollow walls. Arrangements were made to feed
the animals in measurable amounts without disturbing or alter-
ing the environment. When the consumption of food and water
had dropped to zero, one could be certain that the animal was
no longer active and no longer regulating its body temperature.
At intervals of 1 to 3 weeks it was observed that food and water
had been consumed over periods of one to two days, thus indi-
cating that the animal had awakened temporarily. During most
of the intervening fasting period the animal was surely in hiber-
nation, but it is quite possible that partial awakening without
food or water consumption had occurred during these periods.

Animals were taken for experiments at various times during
the hibernating cevele: at three days of hibernation, at 12 days
of hibernation, and then after 32 days total hibernation. 'To
define transitional stages, two additional groups of animals were
studied: animals which had been hibernating but were stimu-
lated to arouse until the body temperature had reached 20°C,
and animals which had been hibernating in the cold but had
spontaneously become active and remained so for some weeks.
Kinally, a group of animals were examined which had not been
subjected to a cold environment but had become lethargic at
room temperature durine the winter (ordinary hibernating )
season,

The seiatie nerve, used in these experiments, was prepared as
follows: removed from the hibernaculum and weighed, the animal
was immediately decapitated and the body temperature taken ;
the careass was dissected at 5°C in a cold room. With a blunt
elass probe the sciatic nerve was exposed from its insertion in
the gastrocnemius to its origin at the spinal roots. All side
branches were severed, the proximal and distal ends were tied
off and the nerve eut free. Durine dissection the nerve was
swabbed with cold) Locke’s solution.

The freed nerve was placed in a nerve chamber (Plate) so
that one end law between two stimulating electrodes and the
remainder rested on a series of pickup electrodes spaced 5 mim
apart. With three selector switches any pair of pickup electrodes
could be chosen for reeording and any third for a ground.
The eleetrodes were enelosed in a lueite housing through whieh
water from a constant temperature bath cireulated. Between
measurements the chamber was flooded with Locke’s solution.

1960 MAMMALIAN HIBERNATION 389

During a typical experiment the nerve was placed in the nerve
chamber at 5°C and allowed to equilibrate for 10 minutes before
the measurements for this temperature were made, The constant
temperature bath and the nerve chamber were then warmed
through 2.5°C and measurements made again after a 10-minute
equilibration period. In this way measurements were made
every 2.5 degrees up to 20°C, then every 5 degrees from 20 to
30°C, and finally at 37°C.

A Textronix,” Model 532, oscilloscope with a DuMont oscillo-
scope camera” was used to record the action potential photo-
graphically. The stimulus was produced by a Grass stimulator
(Model S4C)* through a stimulus-isolation unit.

In measuring conduction velocity a maximal stimulus of
0.1 msee duration was used. This represents the lowest stimu-
lus that will just excite all the fibers in the nerve. Excitability
was determined at stimulus durations of 0.1 and 0.01 msee.
The threshold was measured at the maximum: sensitivity of the
oscilloscope (20em/mV) by observing the voltage at which the
action potential just disappeared.

The refractory period was measured by shortening the time
between two successive stimuli (0.1 msec) until the action
potential which resulted from the second stimulus showed a
deerease in spike height. This interval is a relative refractory
period and relates to the behavior of the least excitable fiber
groups. The time between two stimuli at which the second action
potential just disappeared was also measured. This is taken
as an absolute refractory period and is dependent upon the most
excitable group of fibers. The stimulus-streneth for the meas-
urement of absolute refractory period was about 10 times the
threshold.

Results

Conduction velocity. Conduction velocities from a typical ex-
periment are plotted in Figure 1. These data show a linear
relationship of conduction velocity with temperature which was
characteristically seen in this study. This particular experiment
Was unusual in providing two measurable spikes. Here, the faster
fiber-group is taken to be the alpha, with velocity at 37°C of
100 M/see. The slower fiber-group with velocity of 30 M /see

2 Textronix Inc., Portland, Oregon.
“Aten B. DuMont Laboratories Ine., Clifton, New Jersey.

Grass Instrument Co., Quiney, Massachusetts.

390 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

at 37°C was judged to be the beta. Only in animals whieh have
just entered hibernation was it possible to obtain clear-cut re-
sponses from the slower fibers. Although a beta spike could be
seen in nerves from other groups, it was too close to the alpha
spike to be accurately measured.

oak CTN 9:11-22-57
15 DAYS TOTAL HIB
_ 80Fr
e)
uw
n”
Ss:
=
= 60r
:
ro) aa
3 4ob we
= °
8g °
° a ee 08 Oa
ofr °
20 ig : A
fo) geo 8
a =: o90——O 7
geo (oo
s ! ee ee, ee es (ee ee eee hare en Seen eee eee
(0) 5 10 6 20 25 30 3D 40

TEMPERATURE IN °C

Fig. 1. Conduction velocity as a function of temperature. Both alpha
(upper group) and beta (lower group) components are shown. This run
is an example of an animal in the transitional stage.

Kigure 2 demonstrates the stability of the isolated nerve
preparation by comparing measurements made at 2, 9, 24, 48
and 72 hours after decapitation. There was excellent repro-
ducibility over this entire period, during which the nerve was
held at 5°C, with, however, some decrease in excitability even
after 24 hours.

The two parameters describing a linear function are the slope
(dV /dT) and the intercept (at V=0). The average values for
these conduction-velocity increments and intercepts in the several
groups of animals are tabulated in Table I and plotted in Figure
3. It may be seen that the conduction-velocity increment for the
active animal is considerably greater and the intercept is higher
than for the hibernating animal. The intercept values for the
active ground squirrels are the same as those noted by Chat-
field ef al. (1948) for the hamster. The slopes appear to be of

1960 MAMMALIAN HIBERNATION 39]

the same order of magnitude, but unfortunately cannot be exaetly
eompared. Accordingly, in the thirteen-lined ground squirrel
there is an adaptation during hibernation to facilitate conduction
at lower temperatures than would be possible in the active ground
squirrel.

30
e
o
O PA
a Oo 2HR rf
= 2 9HR of
= @24HR. ;
> 20F O 48-72 HR. /e
’
e /
rs fy
=i PA
> /g0
Zz a)
° a’
a e
= 10} a/
a fe)
iz.
S 0
[o)

CTN-16: 1/17/58
48 DAYS TOTAL HIB.

O 5 10 15 20
TEMPERATURE IN °C

Fig. 2. Conduction velocity versus temperature in a series of measure-

ments extending over a period of 72 hours.

The average curves for animals that had just entered hiberna-
tion (3 days and 12 days) (Fig. 3) show a progressive lowering
of the intercept with no change in slope. Consequently, the
nerve has maintained the same ability to respond to changes in
temperature, and yet has adjusted so as to function at tempera-
tures that would otherwise block conduction. As hibernation
is extended Irom 12 to 52 days or longer, the slope is reduced by
about one-half its former value with little change in intercept.

In animals sacrificed during the awakening process (Tp;=20°C )
the nerve maintains unchanged its ability to conduct at low
temperatures, but the conduetion-velocity increment has in-
creased during this brief period (14 hour) to a value halfway

392 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

between those for the active and hibernating animals. The
average curve for the animals that had awakened spontaneously
from hibernation and were active at 5°C is essentially identical
to that for the active animals.

CONDUCTION VELOCITY IN M/SEC.

AT 25°C
ACTIVE

TEMPERATURE IN °C

Fig. 3. Mean curves for conduction velocity versus temperature for the

yarious groups of animals used in this study.

Lerertability. The reciprocal of the threshold voltage may be
used as a measure of excitability. Our data show that a linear
relationship exists between excitability so measured and tempera-
ture (Fig. 4.) Mean values for slopes and intercepts are given
in Table II and plotted in Figure 5. The excitability increment
for animals hibernating for a long time (32 days) is lower than
for active animals and the intercept is also lower for hibernating
animals. Thus, the nerve of a hibernating ground squirrel is

1960 MAMMALIAN HIBERNATION 393

excitable at lower temperatures than the nerve of an active
ground squirrel; any change in temperature will have a smaller
effeet on it. The changes in slope and intercept for excitability
are exactly analogous to those found for conduction-velocity.

TABLE |

Slopes and Intercepts of the Temperature Functions of
Conduction (M/see. vs. °C) in the Seiatie Nerve of the 13-lined
qround Squirrel under Various Conditions.

Slope in M/see-1 °@-1

Group Number Mean S.E. Mean - S.E.
Active
at 25°C 8 2.30 16 4.8 68
(summer )
Active
at 5°C 3 2.29 6.0
Active
at 25°C 3 1.84 4.0
(winter )
Hib. at
5°C
3 days 2 2.38 2.6
12 days : 2 2.71 0.0
32 days 19 1.43 06 1.8 233
Hib. at
256 6 1.61 13 2.7 De
Awake
5—20°C 3 2.29 6.0
ate ee Slope Intercept
Conduction Velocity T-Value T-Value
I. Active vs. hib. both at 25° 2.1 4.9
II. Hib. 5° vs. awake to 20° 0.4 0.4
III. Hib. at 25° vs. active at 5° 1.3 0.3
IV. Active at 25° vs. active at 5° 1.8 3.0

V. Winter active vs. summer active 2.9

If one compares the intermediate stages, quite a different
picture appears (Fig. 5). In animals just entering hibernation,
atter 3 days the average slope and intercept differed but little
from the active animal, but after 12 days the average slope and

394 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

increment were at the hibernating level (32 days). The lowest
intercepts were observed in animals which had become active at
)°C following hibernation. The curve obtained for animals in the
process of arousal was essentially the same as the curve for active
animals at 9°C.

3 : 3
24 HR. 2HR.
;
o——————— ¢__.. 6
se
| v4 ik
s

THRESHOLD IN VOLTS AT 107% SEC
if
_
NN
pe
EXCITABILITY IN VOLTS7~! AT |lo7~4 SEG

Oo 10 20 30
TEMPERATURE IN °C

Fig. 4. Representative results of excitability versus temperature indicat-
ing reciprocal threshold voltage 2 hours after the animal was decapitated
and 24 hours after decapitation. Other symbols are thresholds measured
over a period of 48 hours. The discontinuity at 17.5°C was seen in several
other experiments but had no correlation with the state of the animal.
Only the linear portion of the plot to 20°C was used to derive the mean.

Refractory period. The refractory period represents a measure
of the time required for completion of some recovery process in
the nerve fiber. Its reciprocal, which is proportional to the rate
of this reaction, was not found to be a linear function of tempera-
ture, as were conduction velocity and excitability (Fig. 6).
When the logarithm of the reciprocal of refractory period was
plotted against temperature, a linear function resulted. There
Was a significant shift in the Q,»9 of recovery in the hibernating

1960 MAMMALIAN HIBERNATION 395

eround squirrel nerve as compared to that in the active (Summer )
animal (4.0 to 3.1) and this ehange was not shared by any other
group (Table II1). Amberson (1930) gave a value of 3.0
for the Q,» of the absolute refractory period in the sciatic
nerve of Rana esculenta. It may be significant that high tempera-

PIE
AT 25°G
fo

ACTIVE

EXCITABILITY IN VOLTS~! AT 107% SEC
ime)
is

O 10 fe) 20
TEMPERATURE IN °C

Fig. 5. Mean curves for excitability versus temperature for various
groups of animals used in this study. Animals ‘‘active at 5°C’’ had
previously hibernated.

ture coefficients are also observed in the overall changes of
metabohe rate (Kayser, 1940; Hock, 1951). On the other hand,
much lower temperature coefficients are found for isolated
tissues including brain (South, 1958; Meyer and Morrison,
1960).

The linear relationships between temperature and conduction
or excitability suggest ‘* physical changes’’ in the nerve, perhaps

396 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

not inappropriately in these instances. However, the logarithmie
funetion deseribing the refractory period is suggestive of some
changed ‘‘chemical’’ process supplying energy for recovery.
Henee, a van’t Hoff or Arrhenius funetion is in order. The
higher Q,,» exhibited by the nerve of the hibernating ground
squirrel would suggest that this nerve is better able to respond
metabolically to changes in temperature.

TABLE II

Slopes and Intercepts of the Temperature Function of

} ] i

Excitability in the Sciatic Nerve of the 13-lined Ground Squirrel
under Various Conditions.

Slope in V-1 °C-1

Intercept in °C

Group Number Mean S.E. Mean S.E.
Active
at 25°C 6 0.29 063 6.8 59

(summer )

Active
at 25°C 4 0.18 .033 2.9 1.16
(winter )
Active
at sec 2 0.106 31 0.3 Zl:
THhib.
3 days 2 0.25 5.0
12 days 2 0.10 3.0
32 days 15 0.18 19 0.3 46
Hib. at
25°C 6 0.14 03 1.0 15
Awake to
20°C 4 0.107 05T 0.9 1.5
aa Slope Intercept
T-Value T-Value
I. Active vs. hib. both at 25° 3.5 3.3
Il. Hib. 5° vs. awake to 20° 2.3 1.6

TII. Hib. at 25° vs. active at 5 0,3 O.4

Discussion

Conduction and excitation in the sciatic nerve of the thirteen-
lined ground squirrel have been shown to be lnear functions of
temperature. Such functions are suggestive of a ‘‘physically’’

REFRAGTORY PERIOU IN MoOEYU.

1960 MAMMALIAN HIBERNATION 397

dependent reaction, whereas the logarithmic function deseribing
the refractory period is suggestive of a chemical reaction perhaps
supplying energy for recovery. Amberson (1930) showed such
a function in the sciatic nerve of the frog. His value for the Q,
of the reciprocal refractory period (3.0) was identical to that
found here for the active ground squirrel.

O 10 20 30 40

TEMPERATURE IN °C

Fig. 6. Refractory period versus temperature for the various groups
of animals used in this study. Open circles show direct plot (in msec),
half circles show reciprocal plot (in msec!) and solid cireles show the log
reciprocal plot (log msee—! ),

The normal ground squirrel nerve has a temperature function
quite similar to that obtained by Chatfield et al. (1948) for the
golden hamster. However, these investigators found no differ-
ence between hibernating and active hamsters, and compared
the data from the hamster nerve with data from the albino
rat nerve. One must keep in mind that the ground squirrel is an
‘‘obligate’’ hibernator and not a ‘‘permissive’’ hibernator as is
the hamster. Therefore, one may expect to find physiological
adaptation occurring in ‘‘obligate’’ hibernating animals which
may not be demonstrable in the ‘‘permissive’’ hibernators.

0.0

=—2.0

LOG

398 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

TABLE IIT

Temperature Coefficients for Recovery of Sciatic Nerve in the
13-lined Ground Squirrel.

Relative Refractory 1

Absolute Refractory 2

Period Qio Period Qo
Group Number Mean S.E. Number Mean S.E.
Active
at 25°C ] 3.10
(summer )
Active
at 25°C ff 3.10 26 4 3.08 Of
(winter )
Active
at 5°C 4 3.21 36
Hib.
at 25°C 6 32b2 09 6 3285 a)
Hib.
at 5°C 12 3.95 Qi 4 4.29 1.33
Awake
520°C 4 3.09 4]

lL iteciprocal relative refractory period. Represents the least excitable fiber
group: time between pairs of maximal stimuli to provide measurable diminution
of second spike.

2 Absolute refractory period. Most excitable fiber group; time between pairs
of maximal stimuli to just eliminate second spike.

Significance: Active at 25° vs. hib. at 5° : T= 2.5
Hib. at 25° vs. hib at 5° 20° "320

‘*Transitional’’ Properties

The linear form of the temperature functions and their changes
in slope and intercept showed close analogy between conduction
and excitation in the active summer and hibernating winter ani-
mals. This suggests some common underlying activity, and in-
deed conduction may be considered as propagated excitation.
While it would be attractive to ascribe these functional changes
to some single underlying condition, examination of results in
the transitional stages shows that the situation is more complex.
Thus, although usually correlated, a shift in intercept (rate con-
stant) was not always accompanied by a change in slope (tem-
perature coefficient) and conversely. Further, in transitional
stages, observed changes in the eonduction funetion did not
always parallel the changes in exeitability.

1960 MAMMALIAN HIBERNATION 399

The shift in the Q, 9 of recovery in the hibernating (382 days)
ground squirrel nerve as compared to the active ground squirrel
nerve (4.0 vs. 3.1) has the opposite sign from the Q,os for con-
duction and excitation, ie., the temperature coefficient is im-
ereased in hibernation rather than decreased. However, this
change in recovery function corresponds to the change in tem-
perature function seen for tissue respiration (Meyer and Mor-
rison, 1960).

It may be significant that high temperature coefficients are
also observed in the overall changes of metabohe rate seen in
hibernators (Kayser, 1940; Hoek, 1951). On the other hand,
much lower coefficients are found for isolated tissues including
brain (South, 1958; Meyer and Morrison, 1960).

The underlying causes of these modifications in nerve function
are of much interest. It is known that in the hibernating mam-
mal there is a general involution of the endocrine glands (John-
son, 1931; Kayser, 1940), so that some hormonal influence may
be involved either directly or indirectly. Sueh a notion is sup-
ported by the fact that the endocrine involution is seasonal,
that is, it precedes actual hibernation. Ground squirrels which had
not entered hibernation during the winter showed a modification
in conduction-velocity Increment and exeitability increment simi-
lar to that seen in hibernating animals (Tables I and IT; active
at 25° (winter) vs. hib. at 5° (82 days)). More direct evidence
of hormonal involvement in nerve function is provided by experi-
ments on nerves from hypophysectomized albino rats) (unpub-
lished observations with R. Kk. Meyer) which show lower slopes
and intercepts for both conduction and excitability functions.
Since this transformation exactly parallels the change in the
hibernating ground squirrel, it stronely supports the concept
of a control of axonal function through hormonal levels mediated
through the hypophysis.

REFERENCES

AMBERSON, W. R.
1930. The effect of temperature upon the absolute refractory period
in nerves. J. Physiol., 69:60-66,.

CHATFIELD, P. O., A. F. Batrisra, C. P. LyMAn AanpD J. P. GARCIA
1948. Effects of cooling on nerve conduction in a hibernator (golden
hamster) and a non-hibernator (albino rat). Am. J. Physiol.,

155:179-185.

400 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Hock, R. J.
1951. The metabolic rates and body temperatures of bats. Biol. Bull.,
101: 289-299.

JOHNSON, G. E.
1931. Hibernation in mammals. Quart. Rev. Biol., 4:439-461.

IXAYSER, C.
1940. Les échanges respiratoires des hibernants. Theses, Univ. Stras-
bourg. 364 pp.

Meryemr, M. P. AND P. MORRISON
1960. Tissue respiration and hibernation in the thirteen-lined ground
squirrel, Spermophilus tridecemlineatus. (This volume, Pp.
405-420.)

SoutuH, F. E.
1958.) Rates of oxygen consumption and glycolysis of ventricle and
brain slices, obtained from hibernating and non-hibernating
mammals, as a function of temperature. Physiol. Zool., 31:6-15.

DISCUSSION FOLLOWING KEHL’S PAPER

GRIFFIN remarked that KEHL was in effect measuring a
number of synchronized potentials in a nerve trunk. KEHL
agreed, and pointed out that the refractoriness of the most
excitable fibers was determined by a two-stimulus sequence using
a stimulus at ten times threshold in a situation in which the time
was shortened between stimuli until the second spike did not
appear. The usefulness of the stimulus of 10 x threshold was that
it maintained the Q,, at a constant level.

FISHER inquired about size of nerve and electrode size in
measuring threshold, for these would presumably affect the
threshold as KEHL measured it. KEHL rephed that the experi-
mental procedure was strictly relative, that electrodes were of
constant size, and that he always presumed the nerves used were
of constant size.

BULLARD asked whether the curves were extrapolated or if
he could actually measure a spike at low temperatures where
there is no conduction. KEHL rephed that he could not make
such a measurement. Ile made the additional comment that
temperatures as low as 4.5°C in the hibernating animal cause
problems in measuring conduction velocity.

BARTHOLOMEW asked how velocities in hibernating mam-
mals compared with what would be found in the lizard. KEHIL

1960 MAMMALIAN HIBERNATION 40]

replied that he did not know. FISHER asked how the hiber-
nating nerve compared with that of the frog. KEHL replied
that the hibernating nerve had a temperature coefficient quite
similar to that of the frog.

BISHOP remarked that the picture looked as though the
phenomena observed could be obtained by raising the frog
temperature coefficient up about half way or lowering the mam-
mal nerve coefficient down about half way; therefore, the hiber-
nating nerve seemed to stand between these two.

SCHONBAUM asked if anything was known of the biochem-
istry of nerves during hibernation as compared to non-hiberna-
tion. KEHL replied he was not qualified to answer. SCHON-
BAUM inquired whether anything was known about the effect of
insulin or other hormones on nervous activity. KEHL remarked
that his data were not in agreement with those of SUOMALAI-
NEN, whose paper was presented earlier. He felt the differences
may be clarified by doing hormone titers.

SOUTH noted that he would like to get more detail from
KEHL on the design of his experiments: how many animals
comprised a group at one temperature, whether he used mean
values obtained from different nerves for his data. KEHI,
rephed that in summer animals he did use mean values for ten
samples, that the data agreed with P. O. Chatfield e¢ al. (Am. JJ.
Physiol., 155:179, 1948). In the case of winter animals, a total
of 30 animals was used; all nerves were run through a series
of temperatures. SOUTH noted further that he had found that
nerves do recover from a sojourn at a given temperature but they
behave differently. He also stated that he used different pro-
cedures in threshold calculations. He asked whether voltage or
milliamperage was being recorded, and whether it was monitored.
KEHL replied that they used a single duration in measuring
threshold, and used several times threshold at 0.1 msee. with a
Grass stimulator which had been reealibrated. SOUTH suggested
they use another beam on an oscilloscope as a monitor.

FISHER then asked what parameters were really measured.
He remarked that if the duration of the stimuli were long enough
and constant in length, the differences noted might conceivably
be related to accommodation. KEHL rephed that the stimulus
duration was constant. FISHER then said that the variation
in threshold might have to do solely with the size of the nerve.

402 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

KEHL replied that a single nerve was used in each case, hence
size could not be a factor. FISHER remarked that variation in
the water and salt content and hence the resistivity might also
affect the threshold as measured, so that a ‘‘true’’ variation in
excitability might not be involved at all. KEHL remarked that
the change in temperature coefficients seen in peripheral nerves
with the advent of hibernation seems to be associated with a
decrease in hypophyseal secretory activity.

nite WW ASC ee es 29 28802 6 88 22 Tey

PLATE

Nerve Chamber. The stimulating electrodes are at the extreme left.

XX]

TISSUE RESPIRATION AND HIBERNATION
IN THE THIRTEEN-LINED
GROUND SQUIRREL,
SPERMOPHILUS TRIDECEMLINEATUS'

By Marton P. Meyer and Perer Morrison
Departments of Zoology and Physiology
University of Wisconsin
Madison, Wisconsin

Animals which enter hibernation demonstrate a considerable
decrease in metabolism along with a profound drop in body
temperature. The total animal metabolism is, of course, a reflec-
tion of the respiration levels of the constituent tissue, and Martin
and Fuhrman (1955) have shown that the summation of meas-
ured tissue respiration levels can give values reasonably close
to observed basal metabolic rates. The relationship of the basal
or intrinsic tissue respiration to the total animal metabolism is
not completely understood, however, and the question arises as
to whether the low metabolic rate observed during hibernation
can be accounted for simply in terms of the temperature co-
efficients of the constituent tissues. In order to answer this ques-
tion, the respiration levels and temperature coefficients of the
tissues must be examined. The comparison of the hibernating
animal in these respects with the non-hibernating animal of the
same species and with mammals which do not hibernate will
provide evidence as to possible intrinsic metabolic adjustments
or adaptations.

In 1941, Hook and Barron, in their study of the role of brown
fat in hibernation, measured the O» consumption of brown fat
and kidney tissue from ground squirrels. In substrate-free
media, the levels of respiration were the same in the hibernating
and non-hibernating animal. The inclusion of various substrates
considerably augmented the respiration level of kidney at 38°
but not at 8°C and appeared to increase kidney respiration in
the active animal slightly more than in the hibernating one.
The Q1o of kidney was 1.9, and of brown fat, 1.4, when substrate

1 Our program of studies on hibernation receives continuing support from the
Wisconsin Alumni Research Foundation. This study was begun under USPH
stant no. H-2095, assisted by the Wisconsin Heart Association and completed with
aid from the American Cancer Society (Inst. grant to U. W.).

406 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

was not added; with suecinate, the Q,o for kidney was 2.6, and
for brown fat, 1.7.

Kayser (1954a,b) measured the Os consumption of kidney
shees from hibernating and non-hibernatinge hamsters and white
rats at a series of temperatures from 5° to 38° in a medium
containing @lucose. The data gave Q,,9 values of 2.3 for the white
rat and 1.9 for both the hibernating and non-hibernating ham-
ster. The hibernating hamsters, however, showed a uniformly
lower oxygen consumption throughout the temperature range.
Fuhrman and Field (1942) found a Qo of 2.2 in the white rat.
Thus, in the kidney of the hamster and ground squirrel, the
temperature coefficient appears to be the same for the active and
hibernating animals although the coefficient for the hamster
seems less than for the white rat. The hibernating hamster
showed a uniformly lower respiratory level than the active
animal.

South (1958) published the results of oxygen consumption
ieasurements at a series of temperatures from 5° to 43°C on
heart ventricle and cerebral cortex of the white rat, active and
hibernating hamster, and torpid bat. The temperature coef-
ficients were not constant over this range for heart slices. Values
were highest for the white rat, lower for the active hamster, and
still lower for the hibernating hamster and torpid bat. On the
other hand, values for oxygen consumption of brain slices indi-
cated a Q,,9 of about 1.8 to 1.9 for all the four groups with a
shehtly lower level of metabolism in the hibernating hamster as
compared to the active animal. Field et al. (1944) found a
higher Q,» of 2.1 for cerebral cortex in a large series of white
rats. Weiss (1954) gave a value of 2.2 for rat brain. Thus, while
the brain showed no adaptation in the temperature coefficient, the
heart of the hibernating hamster and bat appears to have a
lower temperature coefficient resulting in a higher metabolic rate
at the lower temperatures.

The bat and the hamster have been the principal hibernators
studied in regard to tissue respiration. Bats hold a unique posi-
tion in regard to hibernation in that they fall into a torpid state
daily upon the cessation of activity, in contrast to the seasonal
hibernation of other species. The hamster, on the other hand.
might be termed a ‘‘permissive’’ hibernator since it stores food
and may withstand low environmental temperatures for long
periods of time without lowering the body temperature. Until
now no extensive respiration measurements have been made upon

‘

any ‘‘obligate’’? hibernator which must almost always enter

1960 MAMMALIAN HIBERNATION 407

hibernation in the fall and winter season. Aceordingly, the pres-
ent study of tissue respiration in the thirteen-lined ground squir-
rel was undertaken. And since each of the various body tissues
thus far examined seems to present its own characteristic meta-
bolic relationship to temperature, it was deemed desirable to
examine a variety of individual tissues. By this means also we
are able to arrive at an over-all metabolic picture of the influence
of hibernation on tissue metabolism.

Materials and Methods

Thirteen-lned ground squirrels (Spermophilus tridecemlinea-
fus) were captured during the summer in the vicinity of Madison,
Wisconsin. They were kept in small cages with access to water
and rat ration and with supplements of greens and fruits. In
the fall, a group of these animals were placed in individual cages
in a hibernaculum—a bank of units individually isolated
against light and noise and maintained at a temperature of 6°C
by the circulation of refrigerated water through hollow ‘‘cold-
walls.’” Each animal was supplied with cotton bedding material,
water, and rat pellets. The period of hibernation was defined
as the number of days since the last food or water was taken.
This will represent a maximum period of hibernation, since the
animal may have wakened from hibernation during the interval
but not eaten. At this time it is not known which of these inter-
vals is more significant. It is even possible that the total hiber-
nation time during the season may be of greater importance.

Animals were removed from their cages, and rectal body
temperature taken immediately after weighing and decapitation.
The hibernating animals were dissected rapidly in a 5°C room.
The organs were removed, weighed and eut into small pieces.
They were then placed on filter paper moistened with inorganic
Krebs medium in covered petri dishes which were kept on ice.
The standard Warburg procedure for measuring the oxygen
consumption of tissue slices was followed. Krebs medium IIT
with phosphate buffer and glucose, pyruvate, glutamate and
fumarate substrates and 1 per cent albumin was used to provide
optimum conditions im vitro.

Measurements were carried out at 37.5° and 15°C on liver,
kidney (cortex), spleen, lung, brain (cerebral cortex), diaphragm,
heart (ventricle), and skeletal muscle (limb) of hibernating and
active thirteen-lined ground squirrels. A few measurements were
also carried out at 5°C. Also, several animals in intermediate

408 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

states of hibernation were studied. Two animals were cooled in
an ice bath until their rectal body temperature dropped to 23°
before decapitation. Another two animals were kept in the hiber-
naculum at 6° for several weeks after they had come out of hiber-
nation in the spring. Two hibernating animals were allowed to
warm spontaneously to 20°C before decapitation.

tesults

The percentage of body weight which each organ represents
is presented in Table I. These values are not given on a basis of
fat-free body weight since, in most cases, the total fat contribu-
tion was not determined. There is considerable variability owing
to the large weight changes which ean occur with changes in fat
deposit in these animals. The large standard deviations indicate
this and make comparisons between the two groups difficult. How-
ever, the enlargement of the spleen known to oeceur during
hibernation is apparent (Mann and Drips, 1917; Lidiecker and
Davis, 1955).

TABLE I

Organ Weights in Spermophilus tridecemlineatus as
Percentage of Body Weight + Standard Deviations ®

Organ : hea ipa h : Dormant ¢
Liver 4.20 + 1.03 449 + 0.93
Kidney 0.85 + 0.25 0.90 + 0.22
Spleen OL 2-046 0.29 = 0.15
Lung 0.73 = 0.20 0.95 <= 0.25
Brain 1.58 + 0.45 1.99 = 0.43
Diaphragm 0.44 4 0.07 0.45 = 0.09
Heart 0:53 SE 0:35 0.638 =e 0.15
Stomach 0.96 2+ 0.24 0.98 = 0.19
Adrenals 0.016 = 0.005 0.014 += 0.005
Brown Fat (0.32) (1.68)
White Fat (24.) (13)
Skin (14) (15)

Sk. Muscle (38)

a Parenthesized values for only a few animals.
b23

23 non-hibernating animals ranging from 102-284 gm and averaging 155 gm.

¢20 hibernating animals ranging from 98-208 gm and averaging 127 gm.

1960 MAMMALIAN HIBERNATION 409

A summary of the results of oxygen consumption measure-
ments is presented in Tables I] and TIT. The value at 37.5° for
the kidney cortex of active ground squirrels, 30.8, approximates

Tasue Il

Oxygen Consumption of Tissue Slices from Non-hibernatine
Spermophilus tridecemlineatus.

ce oxygen per dry gram hour (QOzs)

Th = 23°C" T, = OC?
52C 15°C By Gana & Fy eta oie 15:2:C
Tissue No. Mean No. Mean S.D. No. Mean S.D. No. Mean No. Mean
Liver 3 2.1 5 1.7 A 14 4.6 1.3 2 5.9 2 2.6
Kidney 3 2.4 a) Dal 3 14 30.8 5.3 2 23.1 2 9
Spleen | 0.57 2 0.93 1 10 86 12 J 8.0 | 1.5
Lung 2 0.35 } 0.76 02 7 4.8 0.8 2 d.1 ] 0.95
Brain 3 0.89 5 2.3 oO 14 -12.5 2.3 2 14.1 2 2.5
Diaphragm 1 0.86 2 1.9 4 #10 26 #O8 1 3.6 | 2.4
Sk. Muscle 2 1.0 3 1.6 2 6 2.8 06 — —- 1 1.6
Heart 3 2.3 5 3.6 a 6 40 06 2 7.4 2 6.4
a Active animal cooled in ice water to 23°C body temperature.
bh Active animal kept at 6G°C following termination of hibernation.
TaBLeE III
Oxygen Constumption of Tissue Shees from Hibernating
Spermophilus tridecemlineatus.
ce oxygen per dry gram hour (QOs)
Te = 205'Ca
HC 15°C Ea a Oe LOE Sede
Tissue No. Mean No. Mean S.D. No. Mean S.D. No. Mean No. Mean
> 75 22
Liver } 1.9 13 2.2, 0.5 - ~ il 3.1 2 3.8
ee a S
Kidney 1 1.5 11 4.7 0.6 10 28.7 3.4 1 4.3 2 28.5
Spleen ] -— } 0.82 0.2 ) 8.6 1.6 -

Lung l 0.22 7 0:72 0.2 7 5.3 0.9 — — 1 5.3
Brain ] O.79- Th 2.4 0.5 VO TAG 2.8 il 2.6 2 13.9
Diaphragm 1 0.89 7 2.0 0.4 8) 5.85 ps 2 4.1 1 5.5
Sk. Muscle ] -— 4 15 0.1 4 4.4 0.6 1 2.4. 2 4.8
Heart 3 2.4 13 4.4 1.2 6 6.8 2.4 1 5.8 2 7.4

a Hibernating animal warmed to 20°C body temperature,
» Hibernating less than 4 days.
° Hibernating more than 4 days.

410 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

the value of 27 cc/dry gm hr (QO) found by Hook and
Barron (1941) who used just pyruvate as substrate. The meta-
bolic levels for the tissues of the active thirteen-lined ground
squirrel appear to be comparable to the levels found in the white
rat, except in the case of muscle tissues. Our measurements
using the same medium gave values of 5.5 for liver and 25.5
for kidney in the white rat, comparable to 4.6 in liver and 30.8
ce/dry gm hr in the kidney of the ground squirrel. On the
other hand, rat diaphragm had a value of 6.4 as compared to
2.6 ee/dry gm hr in the active ground squirrel. Krebs (1950),
using this medium, found values three times higher for hver and
50 per cent higher for kidney of the rat. Most investigators have
used media containing only glucose for substrate and have found
values slightly lower than those reported here, except in kidney
tor which literature values are about one half (Fuhrman and
Field, 1942; Weiss, 1954). The difficulties in comparing tissue
respiration values from various investigations are well known.

The muscle tissues measured in the ground squirrel seemed
to have a lower respiration rate relative to other tissues than in
a non-hibernator, the white rat. It should be noted that in the
eround squirrel the respiration rate of muscle declined during
the measuring period. Thus the initial rate of respiration would
be higher than the mean value found for the hour of measure-
ment. Extrapolations of the steady decline back to the time the
flasks were placed in the constant temperature bath have been
made. The decline was linear in the ease of diaphragm and skele-
tal muscle and logarithmie for the eardiae tissue. The values
derived by extrapolation, then, are considerably higher and
equal to the normal rat values (which show less decline during
measurement). However, errors due to changing rates of decline
and extrapolation ereatly increase the variability in duplicate
measurements among the animals and make interpretation dif-
ficult.

At 15° none of the tissues studied from hibernating animals
were significantly different from those from active animals, as
is indieated in Table [V. The liver showed a slightly lower, and
the kidney a slightly higher, respiration in the hibernating
animal (i< P=—.2):

At 37.5°C the respiration of kidney, spleen, and lung from the
hibernating animal was the same as from the aetive animal. The
brain showed a significant, though small, increase of 20 per cent
in hibernation (P<.05). The muscle tissues, on the other hand.
increased their rate by 60 to 120 per cent in hibernation, so that

1960 MAMMALIAN HIBERNATION 41]
they were equal to that of the white rat at 37.5°. If the ex-
trapolated values were considered, however, the respiration rate
would be higher than for rat musele. At 37.5° there was a
transient increase in liver metabolism during the first few days
of hibernation. Figure 1 presents values for liver in relation to
the number of days since the animal! last took food. During the
first three days of hibernation the liver respiration was about
60 per cent higher than in the active animal. It then dropped
back to the non-hibernating level. At 15° there was a_ possible

: |
D
“6! LIVER
a 2 ee
tee
@ '
@ as} O
6 Of i q
Qo
2 6
4r '
@
P | i a
C3}
a sie

ce aoe ee ee —,| fe
10 20 35
DAYS SINCE LAST ACTIVITY

Pig, 1. Oxygen consumption of liver slices of the thirteen-lined ground
squirrel in relation to time in hibernation. Numbers are days since first
entering hibernation; vertical lines connect measurements on the same indi-
vidual; horizontal lines indicate mean values; diamonds on ordinate show
means for non-hibernating animals at 37.5° and 15°C.

shght increase in liver respiration throughout hibernation. The
total number of days that the animal had been in hibernation
during the season is given at each point in the early period. The
transient rise in metabolism appears less evident in animals
which have been hibernating for extensive periods before this
last awakening and re-entry.

Table LIV allows a comparison of tissue respiration in hibernat-
ing and non-hibernating animals in terms of the ratio of these
values. The few measurements at 5° indicate a lower rate in

412 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY YVol, 124

hibernating animals except in the ease of the muscle tissues which
did not change. More data is needed to verify this, however. At
15° the tissues studied showed the same respiratory rates in the
active and hibernating animals. At 37.5°, however, the liver
(during the first three days) and the muscle tissues demonstrate
a considerable increase in respiration in the hibernating animal
over the active one.

The values from two active animals whieh were foree-cooled
to 23° indieated an increase in the respiration of eardiae muscle
to the hibernating level or higher, and a lesser inerease in liver
and diaphragm (Table IT). The kidney apparently decreased

TaBLE IV

Influence of Hibernation on Oxygen Consumption of
Tissue Slices (H:N-H ratios)

ahd 15° B25" 37.5°
(Te = 202)
Liver 0.91 1.3** 1.6* <4 days 1.27
1.0 > 4 days 8]
Kidney 0.62 0.92** 0.93 1.2
Spleen = 0.88 1.0 11
Lung 0.63 1.1 1.1 1.0
Brain 0.89 1.1 L.2a* 1.0
Diaphragm 1.0 1a 2.2* 1.6
Sk. Musele — 0.95 1.6* —
Heart 1.0 1.2 Ie 0.92
*P < .05
*05 <P < 2

in oXygen consumption. These changes may well be the result
of immediate hormonal variations under this stressful situation.
It is of interest that the muscle tissues and liver were again the
organs which showed increased metabolic activity.

Another two animals were maintained in the hibernaculum
at an ambient temperature of 5° for several weeks after their
natural awakening from hibernation in the spring. The values
presented in Table II indicate increased metabolic levels in liver,
diaphragm and most particularly heart when measured at 15°.
Such changes at the lower temperature were not found in hiber-
nation; again muscle and liver are the tissues indicating changes.
Weiss (1957) has shown that these are the tissues which show

1960 MAMMALIAN HIBERNATION 413

increased metabolism in white rats that have been cold-adapted.
Thus, these preliminary experiments indicate that a hibernator
when not in hibernation may utilize similar mechanisms for cold
adaptation as non-hibernators.

The tissue respiration levels of two hibernating ground squir-
rels which were allowed to warm to 20° during arousal indicated
an increase in the metabolism of muscle tissues and liver at 15°,
as in cold adaptation (Table III). However, at 37.5° there ap-
peared to be no changes. The results of these preliminary ex-
periments involving only one or two animals are, of course, not
definitive. However, they do show the adaptability of muscle and
liver tissue as compared to the relatively unchanging metabolic
levels of spleen, lune, brain, and kidney.

TABLE V

Temperature Coefficients for Spermophilus tridecemlineatus: Qo

‘Non-Hibernating / Hibernating
7 “Organ _ 5°-15° ; 15°-87.5°. Fh ih 415°) co 45° 37.5° i
Liver 0.8] 1.56 4.16 2.37 < 4 days
1.538 > 4 days
Kidney 2,12 2.22 3.13 2.23
Spleen 1.65 2.70 - 2.84
Lung e O8ai 9.29 3.73 2.42
Brain 2.60 2.12 3.04 2.24
Diaphragm 2.2) L.15 2.23 LoL
Sk. Muscle 1.60 1.28 —— 1.62
1.22

Heart 1.56 1.05 L.83

Q1o values as determined between 5° and 15°, and 15° and
37.9 are presented in Table V. The Q,,9 values between 15°
and 37.5° for liver, kidney, and brain in hibernating and active
anunals are in the range of expected values based upon white
rat studies (Fuhrman and Field, 1942; Field et al., 1944; Fuhr-
man and Field, 1945; Fuhrman et al., 1950; Kayser, 1954a;
Weiss, 1954). South (1958) found also that there were no
differences in temperature coefficients of brain from the white rat,
torpid bat, and hibernating and active hamsters. No reports
were found in the literature on temperature coefficients of
metabolism in lunge and spleen. The Qj,, of 2.7 for spleen is
appreciably higher than the values for other tissues. It would be

414 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

of interest to know whether this high Q,,9 in the ground squirrel
is typical also of splenic tissue from non-hibernating mammals.

The Q,,9 values in the 5° to 15° range are based upon only 1 to
) measurements at o° and therefore should be considered as pre-
liminary indications of a higher temperature coefficient at the
lower temperatures in the liver, kidney, lung, and brain of the
hibernating animal. In the active animal the Q,9 appears to be
lower in the liver and spleen, about the same in kidney and
lung, and higher in the brain at this lower temperature interval.
Throughout this group of tissues the temperature coefficients of
the hibernating animals were higher than the active. South
(1958) found in brain a higher temperature coefficient for both
the hibernating and non-hibernating hamster between 5° and 10°,
as was found here for the ground squirrel. However, the active
hamster showed the greater increase, whereas in the ground
squirrel the hibernating animal increased more.

At the higher temperature interval, the active animals showed
very low Q,o values of 1.0 to 1.3 for musele tissue, as might be
expected from their low respiration values at 37.5°. That these
values may be below the true respiration level due to declining
rates during measurement has already been indicated. At 15°
also there is a decline in respiration in the muscle tissues. How-
ever, the relative rate of decline, particularly in the heart, is less
than at 37.5° and thus the Q,, would actually be a little greater
than expressed here. Even using these extrapolated values,
the Q,o for heart is only 1.2. Somewhat higher Q, 9 values of
1.7 (Weiss, 1954) for diaphragm, and 1.5 (Weiss, 1954) and
1.7 (South, 1958) for heart have been found in the white rat.

The respiration of diaphragm and limb muscle of the hiber-
nating ground squirrel gave Q,,_ values of 1.6 (1.8 extrapolated )
which approximate the 1.7 for the white rat and are higher than
the non-hibernating values. The Q,,9 of 1.2 for cardiae muscle is
raised to 1.6 by the use of extrapolated values. This value falls
between the 1.5 found by Weiss and the 1.7 by South for white
rats and is higher than South’s extrapolated value of 1.36 for the
hibernating hamster. The Q,,) for muscle then is about the
same in the hibernating ground squirrel as in the white rat, while
in the non-hibernating animal it is lower. South found the Qy5
of heart from the active hamster to be lower than that of the rat.
Ilowever, the heart Q,o of the hibernating animal was still lower.

RNATION

.
4

MAMMALIAN HIBE

1960

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416 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Discussion

Table VI presents a summary of preceding results in order
that differences and similarities among these groups and these
conditions may be kept clearly in mind. The tissues studied
fell into three categories in regard to metabohe changes under
various conditions,

First, the kidney, spleen, lung, and brain of the ground
squirrel showed respiration levels and Qj, 9 values equivalent
to those of the white rat; moreover, no alterations occurred dur-
ing hibernation except for a possible increased Q,9 at low
temperatures. Accordingly, these tissues appear to be unmodified
in respect to hibernation function.

Second, liver, which differs from all other tissues, shows a
transient increase in metabolism in the hibernating animal at
37.0°, and possibly a sheht increase at 15° throughout hiberna-
tion. In the early hibernation period Zimny and Tyrone (1957)
found a transient elevation of elvcogen levels which then
dropped to non-hibernating levels after 38 days. Thus it appears
that there are metabolic adjustments in the liver during the first
few days of hibernation. The arousing animal (at 15°), the force-
cooled active animal (at 37.5°), and the cold-adapted animal (at
15°) all showed increased liver respiration.

The third group, muscle tissues (heart, diaphragm, and
skeletal muscle), demonstrated metabolic rates in the non-hiber-
nating animal below the expected values for the white rat at
37.5° but not at 15° or 5°. The values for hibernating animals
approached those for the normal rat at all temperatures. Thus
the low Qy,o9 in the active animal increased during hibernation
to the level of the white rat.

The first question which comes to mind in interpreting these
results on tissue respiration is their relationship to the changing
metabolic rate of the intact animal: Can the low metabolic
rate of the animal during hibernation be accounted for in terms
of the temperature coefficients of the constituent tissues? The
data of Tlocek (1951) on metabolism of the little brown bat
(Myotis lucifugus) yield a single Q,9 of 3.7 between 2.0 and
37° when minimum values at each temperature are used. This
uniform temperature coefficient over a wide range suggests that
the intact animal may be treated as a single system in regard to
its response to temperature. The bat lends itself to such analysis
since it may assume a hibernating ‘‘posture’’ at any point over
the whole temperature range. Data over more limited ranges

1960 MAMMALIAN IIIBERNATION 417

are available for other hibernators, but temperature coefficients
may be calculated from the basal metabolic rate and values at
)-10°C. Thus from the data of Kayser (1940) a Q1»9 of 3.7-3.9
may be calculated for the spermophile, S. citellus, and the dor-
mouse, Glis glis. The temperature coefficient of a North American
spermophile, S. widulatus, was 3.55 (Morrison, 1960).

It is apparent that the Q,o’s of all tissues are much lower
than this. The muscle tissues which contribute about one-quarter
of the total metabolism have a Q,9 of 1.3. The overall average
Qio (weighted) then is less than 2.0 in the 15° to 37.5° interval.
The greater temperature coefficient in the 5° to 15° interval,
however, would bring the average Q,, to about 2.8. This is still
considerably lower than the values for the total animal metabol-
ism (by a factor of 3.4 at 5°). Extrinsie controls or influences are
thus necessary for the regulation of tissue metabolism at the
reduced levels observed in hibernating animals.

Upon first approaching the study of hibernation, one tends
to look for specializations in the animal during hibernation since
this is considered the unusual condition. It may be, however,
that the most difficult problems are met during the transitional
periods of entranee into and arousal from the hibernating state.
And the low Q,9 of muscle tissues in the non-hibernating ground
squirrel might be an adaptation to maintain function in the
heart and diaphragm during entrance into hibernation. Thus,
in cooline to 5°, cardiae metabolism only dropped to about 60
per cent of the value at 37°C.

On the other hand, the modification which takes place during
hibernation and which results in a greater temperature coef-
ficient, but in no change in the 15° level, would not influence
hibernation itself. It would, however, be advantageous in faeili-
tating the awakening process during which there is a tremendous
demand for energy necessary for the rapid warming of the
animal. The cardiac and skeletal muscle are considered of major
importance in this rewarming process (Lyman and Chatfield,
1955). However, the Q,9 of metabolism during arousal is still
much higher. Thus, although the Q,, of muscle metabolism in-
creases during hibernation, it is still completely imadequate to
account for the high temperature coefficient of the whole animal
on awakening. Obviously, extrinsic hormonal and/or nervous
regulation are superimposed upon the basal tissue respiration in
order to achieve this maximal heat production. The muscle
metabolism of animals partially aroused appeared to increase

418 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

over the hibernating value at 15°. During arousal from hiberna-
tion the animal may be considered to be under stress, as during
force cooling and cold adaptation. And in each of these cases
the muscle metabolism showed increased respiration rates
although, of course, insufficient to aecount for the rise in the
intact animal metabolism.

Summary

The respiratory rate and Q,9 values of kidney, spleen, lung,
and brain from the thirteen-lined ground squirrel showed no
evidence of adaptation either in the active or in the hibernating
animal, except for a possible decrease at 5° in hibernation. The
liver, on the other hand, demonstrated a transient inerease in
metabolism early in hibernation and an increase in several stress-
ful situations (arousal, force-cooling, cold adaptation). The
muscle tissues all showed reduced temperature coefficients in the
non-hibernating animal. These rose to standard values (white
rat) during hibernation. Moreover, the muscle tissues, and
particularly the heart, showed increased metabolie rates during
the stress situations. Thus, the muscle tissues show metabole
adaptations appropriate to hibernation: a reduced temperature
coefficient in the active animal which might allow suecessful
functioning of the heart upon entering hibernation, and an in-
creased coefficient during hibernation which might facilitate the
awakening process. These intrinsic temperature coefficients do not
in themselves account for the pattern of changing metabolism
in the intact animal, but must interact with extrinsie nervous
or hormonal factors.

REFERENCES

FIELD, J. 2ND, F. A. FUHRMAN AND A. W. MARTIN
1944. Effect of temperature on the oxygen consumption of brain
tissue. J. Neurophysiol., 7:117-126.

KUHRMAN, F. A. AND J. FIELD, 2ND
1942. Influence of temperature on the stimulation of oxygen con
sumption of isolated brain and kidney by 2-4 dinitrophenol.
J. Pharmacol., 75:58-63.
1945, Faetors determining the metabolic rate of excised liver tissue.
Areh. Biochem. Biophysies, 6:337-349.

FUHRMAN, G. J., F. A. FUHRMAN AND J. FIELD, 2ND
1950. Metabolism of rat heart slices, with special reference to effects
of temperature and anoxia. Am. J. Physiol., 163:642-647.

1960 MAMMALIAN HIBERNATION 419

Llock, R. J.
L951. The metabolic rates and body temperatures of bats. Biol. Bull.,
101 :289-299.

Hoon, W. E. AND E, S. G. BARRON
1941. ‘Phe respiration of brown adipose tissue and kidney of the
hibernating and non-hibernating ground squirrel. Am, J. Phy-
siol., 133 :56-63.

KAYSER, C.

1940. Les échanges respiratoires des hibernants. Théses, Univ. Stras-
bourg, 364 pp.

1954a. L’incrément thermique critique de la respiration, in vitro, du
tissu rénal de Rat blane et de Hamster (Cricetus cricetus).
C. R. Acad. Sei. (Paris), 239:514-515.

1954b. L’incrément thermique eritique de la respiration in vitro de tissu
rénal de Hamster ordinaire (Cricetus cricetus) réveillé en
été et en sommeil en hiver. C. R. Acad. Sci. (Paris), 239:554-556.

KREBS, H. A.
1950. Body size and tissue respiration. Biochim, Acta, 4:249-269.

LipicKsEr, W. Z., Jk. AND W. H. DAVIS
1955. Changes in splenie weight associated with hibernation in bats.
Proe. Soc. Exp. Biol. Med., 39:640-642.

LYMAN, C. P. anp P. O. CHATFIELD
1955. Physiology of hibernation in mammals. Physiol. Rey., 35:403-

425.

Mann, F. C. anp D. Drips
1917. The spleen during hibernation. J. Exp. Zool. 23:277-285.

Martin, A. W. AND F. A. FUHRMAN
1955. The relationship between summated tissue respiration and meta-
bole rate in the mouse and dog. Physiol. Zool., 28:18-34.

MorRISON, P. R.
1960. Some interrelations between weight and hibernation function.
(This volume, Pp. 75-91.)

SourH, F. E.
1958. Rates of oxygen consumption and glycolysis of ventricle and
brain slices, obtained from hibernating and non-hibernating
mammals, as a function of temperature. Physiol. Zool., 31:
6-15.
Weiss, A. K.
1954. Adaptation of rats to cold air and effects on tissue oxygen con-
sumption. Am. J. Physiol., 177:201-206.

1957. Tissue responses in the cold exposed rat. Am. J. Physiol., 188:
430-443.

420 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

ZIMNY, M. L. AND V. TYRONE
1057. Carbohydrate metabolism during fasting and hibernation in
the ground squirrel. Am. J. Physiol., 189:297-300,

DISCUSSION FOLLOWING MEYER’S PAPER

ZIMNY commented that in studying liver metabolism one
should not worry about large variations in results. For example,
if one looks at glycogen levels in the liver, one finds this organ
‘will do whatever it can for other organs.’’? Studying the com-
pounds of intermediary metabolism showed that the liver aided
the heart and skeletal musculature, and that this caused great
variation in the levels of compounds in the hver. MEYER
assented,

BULLARD asked whether the metabohe rates taken on indi-
vidual tissues as related to weights for the organs from which
those tissues were obtained equalled in sum the metabolic rate
values for the whole animal. MEYER replied that this had been
done by A. W. Martin and F. A. Fuhrman (Physiol. Zool., 28:18,
1955), who found that the summed respiration rates of the organs
and tissues for the mouse and the dog were reasonably close to
the observed metabohe rate of the intact animal. However, the
level of the metabolic rate of tissues 77 vitro is In part a function
of the system used in measuring it, so much so that the many
factors Involved make it difficult to know the actual rate compar-
able to that of the tissue 7m situ. In tissue studies, the best one
can realize is a comparison of metabolic rates and this compara-
tive picture is what is obtained here.

XXIT

THE INTERNAL ENVIRONMENT
DURING HIBERNATION

By Marvin L. Rtepesen
Department of Occupational Health
Graduate Sehool of Public Health

University of Pittsburgh, Pittsburgh, Penn.

Lowered body temperature and reduced metabolic rate for an
extended period of time are ineluded in the simplest description
of hibernation. Recently, we have learned to expect numerous
biochemieal changes during hibernation. Biological processes
are recognized as being subject to modification by these three
factors: temperature, time and chemical environment. Before
considering the biochemical changes which occur with hiberna-
tion, let us consider briefly some examples of the interdependence
of these factors. The interdependence of time and chemical
environment in biological processes is demonstrated by the
catalytie action of enzymes and ions. The interaction of tempera-
ture and chemical environment is illustrated by Bachrach’s
studies (Bachrach, 1946). He observed a drop of 17°C in the
optimum temperature for contraction of the snail (/Telix aspera
Mull) heart when Kreb’s solution was replaced by isotonic mag-
nesiim chloride. The optimum temperature for contraction in
isotonic potassium and sodium ehloride was intermediate. Other
demonstrations of the interdependency of chemical environment
and temperature were made by Conway and Geoghegan (1955)
and by Adolph and Riehmond (1956). They reported a gain in
weight when tissues were suspended in cold isotonic solutions.
Adolph and Richmond have suggested a redefinition of isotome-
ity in terms of temperature and time. Interdependence of tem-
perature and time is demonstrated by the effect of time upon
the optimum temperature of enzymes and enzyme systems (Spee-
tor, 1956; Dixon and Webb, 1958). The importance of time in
defining tolerance limits of animals to heat and cold is another
example of the interdependence of time and temperature. It
appears that a study of biological processes during hibernation
must consider the interdependence of temperature, time and
chemical environment.

422 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Serum Electrolyte Levels During Hibernation

The relation of the magnesium ion to temperature regulation
has given particular interest to the elevation of serum magnesium
during hibernation. Three types of studies other than hiberna-
tion have related the magnesium ion to heat loss. Parenteral in-
jection of magnesium has been demonstrated to facilitate hypo-
thermia (Schutz, 1916; Heagy and Burton, 1948; Hall et al.,
1951) and elevated serum magnesium has been reported to be
produced with hypothermia in a number of animals (Stead-
man et al., 1943; Platner, 1950; Platner and Hosko, 1953). The
antipyretic action of magnesium has been described in experi-
mental animals and in human beings (Barbour and Winter,
1928; Sollman, 1957).

TABLE I

Reports on Serum Magnesium during Hibernation

Common Scientific Increase over

Name Name Controls INV ESPARALOS
Thirteen-lned Citellus tridecem- 65% Riedesel and Folk (1957 )
ground squirrel lineatus
Woodchuck Marmota monax O37 McBirnie ef al. (1953)
Golden hamster MJesoericetus 25% Riedesel and Folk (1957 )

auratus

Little brown bat JALyotis lucifugus 62% Riedesel and Folk (1957)
Big brown bat Lptesicus serotinus 53% Riedesel (1957)
Iledgehog Erinaceus europacus 92% Suomalainen (1939)
Hedgehog Hrinaceus europaeus none Biorek ef al. (1956)

Klevation of the serum magnesium appears to be a character-
istic of hibernation. Reports of elevated serum magnesium have
been made on a large sample of the hibernators by several inde-
pendent laboratories (Table I). The extent of the increase in
serum magnesium is dependent upon the species of the hiber-
nator. Some of the details of the change in serum magnesium
were determined in studies on the little brown bat. There was no
significant Increase in the magnesium when esophageal temper-
ature had lowered to 17°C, but an elevation of serum magnesium
was observed when the esophageal temperature had dropped to
13°C (Fig. 1). The elevation of serum magnesium appears to

1960 MAMMALIAN HIBERNATION 49:

be dependent upon cooling of cells. Bats raised their body tem-
perature to the semiaetive level of 18°C in four to eight minutes
without altering the hibernation level of serum maenesium (Fig.
2). The magnesium level appears to be independent of tempera-
ture during arousal from hibernation. Then, when the body
temperature was raised further by exposure in a warm room for
one hour, the serum magnesium was back to the basal level of

‘op) “N

BAS

SERUM MAGNESIUM (mg.%)
after 48hrs Hibernation

Fig. J. Serum magnesium values in bats cooled to progressively lower
body temperatures. (Lightest shaded area represents two standard deviations

above and below the mean. )

active bats. The elevation of body temperature without a reduce.
tion of the serum magnesium contraindicates the role of magne-
slum as a causative factor in hibernation. This argument is
weakened by the fact that awakening from hibernation appears
to be a very different process from ‘*‘going into’’ this state.
Reports on serum clectrolytes other than magnesium deseribe
primarily homeostasis of potassium, sodium and calcium during
hibernation. There are some exceptions. Depending upon the

4?4 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

species studied, the serum potassium remains near control levels
or iInereases during hibernation. An 82 per cent increase in the
serum level of potassium during hibernation of the woodehuek
has been reported (MeBirnie ef a/., 1953) (Table IL). A eon-
sistent increase in serum potassium has been observed with
active hamsters, ground squirrels and bats in a cold environ-
ment (Riedesel and Folk, 1958). The cause and effect relation-
ship of the changes in serum potassium are not apparent, but

0) >

wy) W

SERUM MAGNESIUM (mg. ve 100 ml.)
= S

5° 10° 5° 20° 25° 30°
BODY TEMP, °C. (Esophageal) site

Mig, 2. Serum magnesium levels during arousal from hibernation.

the changes in serum potassium during hibernation may be
effected by adrenal cortical activity or utilization of glycogen.
Fenn and Asano (1956) have reported an increase in intracel-
lular potassium with deposition of glycogen. Thus, the increase
of serum potassium may result from the reduction in glycogen
stores which occurs during hibernation (Zimny, 1956). The
values of serum sodium during hibernation of the hedgehog were
similar to those found in active animals (Suomalainen, 1939,
1953; Biorek ef al., 1956). Reports of serum calcium concentra-
tion during hibernation are controversial. No change has been
reported with hibernation of the ground squirrel, hamster and
hedgehog, whereas hypocalcemia has been observed with hiber-
nation of the little brown bat and European marmot. An 88

1960 MAMMALIAN HIBERNATION 42%

per cent inerease in the serum ecaleium was reported with hiber-
nation of the European marmot (Ferdmann and Feinsehmidt,

1932). A more detailed study of the little brown bat has given

rise to interesting speculation. The data in Figure 3 suggest that

the serum levels observed during hibernation may vary with
the body temperature and/or depth of hibernation. The lower-
ing of serum ealeium with reduced body temperature does not
appear to be large, but it is consistent. The lowering of serum

TABLE II

Reports on Serum Electrolytes during Hibernation

Hlectrolyte Concentrations

Animal as Compared to Controls Investigator
Potassium Calcium Sodium
W oodehuek increased McBirnie et al. (1953)
Kuropean marmot decreased Adler (1926)
decreased increased Ferdmann and

Feinsechmidt (1932 )

Thirteen-lined no change no change Riedesel and Folk (1958 )

ground squirrel

Hamster nochange no change Riedesel and Volk (1958

Little brown bat = increased* — decreased* * Riedesel and Folk (1958

Big brown bat no change Riedesel and Folk (1958

Hedgehog decreased nochange nochange Suomalainen

(1939) (1953

nochange uochange wuochange — Bidrck et al. (1956)

* The increase was consistent but not statistically significant.

** The serum calcium appears to vary with depth of hibernation.

calcium with hibernation of the little brown bat receives further
support from the observation of a 54 per cent reduction in
serum calcium in bats which had hibernated for ten weeks and a
34 per cent decrease after nine weeks of hibernation (Riedesel,
1957). The low and apparently fluctuating calcium levels with
extended hibernation cannot be explained on the basis of the data
available. The calcium level may cycle during long-term hiber-
nation, and the data cited here may have been taken at times
when the calcium level happened to be at a low ebb of the eyele.
Such a eyele may account for the contradictory reports on the
caleium content of the serum during hibernation which appear

426 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

in Table I]. It may be that during hibernation the cireulation
to caleium stores and/or kidney funetion is not consistently ade-
quate to meet the tissue demands for calcium, and at the same
time maintain a constant serum ealeium level.

CALCIUM (Mg %)
Bey
Sm OS | AR GD a

ACTIVE COOLED COOLED WARMED WARMED ACTIVE
4 Days TO TO TO TO | HR.
17-20°C 11-13°C i3°C fa°c
STAGES OF HIBERNATION

Fig. 3. Serum caleium measurements of Myotis lucifugus in stages of
hibernation. (Shaded area represents one standard deviation above and
below the mean. )

Body Fluids

Kew measurements of body fluid have been made during
hibernation. The susceptibility of the hibernating animal to
manipulation limits the success of such measurements. Small de-
creases in plasma volume are indicated by blood volume and
total serum protein measurements. The increase in blood vol-
ume/body weight during hibernation of the hamster was largely
explained by loss of body weight (Lyman et al., 1957). An in-
crease In serum concentration is indicated by the 21 per cent
increase in total serum proteins during hibernation of the ham-
ster (South and Jeffay, 1958). Actually, information regarding
blood and plasma volume is limited to hematological observa-
tions. The hematological studies show inconsistent results (Table
I11). The data on the golden hamster and European marmot
indicate concentration of the blood, if not the plasma. Cyclic

1960 MAMMALIAN HIBERNATION 427

shifts of body water may account for the controversial hema-
tologieal data. The electrolyte data cited earlier indicate homeo-
stasis with regard to body fluid compartments since magnesium,
potassium and ealcium concentrations change independently of
each other; for instance, if the elevation of serum maenesium
during hibernation were due to a decrease in plasma water, one
would expect similar increases in serum potassium, calcium
and sodium values. The urine of hibernating ground squirrels
has been reported to be of very small volume and dilution

Tasue [IT

Indices of Hemoconeentration during Hibernation

Change with Hemo-

Animal

Criteria

‘Thirteen-

Erythroeyte count

Hibernation

decreased

concentration

Investigator

Stuckey and Coco (1942)

lined Erythrocyte count increased +- Svihla and Bowman (1953)
ground Hematocrit sl. increase + Svihla and Bowman (1953)
squirrel Hematocrit no change 0 Riedesel (1957)
Serum sp. gr. no change 0 Riedesel (1957)
Woodehuck Erythrocyte count decreased = Rasmussen (1916)
Hematoerit increased + MeBirnie et al. (1953
Hemoglobin no change 0 Rasmussen (1916)
Blood sp. gr. no change 0 Rasmussen (1916)
European Blood sp. gr increased + Dubois (1896)
marmot Erythroeyte count increased + Dubois (1896)
Water content of decreased + Aeby (1875)
blood Dubois (1896)
Hamster Erythroeyte count increased -f- Lyman et al. (1957)
Hematocrit increased + Lyman et al. (1957 )
Hematocrit increased -f- Riedesel (1957)
Hematocrit increased ++ South and Jeffay (1958)
Serum sp. g no change 0 Riedesel (1957)
Total Serum increased + South and Jeffay (1958)
proteins
Little brown Hematocrit no change 0 Riedesel (1957)
bat Serum sp. gr. no change 0 Riedesel (1957)
Big brown Hematocrit no change 0 Riedesel (1957)
bat Scrum sp. gr. increased -b Riedesel (1957)
Hedgehog Hematocrit increased 4. Bidrek et al. (1956)
Total serum no change 0 Biodrek et al. (1956)

proteins

428 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

(Hong, 1958). Additional information on kidney funetion would
facihitate an understanding of water balance during hiberna-
tion. No major shifts in body water during hibernation are ap-
parent from existing data.

Other Changes in the Internal Environment

The subjects of pH, pOs and pCO. otf the blood, energy
sources, blood sugar and endoerines have been reviewed recently
by Lyman and Chatfield (1955) and Kayser (1957). These
data accumulated on hamsters, woodchueks and ground squirrels
indicate that hibernating animals maintain oxygenation of the
blood, pH and pCO. near the values of active animals. Data
on the respiratory quotient and blood elucose levels indicate
that fat is the principal source of energy during hibernation.
The role of the endoerines as a causal factor in hibernation is
questionable, but changes in endocrine activity may effect a
pre-conditioning of the animal for entrance into hibernation. The
facility of some animals to hibernate in the laboratory the year
round contradicts the supposition that endocrines have a causa-
tive role in hibernation (Riedesel, 1957).

Theories on the Development of Hibernation

The elevation of serum magnesium and the relation of the
magnesium ion to heat loss in other types of studies suggest
magnesium as a causal factor in the development of hibernation.
We have presented several theories on the development of the
state of hibernation with reference to magnesium (Riedesel, 1957).

The ‘‘Independent-Influence Theory’? imphes that exposure
to a cold environment independently alters the activity of the
hypothalamus and also produces elevated sertum magnesiun.
The serum magnesium level has no causative role in the decrease
in body temperature. This theory receives support from the
evidence that elevation of magnesium and lowered body tempera-
ture are separable. This was observed during arousal from
hibernation when bats raised their body temperature to 20°C
without lowering the serum magnesium.

The ‘‘Direct-Influence’’? theory simply means that cold ex
posure and the resultine cooling of cells directly produee a re-
lease of magnesium by the cells, and the resulting elevated level
of serum magnesium influences the hypothalamus to increase
heat loss. This theory receives support from two observations.

1960 MAMMALIAN HIBERNATION 429

The first is that a high level of magnesium was observed before
the temperatures of deep hibernation were reached. Thus, the
elevated magnesium observed with 13°C esophageal temperature
may facilitate the development of the lower temperatures. The
second line of evidence in support of this theory is that intra-
venous and micro-injection of magnesium ion have been demon-
strated to facilitate heat loss (Schutz, 1916; Heagy and Burton,
1948 ; Hall et al., 1951).

The ‘‘ Additive-Influence’’ theory assumes that exposure to the
cold and elevated serum magnesium both influence the activity
of the heat loss center and have an additive effect. The theory
imphes that cooling of tissues, endocrine and cerebral cortical
activity and other factors initiate the lowering of body tempera-
ture and that the elevated serum magnesium facilitates the
process by affecting the heat loss center. This theory receives
support from the observation that the serum magnesium had not
increased when the esophageal temperature had dropped to 17°C.
This evidence indicates that a factor other than elevated mae-
nesium is required for initiating the decrease in body tempera-
ture.

The author is of the opinion that the ‘‘ Additive-Influence’’
theory is the most acceptable in view of the evidence cited. Ele-
vation of the esophageal temperature to 20°C during the arousal
from hibernation without reduction of the serum magnesium
level represents the major evidence that contradicts this theory.
This argument is weakened by the facet that awakening from
hibernation appears to be a very different process from ‘‘ going
into’’ this state. Arousal from hibernation can be initiated by
discrete external stimul, such as handling. Internal stimuli from
thirst, hunger or a full bladder are also undoubtedly involved
in arousal from hibernation. No such stimuli have been related
to the development of hibernation. Arousal from hibernation
must be the result of neural stimulation of the hypothalamus and
this stimulation must be strone enough to overcome the influence
of magnesium on the heat loss center.

To restate the ‘‘ Additive-Influence’’ theory, the development
of hibernation may proceed in the following manner: Cold
exposure and reduced activity produce a cooling of peripheral
tissues, and a release of magnesium from cells to plasma occurs.
This may start when the animal is asleep. The increase in serum
magnesium affects the heat loss center so that body temperature
and metabolism of the animals drop to hibernation levels.

430 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Questions and Suggestions for Future Research

Questions and suggestions to be considered when planning
research on hibernation include the following :

1. More information is needed regarding the interdependence
of temperature, time and chemical environment in_ biological
processes,

2. The chemical environment and temperature of im vitro
studies should be similar to those described in vivo.

3. Are changes in intracellular electrolyte concentrations a
universal response of cells to cooling?

4. Additional information is needed on water balance during
hibernation; for instance, ‘‘What is the extent of the kidney
activity during hibernation ?”’

5. Additional information is needed regarding the possible
eyelie changes in serum calcium concentration during hiberna-
tion.

6. We need a better definition of hibernation in terms of
species differences and stages of hibernation.

REFERENCES
ADLER, L.
1926. Der Winterschlaf. Handbuch der normalen und pathologischen
Physiologie, 17:105-155,

Axrsy, C.
1875. Ueber den Einfluss des Winterschlafes auf die Zusammensetzung
der verscheidenen Organe des Thierkérpers. Arch. exp. Pathol.
Pharmakol., 3:180-184.

ApoupH, E. F. AaNp J. RICHMOND
1956. Water exchanges of isolated mammalian tissues at low tempera
tures. Am. J. Physiol., 187:487-444.

>\CHRACH, EE.
1946. Facteurs chimiques biothermiques. Arch. Internat, Physiol.,
54:19-29.

Barpour, H. G. AND J. E. WINTER
1928. Antipyretic action and toxicity of combination of Mg with
phenyl cinchoninic acid. J. Pharmacol. Exp, Therapeutics,
35 :425-439.

BiOrck, G., B. JOHANSSON AND S. VEIGE
1956. Some laboratory data on hedgehogs, hibernating and non-hiber-
nating. Acta physiol. scand., 37:281-294.

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CoNWAY, J. E. AND EL. GEOGHEGAN
1955. Molecular concentration of kidney cortex slices. J. Physiol.,
130:4388-445.

Dixon, M. AND E. C. WEBB
1958. Enzymes. New York, 782 pp. (Pp. 150-170).

Dusois, R.
1896. Physiologie comparée de la marmotte. Ann, Univ. Lyon. Paris,
268 pp. (Pp. 82-105).

FENN, W. O. AND T. ASANO
1956. Effects of carbon dioxide inhalation on potassium liberation
from the liver. Am. J. Physiol., 185:567-576.

FERDMANN, D. AND O, FEINSCHMIDT
1932. Der winterschlaf. Ergebn. Biol., 8:1-75.

Hau, V. E., R. GRANT AND W. J. WHALEN
1951. The influence of Mg and pyrogens on temperature regulation.
AF Tech. Report No. 6682 (Wright Air Development Center).

Heaey, F. C. anp A. C. BURTON
1948, Effect of IV injection of MgClo on the body temperature of the
unanesthetized dog, with some observations on Mg levels and
body temperature in man, Am. J. Physiol., 152:407-416,

Honea, 8S. K.
1958. Renal function during hypothermia and hibernation, Am, J.
Physiol., 188:137-150.

KAYSER, C.
1957. Le sommeil hivernal probleme de thermorégulation, Rev. Canad.
Biol., 16:303-389.

LyMAN, C. P. AnD P. O. CHATFIELD
1955. Physiology of hibernation in mammals, Physiol. Rev., 35:403-

425,

LyMAN, ‘C. P., L. P. Weiss, R. C. O'BRIEN AND A. A. BARBEAU
1957. The effect of hibernation on the replacement of blood in the
golden hamster. J. Exp. Zool., 136:471-486.

McBirniz£, J. E., F. G. Pearson, G. A. Truster, H. H. KarRAcHI AND
W. G. BIGELOW
1953. Physiological studies of the groundhog (Marmota monaz).
Canad, J. Med. Sci., 31:421-430.

PLATNER, W. S.
1950. Effects of low temperature on Mg content of blood, body fluids
and tissues of goldfish and turtle. Am. J. Physiol., 161:399-405.

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PLATNER, W.S. and M. J. Hosko
1958. Mobility of serum magnesium in hypothermia, Am, J. Physiol.,
174:273-276.

RASMUSSEN, A. T.
1916.) The corpuscles, hemoglobin content and specific gravity of the
blood during hibernation in the woodchuck (Marmota monax),
Am. J. Physiol., 41:464-482,

RIEDESEL, M. L.
1957. Serum magnesium levels in’ mammalian hibernation, Trans.
IXansas Acad. Sei., 60:99-141.

RIEDESEL, M .L. AND G. E. FOLK, JR.
1957. Serum magnesium changes in cold-exposed mammals. J. Mam-
mal., 38:4238-424,
1958. Serum electrolyte levels in hibernating mammals, Amer, Nat.,
92:307-312.

ScuHvutz, J.
1916. Zur Kenntnis der Wirkung des Magnesium auf die WKorper-
temperatur. Arch. exp. Pathol. Pharmakol., 79:285-290.

SOLLMAN, T.
1957. A manual of pharmacology. Philadelphia, 1535 pp. (Pp. 1057-
1062).

Souru, F. E. Np H. JEPFAY
1958. Alterations in serum proteins of hibernating hamsters. Proc.
Soe. Exp. Biol. Med., 98:885-887.

Specror, W.S8.
1956. Handbook of biological data. Philadelphia and London, 584 pp.
(Pp. 28-29).

SrEADMAN, L. T., I. ARIEL AND S. L. WARREN
1943. Studies on the effect of hypothermia. IV. The rise of serum Mg
in rabbits during the hypothermic states as shown by the spectro-
chemical method. Cancer Res., 3:471-474.

SrTuckEyY, J. AND R. M. Coco
1942. A comparison of the blood pictures of active and hibernating
ground squirrels. Am. J. Physiol., 187:481-435,

SUOMALAINEN, P.
1939. Hibernation of the hedgehog VI. Serum Mg and Ca. Artificial
hibernation. Also a contribution to chemical physiology of
diurnal sleep. Ann, Acad. Sei. Fenn. (A) 53(7) 21-71.
1953. Hibernation of the hedgehog. Proc. Finn, Acad. Sci, Letters,
63:131-144.

1960 MAMMALIAN HIBERNATION 433

Sv1lH.LaA, A. AND H. C. BOWMAN
1955. Stimuli and their effects on awakening of dormant ground
squirrels. Am. J. Physiol., 172:681-683.

ZIMNY, M.
1956. Metabolism of some carbohydrate and phosphate compounds
during hibernation in the ground squirrel. J. Cell. Comp.

Physiol., 48:371-392.

DISCUSSION FOLLOWING RIEDESEL’S PAPER

WIMSAT'T observed that RIEDESEL’S serum measurements
could reflect discontinuity in hibernation, and might also provide
evidence for arousal. If so, one may be able to manipulate mech-
anisms in such a way as to eliminate arousal. He also gave fur-
ther evidence for eyeling of hibernation. In visitine bat caves
every two weeks during three winters he observed that the
clusters of bats moved, and bandine studies showed movements
of animals between caves in the middle of winter. Even in an
artificial hibernaculum there are times when bats are active in
the hibernating season. RIE DESEL thought this was true, and
he felt that the evele may partly reflect temperature changes.
With respect to movements of bats in caves, FOLK (G. KE. Folk,
J. Mammal., 21 :3806, 1940) was cited as having reported changes
in positions of bats in winter, quite a bit of movement being
characteristic.”

ADOLPH then asked if SUOMALAINEN would give his pres-
ent view of the role of magnesium in hibernation. SOUTH also
asked if SUOMALAINEN held to his old viewpoint. SUOMA-
LAINEN remarked that it is a difficult question, but that he was
sure magnesium is a typical indicator of the hibernating state,
whether in a primary or secondary role he could not say.

RILEDESEL remarked that the data cannot be interpreted
readily to mean that magnesium is a factor in initiating hiberna-
tion, and that he did not intend to leave the impression that
magnesium initiates hibernation.

POPOVIC stated that he felt bats were not well-chosen ani-
mals for this work since they differ considerably from ground
squirrels and other hibernators. A hibernating ground squirrel
and an artificially cooled ground squirrel would probably show
a bie physiological difference in respect to magnesium, in con-
trast to hats. Other striking contrasts are seen; for example, the

434 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

blood pressure is low in hibernation, whereas in the artificially
cooled animal, it is 90 mm Hg. Heat production is 5 to 15 times
lugher in cooled than in hibernating animals. He believed that
further experiments should be performed to see if the changes
in magnesium should be attributed to hypothermia or to hiberna-
tion.

RIEDESEL rephed that he had ‘‘heard rumblings as_ to
whether the bat is a good example of a hibernator.’’ He de-
fended the bat as a hibernator, saying that he had spent 10-11
hours in a cold room waiting for bats to go into hibernation to
make study possible. He checked each one individually every
15 minutes. They remained relatively active and kept their body
temperature up. He checked body temperature until it lowered
to 20°C, then drew blood and analyzed it. Sometimes body
temperature rose 7-8°C in 15 minutes, but half an hour later
it would be down again. Concerning changes in body tempera-
ture and magnesium, he believes that on the cellular level hypo-
thermia and hibernation are quite similar. With respect to the
whole ground squirrel put into hypothermia, he felt that dif-
ferences would exist between this condition and hibernation
at system, organ or tissue levels, but not at the cellular level.

SCHONBAUM said that an increase in magnesium in the
hibernator’s blood had to come from somewhere ; did RIEDESEL
have any idea as to which part of the organism gave up mag-
nesium first — body tissues generally or specific organs, such as
muscle? He also wondered what happens to the magnesium
which has entered the serum. RIEDESEL said that he had no
evidence on this, but his impression was that temperature affects
the extracellular :intracellular magnesium ratio which at 37°C
is 20:1. As peripheral cells (skin and muscle) are cooled, this
ratio becomes smaller and the serum magnesium level increases.
Ile believes that the first release of magnesium into the serum
oeeurs in the peripheral tissues where the temperature is lower.
In nature, bats in the temperate zone go in and out of hiberna-
tion at least once a day in the spring and fall, and since there is
no evidence of magnesium deficiency, it is doubtful that extra-
ordinary amounts of magnesium are excreted in the urine. He
believes magnesium does not leave the body during hibernation,
but returns from plasma to cells on arousal from hibernation.

KAYSER then stated that he believes there is a clear relation-
ship between magnesium levels and hypothermia. One sees an

1960 MAMMALIAN HIBERNATION 435

increase of serum magnesium in hibernation and in hypothermia.
IIe believes magnesium increase in the serum is related to (is
perhaps the consequence of) hypothermia. He said he was not
convinced that magnesium had a causal effect on hibernation,
but rather believed that elevated serum magnesium was a con-
sequence of cold.

FOLK remarked that he felt the discussion was difficult be-
cause there was not time enough to go into methods. Relative
to WIMSATT’S and POPOVIC’S comments, he said it should
be emphasized that measurements were made after sacrificing
animals in spontaneous hibernation.

BARTHOLOMEW, extending KAYSER’S remarks, pointed
out that hibernation-hypothermia discussions are impeded by
the chronic problem of causation and correlation. He would like
to separate hypothermia and hibernation. By experience with a
variety of forms, he had noted that hypothermia, per se, is not
an essential causal factor for hibernation. He felt that peri-
pheral cooling is a result rather than a cause. He asked that we
release ourselves from dependency on the idea that cold exposure
is a necessary prior condition to entering hibernation.

RIEDESEL stated that it is difficult to designate magnesium
as a causative factor in initiating hibernation. However, it ap-
pears logical that the elevated serum magnesium (by its effect on
the heat loss center) facilitates loss of body heat and the redue-
tion of body temperature to levels characteristic of deep hiberna-
tion.

XXITI
A STUDY OF THE METABOLISM OF
LIVER, DIAPHRAGM AND KIDNEY IN
COLD-EX POSED AND HIBERNATING
HAMSTERS’

By Arutss DeENYrES and JOAN HAsserr
Biology Department
(Queen’s University
Kingston, Ontario, Canada

Of the many problems in hibernation two basie ones ean be
broadly defined: 1) the elucidation of the mechanisms for en-
trance into and arousal from hibernation, and 2) the nature of
the intermediary metabolism of the tissues of hibernators when
the animals are exposed to varying ¢limatie conditions, when
entering hibernation, in hibernation and when arousing. In
regard to the second problem we are fortunate in having a wealth
of hterature on the intermediary metabolism of the rat for
comparison.

These two problems are related but in terms of an approach
to research in hibernation they should not be confused. Even-
tually, the knowledge of the activities of various organ systems
and the whole animal will be integrated with the facts of inter-
mediary metabolism of cells and the transport state of the cir-
culatory system. At present, in hibernation research, the aetivi-
ties of the systems and the whole animal are far better known
than the intermediary metabolism of the various tissues. It is
important to bring the knowledge of the cellular metabolhsm of
hibernating animals, both in and out of hibernation, to the level
where such integration in the total economy of the animal is
permissible.

We have used the golden hamster, Mesocricetus auratus, for
this study of tissue metabolism. In recent years it has been
recognized that there is not a single type of hibernation but a
spectrum of hibernating types among the mammals. Of these the
volden hamster has its own pecuhar pattern which differs from
that of certain other hibernating mammals such as the wood-
echuek and ground squirrel. Some of these differences are the

1'This research has been supported by the National Science Foundation of the

United States, the Ontario Research Foundation and the National Research
Council of Canada.

438 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

prolonged sojourn in the eold (8-12 weeks) preceding hiberna-
tion for the hamster, and frequent arousals at which time food
intake is necessary for survival if hibernation is entered again
(Lyman and Ledue, 1953).

Our interest in the golden hamster is largely confined to the
investigation of tissue metabolism and the possibilities of meta-
bolic adaptations in cells. The lone pre-hibernation period of eold
exposure provides an opportunity to study the animal through
many weeks of adaptation to cold stress. In a study of the
adaptations of intermediary metabolism to cold stress and hiber-
nation in the hamster we expect to find only an enhanced or
diminished utilization of components and pathways, or alterna-
tive pathways already available to rodents in general. The
oxygen consumption and response to substrates of tissues from
cold-exposed and hibernating mammals is a good foundation for
more detailed inquiry into intermediary metabolism by enzyme
and substrate assay, radioisotope technique and cell particulate
studies.

The Metabolism of Liver Slices from Non-Fasting Hamsters
Exposed to Cold for Twelve Weeks and in Hibernation

Seven groups of male hamsters were studied in regard to body
weight, liver oxygen consumption, and the response of liver
to 2 x 10°2 M. succinate (Table 1). The control group was kept
at room temperature while the cold-exposed and hibernating
eroups lived in a cold room at 5 =1°C. The animals were selected
so that they would be approximately 6 months old when used.
Each animal was weighed before use and killed by a sharp
blow on the head. The liver and diaphragm were removed im-
mediately and placed on moistened filter paper in a chamber
over ice. Liver shces were cut with a Stadie-Riggs hand micro-
tome to a thickness of 0.5 mm and kept cool in a moist chamber
over ice until placed in the Warburg bath. The diaphragm was
simply cut into small rectangles and treated in the same way
as the liver shees. Each Warburg vessel contained 40-50 mg of
tissue ina Krebs-Ringer phosphate buffer with the CaCly reduced
to three parts of a .055 M. solution. The oxygen consumption
is expressed as QOz (microliters of Oz per milligram of initial
dry weight per hour at 37°C). Substrate was added from a
sidearm after 30 minutes of endogenous respiration. For the
determination of dry weight, slices comparable to those used for

439

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440 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

the determination of oxygen consumption were dried to a eon-
stant weight at 99°C. Durine the weeks of cold exposure there
was a progressive loss in body weight. By the end of the first
week, 21 per cent of the imitial body weight was lost. Between
the ninth week and hibernation, a weight loss of 17 per cent
occurred. By the time the animals entered hibernation they had
lost 40 per cent of their initial body weight.

There is no. statistically significant difference in the dry
weight of the liver tissue throughout the weeks of cold ex-
posure and in hibernation (Table I). At the end of one week
at 59°C the endogenous respiration of the liver had inereased
21 per cent over that of the animals kept at room temperature.
The oxygen consumption increased until the ninth week and
then dropped to the level found in early cold exposure. Shortly
after this the animals hibernated. The oxygen consumption of
liver shees taken from hibernating animals that were allowed
to ‘Sarouse”’ at 37°C is not significantly greater than that found
in slices from animals exposed to cold for the first three weeks.
This means that the pattern of metabolism in ‘‘arousing’’ liver
slices is not one which produces an excess of oxidative processes
beyond those already established in the cold-exposed animal. On
exposure to cold, therefore, the oxidative metabolism of the liver
increases 21 to 25 per cent over that of the control animals
and no further imerease is achieved during ‘‘arousal’’ of the
tissue.

At the sixth and ninth weeks the endogenous respiration was
highest, and at this time the addition of sueeinate had the least
effect. The response of liver slices from control animals to the
addition of succinate was similar. Slices from one-week cold-
exposed animals and from hibernating animals gave a greater
response to succinate, namely a 71 and 77 per cent increase. You
and Sellers (1951) found an increase in liver suceinoxidase in
rats exposed to cold for several weeks while Campbell ef. al.
(1958) found a similar increase in coenzyme A content of the
liver of rats exposed to cold. The coenzyme A increase occurred
Within one day and persisted for 24 days of cold exposure. It is
possible that there is an increase of these factors in the liver
of hamsters exposed to cold. This, however, would not answer
the entire problem of the response of liver to succinate since it is
difficult to believe that these factors would increase initially, fall
at twelve weeks of cold exposure and then increase again during
hibernation. It is more hkely that the enzyme and coenzyme
content of the hamster liver remains high during cold exposure,

1960 MAMMALIAN HIBERNATION 44]

and the liver from one-week cold-exposed animals and hibernat-
ing animals is low in Krebs’ eycle substrate. An inerease in
enzyme and coenzyme content of the tissues of ecold-exposed
hamsters should not be unexpected in heht of the great inerease
in food consumption, as shown by Farrand and Folk (1957).
The discussion by Potter (1958) on the nature of feedback
mechanisms in enzyme adaptation describes the general effect of
the inerease of metabolites on the sequenee of DNA to RNA
to enzyme.

The Effects of Fasting on the Metabolism of Liver from
Cold-Exposed Hamsters

In order to demonstrate more clearly alterations in the metabo-
lism of liver from hamsters exposed to cold, an experiment based
on a comparison of fastine and non-fastine was designed. A
eroup of twelve male hamsters was kept at room temperature.
Half of these were fasted for 24 hours before use but not denied
water. Another group of twelve males was kept in the cold
room (5 =1°C) for three weeks. Half of these were fasted for
24 hours in the cold but not denied water. The effeets of fasting,
in these two groups, on the endogeneous metabolism of liver and
its response to 2 x 10-2 M. suecinate and 200 me per cent gli-
cose are presented in Table Il. The data for the six hibernating
hamsters were taken from the previous experiment. The tissue
was treated as described in the previous section and substrates
were added after 30 minutes of endogenous respiration.

There was no significant difference in the amount of dry
material present or in the endogenous respiration of the liver
from fasted and non-fasted control animals. In other words, 24
hours of fasting at room temperature does not affect the level
of oxidative metabolism. The liver from both the fasting and
non-fasting control animals responded to succinate with increases
of 78 and 95 per cent oxvgen consumption. Fasting, therefore,
affected the tissue response to succinate to a slight extent. The
addition of 200 mg per cent glucose to liver from fasted control
animals had no effect on oxidative metabolism. These data sug-
gest that liver from fasted control animals had some adaptation
toward the greater use of a Krebs’ eyele intermediate.

The endogenous respiration of liver from fasted, cold-exposed
animals is significantly lower (P<.01) than that of the non-
fasting cold-exposed animals. The oxygen consumption of the
liver from these fasted animals had fallen to that of the control

Vol. 124

MUSEUM OF COMPARATIVE ZOOLOGY

BULLETIN :

442

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1960 MAMMALIAN HIBERNATION 443

animals. The addition of glucose did not repair the drop in
metabolism. The liver from these fasted, cold-exposed animals
gave the greatest response to succinate (118 per cent) but none
to glucose. These data show that both fasting at room tempera-
ture or fasting after three weeks in the cold room reduced the
level of Krebs’ eyele activity.

The endogenous respiration of liver from hibernating ham-
sters is similar to that from the non-fasting, cold-exposed ani-
mals. The response to succinate, however, is greater and this
suggests a metabolic state intermediate between the non-fasted
and fasted, cold-exposed animals. The animals used in this study
had been in hibernation for only two or three days and were not
very experienced performers. An experiment with one animal
that had been in and out of hibernation regularly for a month
gave different results. In this ease the endogenous respiration
of the liver was lower than that of the inexperienced animals
and the response of the liver to succinate (128 per cent) was
similar to that of a fasted, cold-exposed animal.

The Metabolism of Diaphragm from Non-Fasted Hamsters
Exposed to Cold for Twelve Weeks and in Hibernation

The effect of sojourn in the cold (5 =1°C) on the respiration of
diaphragm from non-fasting animals for a period of twelve weeks
and in hibernation are presented in Table I. These are the same
animals on which the liver work was done and therefore these
tissues can be compared directly. The diaphragm was eut into
small rectangles and treated in the same way as the liver slices.

There was no statistically significant difference in the per-
centage of dry material of the diaphragm throughout the weeks
of cold exposure. Respiration had increased 35 per cent (as com-
pared to 21 per cent for liver) above that of the control animals
after one week in the cold and was at this higher level at the
third week. At the sixth week the respiration fell 12 per cent
below that of the third week. Most of the animals in our colony
become sleepy after six weeks in the cold and a few hibernate.
The drop in respiration may be related to this phenomenon.
This was followed by a marked increase in oxygen consumption
of diaphragm at the ninth week. By the twelfth week respiration
fell below that of the ninth week. After the twelfth week animals
in the colony began to hibernate.

Diaphragm taken from hibernating animals and allowed to
‘arouse’? at 37°C had an oxygen consumption 8 per cent above

4.44 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

the twelfth week prehibernation value, 17 per cent above tissue
from animals exposed to cold for one week and 60 per cent above
the value for control animals. Diaphragm, therefore, is eapable
of an impressive increase in oxygen consumption as the animal
is taken from room temperature to an environment of 5°C. Con-
sidering its high level of oxygen consumption in the chronie
cold-exposed animal it is remarkable that the diaphragm is eap-
able of a further 8 per cent increase when ‘‘arousing.’’

The Effects of Fasting on the Metabolism of Diaphragm trom
Cold-Exposed Hamsters

The diaphragm was taken from the same animals used in
the fasting experiment with liver, and the data are given in
Table III. Fasting did not alter the endogenous metabolism of
diaphragm from animals living at room temperature nor did
the addition of glucose have any effect on oxygen consumption.
Diaphragm taken from fasted or non-fasted cold-exposed animals
had the same initial oxygen consumption. The oxygen consump-
tion of the diaphragm from fasted animals began to fall off at
60 minutes, and by 90 minutes was significantly lower than that
from the non-fasted animals. This fall in oxygen consumption
was repaired by the addition of glucose but not increased beyond
that of the non-fasted, cold-exposed animals.

In the intact mammal there is an intimate metabohe relation-
ship between liver and musele. «A comparison of the respiration
of liver and diaphragm from the fasted, cold-stressed animals
suggests that the liver has supplied the diaphragm with glucose
at the expense of its own oxidative metabolism. These results
support the conclusions reached by Lyman and Ledue (1953)
who used direct analyses for glycogen in liver, muscle and heart,
and glucose in blood.

The Metabolism of Kidney Cortex from Cold-Exposed and
Hibernating Hamsters

The endogenous oxygen consumption and the response of kid-
ney cortex to citrate (7 x 10°? M.), pyruvate (7 x 10-8 M.) and
succinate (1 x 10-2 M.) was determined for four groups of ham-
sters (Tables IV, V). The control animals were kept at room tem-
perature and the groups in the cold (5 £1°C) were studied at
three days and twelve weeks of cold exposure, and in hibernation.
xcept for the hibernating animals, all were fasted for 24 hours
before use. The animals were killed by a sharp blow on the head,
the kidneys removed and deeapsullated and demedullated in a

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446 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

moist chamber over ice. Cortical shees were cut to 0.4 mm thick-
ness by hand and treated in the same way as the liver and dia-
phragm. The medium was a modified Krebs-Ringer solution at
pl 7.3 with Tris buifer in a final concentration of .038 M. Compar-
able tissue slices were used to determine the dry weight and the
nitrogen content. The tissue was dried to a constant weight at
99°C. Substrates were added from sidearms after 30 minutes
of endogenous respiration.

Blood for ion analysis was withdrawn by cardiae puncture
and allowed to clot, and bladder urine was withdrawn by hypo-
dermic at the same time. The concentrations of sodium and
potassium in blood and urine were determined with a Coleman
flame photometer and expressed as milli-equivalents per liter
(Table VI).

The endogenous oxygen consumption of kidney cortex from
control animals is approximately 214 times greater than that of
diaphragm, and 3 times that of liver tissue (Fig. 1). During
cold exposure, the difference between kidney cortex and dia-
phragm is shehtly reduced but this difference is maintained be-
tween the kidney cortex and liver tissue. After three days in
the cold room, the kidney cortex had an endogenous oxygen con-
sumption significantly higher (P<.01) than that of the control,
the twelve week cold-exposed, or hibernating animals. By three
days of cold exposure the dry weight of the cortex was ereater,
the total wet weight of the kidney had increased 25 per cent and
the animals had lost 16 per cent of their initial body weight.
Associated with these changes was a 17 per cent increase in 12-
hour urine volume and an increase in the output of potassium.
There was no change in serum sodium or potassium nor in any
ratio between these two ions except the urinary sodium to potas-
sium ratio which fell from 0.9 to 0.6. The ereatly increased
water load had forced the kidneys to work harder in order to
maintain the blood ion balance. The excess loss of potassium
would be the result of passive removal with the water (Smith,
1956). Hastings ef al. (1952) working with rat liver found that
a potassium-rich medium facilitated the conversion of elucose-b-
PO, to glycogen while a soditun-rich medium was inhibitory to
this reaction. If potassium lost from the blood were beimg re-
placed by a movement of potassium out of tissues with an inward
movement of sodium this would provide a stimulus for glyco-
genolysis. This point should be specifically investigated since
Masoro et al. (1954) found that one day of cold exposure dras-
tically reduced the glycogen level of non-fasted rats.

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448 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

sy twelve weeks of cold exposure the oxygen consumption of
kidney cortex was only slightly higher than the control level.
The animals had lost 26 per cent of their initial body weight
and the wet weight of the kidney had increased 39 per cent over
that of the control animals. The dry weight of the tissue had
returned to control value. After three weeks in the cold the
sodium and potassium ratios were similar to those of the control
animals but the urine volume remained high. The twelve-week
animals may have had a similar ion pattern but we have no data
for them.

The QO» of kidney cortex taken from hibernating animals and
measured ina Warburg at 8°C, which was the internal body tem-
perature of our hibernating animals, was 1.88 =.05 (Table VII).
This is 9 per cent of the oxygen consumption obtained from
kidney cortex taken from fasted, twelve-week, cold-exposed ani-
mals and measured at a temperature of 386°C. During hiberna-
tion the urinary soditm to serum sodium ratio was double that
of the non-fasted, cold-exposed animals. The sodium content of
the serum had fallen to the fasted control level and was high
in the bladder urine. Sodium had been lost from the blood and
had not been replaced. The kidney tubules were probably capable
of little or no sodium return. The serum potassium remained
high in spite of the low activity of the kidney and the high
concentration of potassium in the bladder urime. Obviously, un-
like the sodium, the potassium of the serum was being replen-
ished. Most mammalian cells have a higher potassium than
sodium concentration and this is accomplished both by the Gibbs-
Donnan effect and by active transport of one or both ions.
Cooling the tissue would greatly reduce the active transport
mechanism.

It is interesting to speculate on what this might mean in
regard to the elyeolytic eyele and to the prominence of the
‘’ wave in the electrocardiograms of arousing hamsters (Chat-
field and Lyman, 1950). As the hamster arouses the body tem-
perature rises and the ions would gradually return to their
normal relationships. During arousal, however, a lower potas-
sitan in liver and muscle would encourage glycogenolysis and
the high serum potassium would explain the prominent T wave
of the heart.

The endogenous oxygen consumption of kidney cortex from
hibernating hamsters measured at 36°C was lower than that
from any of the other groups and fell off significantly by 90
minutes. In the intact animal the kidney cortex is therefore

449

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450 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

dependent on exogenous substrate, such as glucose from the
liver, in order to regain the high level of oxidative metabolism
found in the chronic cold-exposed animals.

Since the kidney cortex has a very high oxygen consumption
and accomplishes a great deal of work for the maintenance of
homeostasis in the hamster and yet survives the low body
temperatures of the hibernating animal, its Q,o is of interest
(Table VIL). The Q,9 of tissue from fasted control animals is
the same as that of the hibernating animals. Weiss (1954) sug-
vested that a low Q,, such as he found for heart slices from
rats might render an organ insensitive to changes in externa!
temperature and thus provide it with viability. The hamster
kidney cortex has a Qy9 at the level of rat brain and according
to our in vitro work at 8°C probably slowly oxidizes substrate
during hibernation.

In considering the response of kidney cortex to substrates
(Table V), the effect of the various substrates should not be
compared with one another since their rate of entry into the
tissue is different. There is a fall in the utilization of citrate
at the twelfth week of cold exposure and a sheht increase over
control level in the arousing tissue. The response to pyruvate
remains constant except in the tissue of hibernating animals
where there was a 13 per cent increase over the control or cold-
stressed animals. The response to succinate is slightly higher
in cold-exposed animals and very profound in tissue taken from
hibernating animals. The response of kidney cortex from hiber-
nating animals to these substrates shows that oxidative mech-
anisms were unimpaired but that substrate was not as available
for Krebs’ evele activity as in the active animals.

Changes in Body Weight Related to Oxygen Consumption of
Liver, Diaphragm and Kidney

During prolonged exposure to cold the body weight of the
hamsters decreased drastically, and they usually entered hiber-
nation at weights between 80 and 95 grams. When the oxygen
consumption of liver shees and diaphragm from non-fasted con-
trol, three-week and twelve-week non-fasted, cold-exposed ani-
mals, and animals in hibernation were plotted against their
respective body weights a linear relationship was revealed (Fig.
1). The data on liver metabolism from the nine-week cold-ex-
posed and hibernating hamsters and the data for diaphragm
taken from six-week cold-exposed animals are exceptions. The

1960 MAMMALIAN HIBERNATION 45]

data tor kidney cortex are taken from a different group of fasted
annals and are plotted for control, three days, and twelve weeks
of cold exposure, and for hibernation.

a
== KIDNEY

N
‘@]
oC .8 DIAPHRAGM

LIVER =|
ior Sas

ee OF ANIMALS
(GRAMS)

Fig. 1. The effect of weight loss in the cold on the oxygen consumption
8 8 vs

of liver and kidney cortical slices and diaphragm from hamsters of com-
parable age (+ = mean and standard deviation).
1

Weiss (1994) published similar data on oxygen consumption
of visceral tissues from white rats exposed to cold (5 £1°C)
His results were plotted against increasing weight as a measure
of age, whereas the hamsters were chosen so that they would be
approximately 6 months old when used. He obtained the same
relationships as shown for the hamsters. Weiss attributed his
results to the effect of age on oxygen consumption. With the

452 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

hamsters, the same effects were obtained as a function of weight
loss only, in a similar cold environment. When working with
six-week old rats in the cold for 10 days his results with dia-
phragm are similar to those for hamster diaphragm. On the
other hand, cold exposure did not increase the oxygen consump-
tion of diaphragm from six-month old rats. Rat liver QO»
increased significantly by the tenth day of cold exposure in both
the six-week old and six-month old rats. Hamster liver did not
achieve a QO»o comparable to that of the six-week old rats until
3-6 weeks of cold exposure. Hannon (1958) found that the peak
QOos of rat liver occurred at 4 weeks of cold exposure and then
fell off during subsequent weeks whereas the QO». of hamster
liver increased gradually until 9 weeks of cold exposure.

Diseussion

There is evidence from this work that the general oxidative
response of tissues of rats and hamsters exposed to cold is simi-
lar, but that the timing is different. This difference in timing
may be correlated with an inability of the hamster to maintain
its protein content.

The general interrelationships of liver, diaphragm and kidney
of ceold-exposed and hibernating hamsters are defined by this
work. Although liver shees respond to cold exposure of the
animal the liver does not indulge in exeess oxidative metabolism
when ‘‘aroused.’’ The diaphragm from the same animals, how-
ever, produces not only a high QO». but provides excess. oxidation
during in vitro arousal. The diaphragm, therefore, utilizes its
substrate stores for excess oxidative metabolism, and in the intact
animal one could reasonably expect such extravagance to be
constantly replenished by glucose from the liver.

The kidney is not a glycogen-storing organ and depends con-
stantly on transported substrate. The fact that the QO» of arous-
ing kidney cortical slices is significantly lower than that of the
fasted control animals demonstrates that the kidney cannot
return to an effective oxidative level unless supplied by endog-
enous substrate. The kidney maintains many aspects of mam-
malian homeostasis under extreme stresses. Its metabolism and
function in hibernating mammals should be studied more in-
tensively,

The high potassium and lowered sodium content of the serum
of hibernating hamsters is interpreted here as the result of the
depression of the aetive transport systems of the kidney, the

1960 MAMMALIAN HIBERNATION 453

erythrocytes and all the tissues of the body. It may result largely
from an exchange between erythrocytes and plasma and_ the
actual content of tissues has yet to be determined. If these ions
are shifting their equilibria between tissue and plasma as hiber-
nation is entered and during arousal, temporary but highly
significant effects may be produced in the biochemical and_ bio-
physical state of the animals.

The 40 per cent loss of initial body weight in the prehibernat-
ing hamster is a striking phenomenon. According to Zimny and
Tyrone (1957) Citellus tridecemlineatus loses 20 per cent of
its body weight before entering hibernation. Masoro et al. (1957)
deseribed a 20 per cent loss of body weight for white rats living
at 0-2°C for four months. It seems unlikely that oxidation of fat
could account entirely for the large weight loss of the hamster.
Previous to hibernation the hamster is frail in appearanee and
seems to have drawn heavily on its muscle protein.

It is well known that fats and earbohydrates have a protein
sparing action (Munro, 1951). We have found that chronic
cold-exposed and hibernating hamsters have good fat deposits and
that the liver of the hibernating hamster has 50 per cent of the
lipid content of the control animals. It is possible that in the
hamster carbohydrate and fatty acid oxidation cannot or does
not exert sufficient sparing action.

It is our intent in the future to investigate by radioisotope
technique the interrelationships among fat, carbohydrate and
protein metabolism in the tissues of cold-exposed and hibernating
hamsters. The large loss in body weight suggests an important
contribution through gluconeogenesis to the total economy of
the cold-exposed hamster during its preparation for hibernation.
Furthermore, the hormonal control of the integrated fat, carbo-
hydrate and protein metabolism of the cold-exposed and hiber-
nating mammal will have to be elucidated before a complete
understanding of the metabolic state of hibernating mammals is
achieved. A preliminary step in this direetion would be a quan-
titative estimation of the actual circulating level of various hor-
mones in the cold-exposed and hibernating mammal particularly
from a comparative point of view.

Summary

The QOs of liver slices and diaphragm was increased sie-
nificantly when taken from non-fasted hamsters exposed to cold
for one, three, six, nine and twelve weeks.

454 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The greatest QO» for liver slices and diaphragm from non-
fasted hamsters oceurred at nine weeks of cold exposure.

At six weeks, the QO. of diaphragm of non-fasted hamsters
fell significantly. This may be correlated with early attempts at
hibernation or with changes in heat loss due to some faetor such
as pelage increase.

The QOvs of liver shees and diaphragm of non-fasted hamsters
at twelve weeks of cold exposure fell significantly below their
QO. at nine weeks. Shortly after this the animals hibernated.

The QOs of kidney cortical shees from fasted hamsters was
highest when measured after 3 days of cold exposure and fell
to shehtly above control level at twelve weeks.

The QO. of diaphragm was inereased significantly when
‘arousing’? im vitro as compared to its prehibernation value
whereas the QO. of kidney and liver slices fell below their pre-
hibernation value during 77 vitro ‘Sarousal.’’

Suecinate (2 x 10-2 M.) produced the same inerease (85 per
cent) In oxygen consumption of liver shees from non-fasted
3-week cold-exposed hamsters as with liver slices from fasted
control animals. With liver slices from fasted 3-week cold-ex-
posed animals the oxygen consumption was greatly imereased
(118 per cent) by suceinate. The response of liver from hiber-
nating animals (96 per cent) is below this value for inexperi-
enced hibernating animals or higher (128 per cent) for an ex-
perienced individual.

Liver slices from fasted control and fasted 3-week cold-exposed
animals did not increase their QO. in the presence of 200 mg
per cent glucose. The oxygen consumption of diaphragm from
fasted 3-week cold-exposed animals fell off at 90 minutes of
incubation and was repaired by the addition of 200 mg per cent
elucose. Glucose had no effeet on the oxygen consumption of
diaphragm from fasted control animals.

There is little difference in the response of kidney cortical
shiees from control, 3-day and 12-week cold-exposed fasted ham-
sters to citrate (7 x 10°? M.), pyruvate (7 x 10-? M.) and
succinate (1 x 10-- M.) All these substrates significantly in-
creased the QO» of shces from hibernating animals. The two
Krebs’ cycle intermediates had the most effect on the QO» of
‘arousing’ kidney cortical slices.

The Q,, of kidney cortical slices from fasted control and
experienced hibernating animals is the same and is at the level
of Q19 for brain of the six-month old rat.

1960 MAMMALIAN HIBERNATION 455

At 8°C, in vitro kidney cortical slices from hibernating animals
have a QO» only slightly below that of slices from fasted control
animals at the same temperature. This relationship persisted
when these tissues were raised to 36°C in vitro but was repaired
cither by a substrate of the elycolytie eyele or of the Krebs’ eyele.

At the body temperature of hibernation, the oxygen consump-
tion of the kidney cortex in vitro was no higher than 9 per cent
of that from the active cold-exposed hamster and is likely lower
than this in the intact animal where oxygen may not be as avail-
able as in a Warbure vessel. Its funetion in active transport
of ions would be insignificant. Sodium had accumulated in the
bladder and fallen in the serum to the level of the fasted control
animals. Potassium had both a high concentration in the bladder
and a serum concentration at the level of the active non-fasted
eold-exposed animals. The excess potassium had probably moved
out of the tissues.

There was a linear relationship between the weight loss from
prolonged cold exposure and the QO» of liver and diaphragm
from non-fasted hamsters of the same age. This same relation-
ship was true for rats in the cold whose weight and age were
increasing simultaneously.

ITE FERENCES

CAMPBELL, J., G..R. GREEN, E. SCHONBAUM AND H. Socon
1958. Effect of exposure to cold on the coenzyme A content of liver

tissue. Fed, Proc., 17:22.

CHATFIELD, P. O. AND C. P. LYMAN
1950. Cireulatory changes during process of arousal in the hibernating
hamster, Am. J. Physiol., 163:566-574.

FARRAND, R. L. AND G. E. Fouk
1957. Responses of hamsters during first two weeks of cold exposure.
Fed. Proe., 16:35-36.
HANNON, J. P.

1958. Effect of prolonged cold exposure on ‘fin vitro’
and anaerobic glycolysis of rat liver. Am. J. Physiol., 192:253-

> respiration

or-

Ze hive
Hastines, A. B., C. Tene, F. B. NESBETT AND F. M. SINEX
1952. Studies on carbohydrate metabolism in rat liver slices. 1. The
effect of cations on the media. J. Biol. Chem., 194:69-81.
LyMaAn, C. P. anp E. H. Lepuc

1955. Changes in blood sugar and tissue glycogen in the hamster during
arousal from hibernation. J. Cell. Comp. Physiol., 41:471-492.

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Masoro, FE. J., A. I. COHEN AND S. 8S. PANAGOS
1954. Effeet of exposure to cold on some aspects of hepatie acetate
utilization. Am. J. Physiol., 179:451-456.

Masoro, E. J., J. M. FELTS AND S. S. PANAGOS
1957. Effeet of prolonged cold exposure on hepatic lipogenesis. Am. .J.
Physiol., 189:479-482.

Munro, H. N.
1951. Carbohydrates and fat as factors in protein utilization and
metabolism, Physiol. Rey., 31:449-488.
Porrer, V. R.
1958. Possible biochemical mechanisms underlying adaptation to cold.
Fed. Proe., 17:1060-1063.
SmiryH, H. W.
1956. Prineiples of renal physiology. Oxford University Press, 237 pp.
You, R. W. anp C, A, SELLERS
1951. Increased oxygen consumption and succinoxidase activity of liver
tissue after exposure of rats to cold. Endoerinol., 49:374-378.
Wriss, A. Ix,
1954. Adaptation of rats to cold air and effects on tissue oxygen con-
sumption, Am. J. Physiol., 177:201-206,
ZIMNY, M. L. aNd V. TYRONE
1957. Carbohydrate metabolism during fasting and hibernation in the
ground squirrel. Am. J. Physiol., 189:297-300.

DISCUSSION FOLLOWING DENYES’ PAPER

STRUMWASSER asked if DENYES had any data on the
effect of age per se on the ability of the hamster to hibernate
and perhaps on the ability of the whole animal to show cellular
adaptation to cold. DENYES replied that she did not, although
she had a 2-year hamster that did not hibernate after the first
vear. She also observed that some animals apparently attempt
to hibernate without success and never try again.

SOUTH noted for a fact that, after a prolonged sojourn in
the cold room and after periodic hibernating spells, hamsters stop
hibernating in the cold.

DENYES remarked that she believes the hamster is an animal
in dangerous metabolic balance when in a cold environment,
probably with considerable proteim, fat, and carbohydrate ex-
change involved. She believes there is a great shifting of hor-
monal relationships with a state finally reached conducive to
hibernation. This state may never be reached again.

RIV
PHOSPHATES AS RELATED TO
INTERMEDIARY METABOLISM IN
HIBERNATORS

By Marityn lL. ZIMny
Louisiana State University
School of Medicine

New Orleans, Louisiana

In 1949 Wollenberger gave a review of work on the energy
metabolism of the failing heart and reported his original re-
search. By means of Starling heart-lung preparations in dogs the
intermediary metabolism of the energy-rich phosphate content
was studied under various experimentally induced conditions of
cardiac failure. Later he reported that the rate of utilization
of phosphate bond energy by the heart is a function of frequeney
of beat (Wollenberger, 1951).

Work underway in neighboring laboratories investigating a
possible mechanism of eardiae glycoside action (Proctor et al.,
1955) and the high-energy phosphate content of different areas
of the dog heart (Mulder et al., 1956) stimulated my interest in
phosphates and eardiae metabolisin.

The heart rate, respiration rate, and body temperature of
active and hibernating ground squirrels were compared by John-
son (1931), and those of active and hibernating marmots by
Benedict and Lee (1938). The decrease in heart rate which was
found during hibernation made it seem that the high-energy
phosphate compounds, adenosine triphosphate and phosphocrea-
tine, should be investigated and correlated with the energy evele
of glycolysis as a means of interpreting the metabolic adaptations
of hibernators.

Early reports of analytical work on hibernating mammals per-
tained to blood glucose and liver glycogen (Dubois, 1896; Wein-
land and Riehl, 1908; Weinland, 1925; Endres and von Frey,
1930; Suomalainen, 1935) but relatively little material was avail-
able on phosphate compounds (Ferdmann and Feinschmidt,
1932; Feinschmidt, 19386). Recently, the glyeogen content of
various organs of hibernants has been adequately reviewed by
Lyman and Chatfield (1955), Hisentraut (1956) and Kayser
(1957). There is general agreement that hypoglycemia oceurs
during hibernation, but the degree reached varies from species

458 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

to species, and that reconstruction of cardiac muscle glycogen
during hibernation takes place at the expense of skeletal muscle
and liver glycogen. Our studies of phosphate and carbohydrate
compounds in the thirteen-striped ground squirrel, Citellus tri-
decemlineatus, inelude the heart, skeletal musele and liver.

This investigation during the past eight vears has ineluded
animals that were sacrificed (1) following a period of 3 to 5 days
of uninterrupted hibernation; (2) following a period of 30 days
of uninterrupted hibernation; and (3) following’ stimulated
arousal periods of 7.5, 15 and 30 minutes. The control animals
were maintained at an environmental temperature of 25-27°C
while the experimental animals were kept in an environmental
temperature of 3-5°C. Records of body weight, heart rate (EKG
tracings), respiration rate, and rectal temperatures (thermo-
couple measurements) were kept on several animals for aids in
interpretation.

Glycogen determinations in early phases of the project were
made according to Pfluger’s method as modified by Good ef al.
(1933), and Somogyi’s method as modified by Nelson (1944)
for glucose hydrolyzed from glycogen. As work progressed it was
desirable to analyze the same tissue sample for other compounds
in addition to glycogen. Since the ground squirrel heart is smal]
in size, another elycogen method was needed in order to carry
out this procedure. Therefore, glycogen was determined by the
method of Kemp and van Heijningen (1954). Tissue lactate and
pyruvate have been determined by the methods of Miller and
Muntz (1988) and Lu (1939), respectively. Determinations of
phosphate fractions were based upon the Fiske-Subbarow reac-
tion as used by Wollenberger (1947). The symbols for the vari-
ous phosphate fractions are as follows: adenosine polyphosphate
(APP), adenosine triphosphate (ATP), adenosine diphosphate
(ADP), phosphocreatine (PC), and inorganic phosphate (TP).

Carciae Muscle

Phosphocreatine was deseribed in skeletal muscle by Fiske
and Subbarow in 1929, and in the same year Pohle (1929) re-
ported that muscle adenosine phosphoric acid and also a hexose-
monophosphorie acid had been isolated from the ox _ heart.
Ferdmann and Feinsehmidt (1932) made an extensive study of
the chemical changes in muscle in a small number of hibernating
eround squirrels, Citellus guttatus, and marmots, Arctomys
bobac. They reported that during hibernation the acid-soluble

1960 MAMMALIAN HIBERNATION 459

phosphate of the heart decreased with decreasing ortho-phos-
phate and phosphoereatine. Correlation was suggested between
the activity of the heart and ortho-phosphate content due to the
finding that this compound decreased with decreasing heart rate.

Karly studies relating to phosphoric acid metabolism and the
total metabolism of the heart are reviewed by Cruickshank
(1936). By this time it was apparent that the amount of phos-
phorus produced by the heart was dependent on the rate of
contraction. Although the studies of Lundsgaard (1930) had
shown that muscular contraction derived its energy from phos-
phagen hydrolysis, and Lohmann and Schuster (1935) had
studied the presence of adenylpyrophosphate and adenosine di-
phosphate in heart muscle, it was still questionable whether or
not eardiae muscle metabolism paralleled that in skeletal muscle.
The interest in the relationship of phosphates in cardiae metab-
olism was derived early from studies of the physiology of the
failing heart and later from the pharmacological action of var-
ious drues upon the heart. Szent-Gyéreyi (1947) reported that
the actomyosin obtained from cardiac muscle was indistinguish-
able from that extracted from skeletal muscle but still analyses
of cardiac muscle action based on cellular and molecular changes
were wanting when Wollenberger reported that heart failure can
occur in the presence of normal amounts of ATP and PC in the
myocardium (1947); that the heart has low ability for anaerobic
recovery due to a small PC reserve and low glycolytic power
(1949) ; that the heart resynthesizes ATP at the expense of PC
(1949) ; and that the rate of utilization of enerey-rich phosphate
by the heart is a function of heart rate (1951).

When the phosphate compounds, 1.e., inorganic phosphate
(IP), adenosine polyphosphate (APP), considered in our work
as adenosine triphosphate (ATP), and phosphocreatine (PC),
were first studied in the ground squirrel heart, it was found that
ATP decreased and PC increased approximately to the same
degree and IP remained unchanged (Zimny, 1956). As work
continued it was soon apparent that this was not correct. In
these early experiments procedures involving handling of the
hibernating animal, such as weighing and taking electrocardio-
graphic tracings prior to sacrifice, introduced a time factor
which proved to influence the final results. During later experi-
ments the hibernating animals were sacrificed and the tissue
removed within the first 30 seconds of handling the animal. The
values obtained from these animals, showing a maintenance of
high-energy phosphate and a significant decrease of 20 per cent

460 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

in IP, we believe, are representative of true hibernation (Zimny
and Gregory, 1958b). It is inferred from the work of Bing
(1955) and Wollenberger (1947) that utilization and generation
of phosphate bond energy have reached an equilibrium at a
ereatly reduced heart rate and work load. As hibernation is

MG %

CARDIAC MUSCLE

100

500

Z2QDOO<r@

OMAPIVMNOLV

C SH LH: 7.5. "158530

Fig. 1. Concentrations of glycogen and phosphates in cardiac muscle
following 3-5 days of hibernation (SH); 380 days of hibernation (LH); and

arousal periods of 7.5, 15, and 30 minutes as compared with controls (CONE

prolonged trom an uninterrupted period of 3-5 days to an unin-
terrupted period of 30 days, ATP and PC decrease significantly,
with IP increasing toward the control value (Fig. 1) (Zimny
and Gregory, 1959). Phosphocreatine shows a greater decrease
than ATP because it is maintaining ATP to supply energy for
the slowly beating heart.

Changes in these compounds take place quickly upon stimu-
lated arousal following 3 to 5 days of uninterrupted hibernation,

1960 MAMMALIAN HIBERNATION 46]

especially in the heart(Zimny and Gregory, 1958b). Within
7.9 minutes elycogen decreases 55 per cent, IP increases 88 per
cent, ATP decreases 70 per cent and PC increases 68 per cent.
Within 15 minutes of arousal glycogen and ATP are increasing
while [LP and PC are decreasing, and by 380 minutes the phos-
phate levels are beginning to resemble the control pattern (ig.
1). Cardiae muscle glycogen continues to imerease. Therefore,
the immediate use of ATP for energy is for stimulating elycoly-
sis and is followed by PC transferring phosphate to ATP and
possibly to other metabolic systems for the purpose of glyvconeo-
eenesis.

Cardiac muscle glycogen levels during the hibernating periods
were studied and following five days of uninterrupted hiberna-
tion this compound had increased 60 per cent, indicating that
the heart is storing carbohydrate for the thermogenic processes
of arousal. Although e@lycogen in the heart decreased 36 per
cent upon prolongation of the hibernating period to a month, the
decrease did not influence the physiological state of the animal.

Lactate and pyruvate levels decrease during hibernation but
the laetate-pyruvate ratio remains within the average control
range for cardiae muscle of 9/1 (Zimuny and Tyrone, 1957). The
decrease in these compounds apparently is due to a slower rate of
elveolysis with a decreased metabolic rate.

Skeletal Muscle

The pioneer investigations of intermediary metabolism in rela-
tion to carbohydrate and phosphoric acid were done on skeletal
muscle (Fiske and Subbarow, 1927; Eggleton, 1929; Meyerhof,
1930; Milroy, 1931). In their study of the chemical changes in
muscle of the hibernating animal, Ferdmann and Feinsehmidt
(1932) reported a decrease in phosphocreatine, inorganic phos-
phate, hexosemonophosphate, acid-soluble phosphate and_ total
phosphorus in both the hibernating ground squirrel, Citellus
guttatus, and the marmot, Arctomys bobac. Later studies by
Feinschmidt (1986) revealed a sienificant reduction in ATP
content and an increase in Morganic phosphate and free adenylie
acid during hibernation.

The importance of PC and APP to the contractile system of
musele was emphasized by Kalekar (1941) in a review of the
energetic oxidation-reduction reactions which oeeur during bio-
logical syntheses. Mention was also made that cellular strue-
tures may be acting as phosphate-transfer systems in the living

462 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

cell. Mommaerts (1950) labelled the splitting of PC as the
energetic master reaction in normal muscle, and in 1951 Kaplan
extensively described the thermodynamics and energy mechan-
isms of the phosphate bond. The work of Szent-Gyéreyi (1953)
vives evidence that glycolysis furnishes energy for converting

MG %

SKELETAL MUSCLE

500

ZMDON<F@®

fo)
P 30
H
O
S 20
p
H
A 10
T
E
Ss fo)

C SH. LHS 75. (15 9-30

Fig. 2. Concentrations of glycogen and phosphates in skeletal muscle
following 3-5 days of hibernation (SH); 30 days of hibernation (LH); and
arousal periods of 7.5, 15, and 30 minutes as compared with controls (C).

ADP to ATP whieh breaks down during contraction, and PC
may be rephosphorylating ADP held by contracted myosin, In
1954 Mommaerts stated that the purpose of metabolism is_ to
eenerate ATP as it is used, and the findings of Uchida et al.
(1954) supported the theory that ATP is the immediate source
of energy for contraction, with PC splitting in the initial phase
of muscle contraction. Correlation of the mechanical with the

1960 MAMMALIAN HIBERNATION 463

chemical events which occur during muscle contraction, Meorpor-
ating both the glycolytic and oxidative phosphorylating mito-
chondrial systems has recently been reviewed by Perry (1956)
and by Buehthal ef al. (1956).

Phosphate compounds in skeletal muscle samples from the
early hibernation studies (Zimny, 1956) followed a pattern
similar to those in cardiac muscle. Adenosine triphosphate de-
creased and PC increased to approximately the same degree with
a small decrease in IP. Later investigation (Zimny and Gregory,
1958b) showed that ATP is maintained during hibernation, and
PC increases apparently at the expense of decreasing IP and
glycogen. When the hibernating period is prolonged to 30 days,
both ATP and PC decrease but the ATP-PC ratio remains un-
changed at 1/1 (Fig. 2). Immobility for the extended period
explains this combined decrease (Zimny and Gregory, 1959).

Adenosine triphosphate is used as the immediate source of
energy during arousal with PC affecting resynthesis (Zimny
and Gregory, 1958b). Within 7.5 minutes of arousal PC de-
creases 46 per cent indicating a phosphate transfer forming ATP
which then shows a 114 per cent increase by the end of 30
minutes of arousal (Fig. 2). As might be expected, skeletal
muscle activity lags behind that of cardiae muscle during arousal.
After 15 minutes of arousal the heart rate had increased 192
per cent, a gain of 71 beats, and after 30 minutes, it increased
644 per cent,'a gain of 174 beats. At neither time was the
increase in skeletal muscle activity in any way comparable.
Glycogen increases after 15 minutes of arousal and continues
to do so after 30 minutes. Once again a possible explanation may
he that of elyconeogenesis.

Skeletal muscle glycogen decreases during 3 to 5 days of
hibernation and continues decreasing with increasing length of
the hibernation period. This decrease may be due to increasing
the stores of cardiae glycogen or a metabolic response to low
temperature prior to hibernation.

Lactate and pyruvate decrease in skeletal musele during
hibernation but the ratio between the two compounds remains
within the average control range of 13/1 (Zimny and Tyrone,
1957). The decrease is apparently due to a slower rate of
elycolysis at a decreased metabolic rate.

Liver

Liver phosphate studies are few because the early work on
phosphates was centered upon the chemical chanees associated

464 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124
with muscle contraction. A liver homogenate was used as a
source of adenylpyrophosphatase in studies pertaining to muscle

(Jacobsen, 1951), and studies of changes in liver phosphates as
a result of experimental conditions were related to fasting and

G °
iy ee LIVER

10 PC. 3” \ 2

OmaAa>riwvwnNOoOlLtv
as

C of. LH jboss a9

Fig. 3. Concentrations of glycogen sand phosphates in liver following 8-5

days of hibernation (SIL); 30 days of hibernation (LH); and arousal

periods of 15 and 30 minutes as compared with controls (C

the effects of various diets (Flock et al., 1986). In 1939 Hevesy
found a much larger turnover of labelled phosphorus in the liver,
kidneys, and intestinal tract than in the muscles or brain of
rats. Both Nelson et al. (1942) and Rapoport ef a/. (1943) found
that the concentration of IP increased in the livers of fasted

1960 MAMMALIAN HIBERNATION 465

rats, with a decrease in the easily hydrolyzable fractions. No
explanation for the shift was given. The liver, as stated earlier,
Was among the first organs analyzed for glycogen in the hiber-
nant and early work on carbohydrate metabolism is aptly
reviewed by Cori (1981). More recent work is discussed by
Soskin and Levine (1952).

In the liver of ground squirrels, adenosine triphosphate in-
creases 121 per cent and PC decreases 62 per cent following
3 to 5 days of hibernation (Mig. 3). As the hibernating period
is prolonged, ATP decreases 55 per cent and PC increases 107
per cent so that the values at this time are nearly equal. After
a 15 minute arousal period following 3 to 5 days of uninterrupted
hibernation, ATP has decreased 47 per cent and PC increases
f4 per cent. By 380 minutes of arousal it is apparent that PC
is transferring phosphate for ATP formation. During hiberna-
tion, the lessened metabolic demand allows ATP to accumulate
for the purpose of supplying energy to the many liver functions
which oceur upon awakening (Zimny and Gregory, 1958b, 1959).

Liver glycogen increases during early stages of hibernation,
shows a decrease after 380 days of hibernation and inereases dur-
ing arousal (Zimny and Gregory, 1958b, 1959). For want of
any other explanation, the Increase in tissue elycogen is attrib-
uted to processes of elyconeogenesis. Liver glycogen values dur-
ing hibernation and arousal depend upon several factors. Varia-
tions in respiration rate durine the hibernating period could
influence the @lycolytic rate. The leneth of time the animal had
spent in the cold enviromment responding to calorigenic stimull
before entering hibernation should also be considered in addition
to the composition of the diet and weight loss factors. Lastly,
the method of sacrificme an animal can influence the tissue
elveogen level as well as the levels of other compounds.

Both lactate and pyruvate levels decrease during hibernation,
but as in the heart and skeletal muscle the lactate-pyruvate ratio
remained within the control range of 10-20/1 (Zimny and
Tyrone, 1957 ):

Related Studies

Brown fat. The process of elyeconeogenesis has been men-
tioned as a possible explanation for increased e@lycogen levels im
the tissues studied. With this in mind the interseapular brown

fat pads of hibernated. aroused and control ground squirrels

466 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

were analyzed (Zimny and Gregory, 1958a). Hook and Barron
(1941) found that although in most tissues the metabolism is
reduced to a minimum during hibernation, brown adipose tissue
still maintains one-third of its optimum activity in terms of
respiratory action of tissue slices. Klar (1941) suggested that
activity increases in this tissue in the hedgehog during the winter
because the oxidation-reduction potentials of brown fat are
vreatly reduced during hibernation. In 1953 Karolewiez reported
that brown fat of the hedgehog does not contain nutritious stores
for the hibernation period, but that stored compounds are used
in the pre-ruttinge period immediately after the animal has
awakened. Further study (Karolewicz, 1956) showed that the
elycogen content of brown fat is low in June and high in No-
vember with a daily rhythm during the summer months showing
peaks of activity im the daytime and at 38:00 A.M. Recent
histochemical and microchemical studies on the lipids of the bat,
Myotis lucifugus lucifugus (Remillard, 1958) revealed decreases
in total weight, water content, total lipids, total cholesterol and
rotal fatty acids during hibernation.

Our studies on the biochemical composition of brown fat in
rhe ground squirrel showed an increase in glycogen and pyruvate
and a deerease in lactate during hibernation and showed all three
volmpounds decreasing during arousal (Zimny and Gregory,
1958a). Tissue lactate was high in the control group, lactate-
pyruvate ratio of 56/1, when compared to the ratios in the other
eroups averaging 20/1. In fact the lactate-pyruvate ratio of
brown fat in the control is much higher than ratios ever obtained
for cardiae muscle, skeletal muscle, and liver which are in the
range of 10-20/1. We interpreted this to mean that in the control
animal a high rate of glycolysis supphes lactate as an energy
souree for the metabolie evele. During hibernation elyeogen stor-
age takes place and during arousal all compounds show a
decrease as a result of supplying energy for awakening.

Phosphate values were low for all fractions in both control and
experimental animals. Kither the amounts used for metabolic
conversions are low or the values represent the composition of
the rich vascular network of the brown fat.

Ultrasound. Gersten and Kawashima (1954), by means. of
ultrasound, produced an increase in PC and a decrease in IP
Without any rise in temperature in an isolated frog gastroene-
mius. In our laboratory we applhed clinical dosages of ultra-
sound to hind legs of control and hibernating ground squirrels.
One of our more interesting findines (Zimny and Head in MS)

1960 MAMMALIAN HIBERNATION 467

is that by means of ultrasound the phosphate fractions, [P, ATP,
and PC, in the hibernating animal can be brought to arousal
levels in a relatively short period of time without any change
in the animal’s rectal temperature.

Conelusion

Adenosine triphosphate is the immediate source of energy for
metabolic work involving phosphorylations, svntheses, and other
possible processes utilizine chemical energy. Phosphocreatine,
glycolysis, and biological oxidations can effect a steady resyn-
thesis of this compound. During hibernation, metabolic work ts
greatly reduced; the animal is stationary; the heart rate is
greatly decreased; and digestive processes involving the liver
are lessened. Cardiac muscle glycogen accumulates at the expense
of skeletal muscle and liver glycogen and glyconeogenesis, pri-
marily from fat, feeds metabolites slowly into the e@lycolytic
system. Upon arousal adenosine triphosphate is used as the prim-
ary energy source with the high-energy phosphate of phospho-
creatine maintainine adenosine triphosphate, and these com-
bined reactions stimulatine the energy producing cycle of
elycolysis for the purposes of resynthesis.

Studies of these phosphate compounds in tissue extracts have
been of value for interpreting metabolic adjustments but
studies must now be extended to cellular and ultracellular struc.
tures. Enzymatic phosphate transfers by kinases, phosphatases
and phosphorylases have prompted us to begin studies on the
concentration of ATPase, ATP-creatine transphosphorylase and
cholinesterase in the hibernating ground squirrel. The loealiza-
tion of oxidative phosphorylations in the mitochondria of musele
stimulates interest in possible structural changes of mitochondria
in cardiae and skeletal muscle occurring during hibernation.
Adenosine triphosphate is necessary for the functional integrity
of the contractile elements of muscle, actin and myosin, and for
the proper sarcoplasmic environment to act as a substrate for
the myofibrils. Anabolie processes of cellular metabolism involv-
ing oxidations, CO. fixation, syntheses and secretory processes
of the cell are related to adenosine triphosphate and the mito-
chondrial system.

The hibernant is capable of both lowering and raising its body
temperature in a cold environment. Tissue phosphate compounds
in a hibernating animal can be stimulated by the mechanical

468 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

means of ultrasound to assume levels comparable to arousal with-
out an increase in body temperature. A rise in heart rate often
precedes the rise in body temperature, but the converse is not
true.

Extended investigation into the fields of enzymatic energy
transfer mechanisms as related to ultracellular structure may
not change any specific conversion we now know in the general
scheme of intermediary metabolism in the hibernator but may
show a heart, rich in mitochondria, possessing great oxidative
capacity for the production of ATP, used for immediate energy
when needed and resynthesized by a high-energy phosphate
transfer from PC to ADP. A similar energy cycle may exist in
skeletal muscle with the possibility of ATP production being
more dependent on glycolytic processes of anaerobic metabolism
than oxidative phosphorylation. In either case it appears that
the energy change in the heart is the last link in the chain of
metabohe events when the animal enters hibernation and the
first spark during arousal.

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DISCUSSION FOLLOWING ZIMNY’S PAPER

KRISHER asked what the body temperature was after 30
minutes of arousal. ZIMNY replied that it was between 16 and
17 Cy a rise of about 9°C from hibernating body temperatures.
POPOVIC asked where the thermocouple was placed. ZIMNY
replied that if was in the reetum. FISHER noted that the
core may be at a higher temperature than this. ZIMNY added
that she expects to take temperatures at a higher level and
analyze muscle samples from the forelimbs in the future.

BULLARD asked if she had noted the heart rate at the 30
minute point of arousal. She rephed that it was about 190 beats
per minute, an increase of about 174 beats from the hibernating
heart rate.

1960 MAMMALIAN HIBERNATION 473

JOHANSSON stated that he had also made lactate determina-
tions in the heart muscle of the hedgehog, comparing hibernating
and non-hibernatine states. He found no significant difference
between the two groups. ZIMNY said she used the Miller and
Muntz method with a frozen tissue sample (B. F. Miller and
J. A. Muntz, J. Biol. Chem., 126:413, 1938). The heart must be
removed very quickly before arcusal has a chance to proceed.
If it is not frozen immediately, some lactate may be lost; in fact,
in phosphate determinations, all may be lost if the tissue is not
handled very quickly.

BULLARD asked how changes in ATP and glycogen in the
heart compared with other species. ZIMNY said she knew of
no particular work on phosphates in other small animals that
was comparable.

FISHER remarked that the production of large quantities of
lactate is a common physiological method of getting a lot of
energy ina hurry. ZIMNY agreed that lactate is probably useful
for fast energy needs. She had not done blood work on this.
With respect to high energy phosphates, they are extremely
labile and ean be lost just by putting a needle into the heart.

WIMSATT noted that glycogen lability in the liver also shows
short-term deviations of concentrations. He stated that a stu-
dent of his had found a definite decline in total liver glycogen
in bats occurring over the spread of the hibernating season, with
a substantial drop in the latter part of the season.

Concerning ZIMNY’S high liver ATP values for the ground
squirrel, DENYES noted that liver slices from hibernating ham-
sters have a high oxygen consumption which is increased by the
addition of succinate but not by glucose. This also indicates a
retention of ATP during hibernation.

MUSACCHIA remarked that (adding to WIMSATT’S com-
ments) there is a slow and steady utilization of liver glycogen
in the hibernating turtle in a 4-6 week period, with utilization at
a steady rate even though there is no maintenance of a steady
body temperature.

XXV
SOME METABOLIC SPECIALIZATIONS IN
TISSUES OF HIBERNATING MAMMALS’

By FRANK E. Soutu
Department of Physiology
University of Illinois College of Medicine

Chicago, Illinois

It has long been realized that the hearts, peripheral nerves
and reflexes, as well as other tissues and organs, of hibernators
are physiologically modified to the extent that they are capable
of continued function at temperatures considerably lower than
those of other mammals (ef. Hegnauer ef al., 1950; Lyman and
Chatfield, 1955; Dawe and Morrison, 1955; MeQueen, 1956).
The problem, in paraphrase, is: what general or specific factor
makes it possible for the heart of one animal, a hibernator, to beat
at a temperature of 4°C while that of another animal will go
into asystole or fibrillation at 15° or 20°C? The same question
may be asked in regard to certain reflex patterns (e.g. respira-
tion), nerve conduction and other processes.

Possible answers may range from simple enzyme or substrate
concentration differences to some of the more abstruse concepts
of kinetics and thermodynamics. Indeed, in a recent challenging
paper Brown’ (1956) maintained that the hibernator could sur-
vive at low body temperature due to a higher setting of the
reaction rates of its cellular processes relative to that of the
non-hibernator. This means that while a given rate process
common to both types of animals would have identical Q,9’s, the
rate observed for the hibernator would be higher throughout
the significant temperature range. This is one theory, and it does
fit with some, but not all, of the facts.

Many biologists, including those interested in hibernation,
have taken a clue from Crozier (1924) and attempted to analyze
their data on the basis of either Q;» or the Arrhenius equation,
one form of which is:

Ea=R ln Kr, In Ko
VA ES
or
—Ea
k= CeRT

1 This work was supported by a contraet with the Office of Naval Research,
Contract No. Nonr-1459-05, and by a grant from the National Institutes of Health
(H-53805).

476 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

in which ky, and kyo refer to the velocity constants at absolute
temperatures, T, and Ts; R is equal to the gas constant and Ea
is taken as the apparent ‘‘enerey of activation’’ (uy is also a
common symbol employed here ).?

While such plots of the logarithm of the velocity against the
reciprocal of the absolute temperature often yield straight lines
over a limited range, especially for pure systems, they are more
often curved lines for more complex systems such as heart rates.
Such non-lnear plots have been analyzed by assuming linearity
over a limited portion, as was done by Crozier or by the empiri-
cal method of Kavanaugh (Kavanaugh, 1950; South, 1958).
Although certain of the assumptions of both of these approaches
have been questioned on theoretical grounds, yet when used with
caution and with properly controlled experiments, such analyses
may prove to be extremely powerful tools in the exploration of
the basic phenomena of hibernation.

A rational basis for utilizing temperature analyses is provided
by comparing temperature plots of operationally identical sys-
tems (e.@., rates of tissue oxygen consumptions) of hibernating
and non-hibernatinge animals. By progressive dissection, from
the complex to the simpler, it should become possible to define
those factors upon which the hibernant depends for the main-
tenance of life at low temperatures.

This principle may be illustrated by data from studies which
have been made upon cardiac muscle and upon peripheral nerves
and skeletal muscle.

The first thorough study of this type upon the comparative
effects of temperature upon neural conductivity was that of
Chatfield et al. (1948). Using the tibial nerves of golden ham-
sters and rats they were able to show that hamster nerves
could conduct a spike potential at much lower temperatures than
could those of the rat (means at which conduction ceased were
3.4° and 9°C, respectively) ; the range of incubation tempera-
tures was 2° to 20°C. Of equal significance was the observation
that with descending temperatures the action potential, excitabil-
ity and conduction velocity of rat nerves declined as a steep
funetion. In contrast, the conduction velocity and excitability
of hamster nerves fell off much less with lower temperatures

2Tt is generally agreed that the experimental energy of activation is that
chergy required by the reactants to reach an intermediate complex or configura-
tion which is necessary in order that the reaction may proceed to completion.
According to the most prominent view, enzymes affect reaction rates by reducing
the energy barrier (i.e. the energy of activation) over which the reactants must
pass in order to form products (Glasstone et al., 1941).

1960 MAMMALIAN HIBERNATION 4T7

while the action potential passed through a maximum at 15°C,
declined rather slowly to 5°C, and rapidly thereafter. No dif-
ferences between hibernating and non-hibernating hamsters were
observed.

In a series of experiments, in which phrenie nerve-diaphragm
preparations obtained from rats and from both hibernating and
non-hibernating hamsters were studied, it was found that the
results obtained from the phrenic nerves were qualitatively quite
similar to those of the experiments cited above. One funda-
mental difference did appear, however, in that the excitability
at 5°C was greater in the case of the hibernating hamsters than
for hamsters maintained at room temperature (South, MS in
prep.). The use of the phrenic nerve-diaphragm preparations
proved to be extremely useful in that they provided an oppor-
tunity to study a number of the properties of both nerves and
muscles in relationship to temperature, hibernation, and the
possibility of phylogenetic adaptation. Some of the results will
be discussed below.

One of the more interesting results which were obtained related
to myoneural transmission. It was observed that while all of the
rat phrenic nerves were capable of conducting a spike at a tem-
perature of 10°C there was no recordable isometric muscle re-
sponse in 70 per cent of the instances. At 5°C the rat phrenic
nerves were inexcitable but those of the control and hibernating
hamsters retamed their ability to conduct spikes. However, in
no case was there evident muscular response to indirect stimula-
tion on the part of preparations obtained from control hamsters,
although those obtained from hibernating hamsters always re-
sponded to such stimuli at 5°C. That these observations were
dependent upon the functioning of the myoneural junction could
be shown by the ability of the muscles of all three groups to
respond to direct stimulation, although the necessary stimulus
varied widely, and by appropriate tests with tubocurarine. These
observations are now being subjected to further study.

Considering the differences in irritability of peripheral nerves,
which have been summarized above, it is not surprising that an
analogous situation should be found to hold true for diaphragm
muscle. The threshold for direct stimulation of rat muscles rose
to relatively high values below 17°C. The hamster thresholds
rose to a lesser degree and were quite similar to one another down
to 10°C. Below this point the threshold for the muscles of control
hamsters became very high relative to that of diaphragm obtained
from hibernating hamsters.

478 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

The contractile responses of the diaphragms were measured
isometrically, the muscles being stimulated at rest length. The
rates of tetanic contraction and relaxation were treated by plot-
ting the reciprocal of the ‘‘half-time’’ for either process against
the reciprocal of the absolute temperature (Figs. 1, 2).

0.0

1
fe)
Oo

Ea(kcal)
5°-17° 109-3)

= Rai 35.0 161

+ Normal 286 12.0
hamster

4 Hibernating
hamster 20.2 13.3

0,00

0.002

9°
ro)
o

Tetanus-Contraction (ti)

3l°

Fig. 1. Relationship between isometric contraction rate of diaphragm
muscle, in vitro, and temperature. Semilogarithmic plot. Each point repre-
sents an average value of 6 to 10 separate experiments, pH = 7.4.

The use of the half-time under these conditions is based on the
treatment of Ramsey (1944), whose equation follows first-order
kinetics and describes the rate of tension production from rest
length with fair precision. Therefore the reciprocal of the con-
traction half-time would be proportional to the specific first-order
rate constant. Such a treatment is sufficient for our purposes.

1960 MAMMALIAN TIIBERNATION 179

Proceeding on these assumptions, it is immediately apparent
that the rate of rat muscle tension production is more tempera-
ture dependent than that of hamsters (Fig. 1). Straight lines
fitted to any portion of the curve (e.g. the fairly linear range,
10°-381°C) indieate a higher Ea for the rat than for the hamsters.

.05

S217° 10% 3)
Beeed stig ste
€ hamster 23.8 173
= AHibernating aig = 164
eae hamster
.0005

lo 36

ig, 2. Relationship between relaxation rate from isometric tetanus,

in vitro, and temperature. Semilogarithmic plot. Each point represents an
average value of 6 to 10 separate experiments. pH = 7.4.

At 59°C the rate of contraction is less than one-third that of the
hibernating aninal. It is also noteworthy that although the
curves obtained from the hamsters lie very close to each other
Within the range of 10°-38°, they become divergent below 10°C,
This result mimics to some extent the irritability differences
noted above.

480 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

Plotting the rate of relaxation from tetanus (Fig. 2) reveals
similar comparative results for the rat relative to the hamster
eroups, the difference being especially marked in the low temper-
ature range (5-17°C). It also may be noted in Figure 2 that there
is a fairly good indication of a lower value of Ea for the data
obtained from hibernating hamsters as compared to their con-
trols. The differences in these curves could be made more
dramatic by plotting the data of the ordinate as per cent of
maximum,

“

b ]

= hamster a =.
= 4 B Hibernating ; ———_p~—

i= hamster

uv“

Isometric Tension

5 10 17 24 3! 38
Temperature ( O°C)

Fig. 3. Isometric tension production as a funetion of temperature.
Rectangular coordinates. pH = 7.4.

While the application of first-order reaction kinetics to the
relaxation period can be questioned on theoretical grounds,
this treatment does serve to illustrate the relative temperature
dependencies.

The relationships between tension produced by isometric tet-
anus and temperature (Fig. 3) in the three groups of animals
reveal the rather surprising result that the control hamster
diaphragms were capable of the greatest tension production,
followed by the hibernating hamster and finally the rat. In
this instance the rat muscle differs from that of the hamster not
only in the lower tension production but also in a somewhat
slower decline in tension with temperature. Arguing teleologi-
cally, one might be led to expect that the tension production by
hamsters would be less affected by temperature than is the case
in the rat. This is clearly not so.

1960 MAMMALIAN HIBERNATION 48]

The general pattern which emerges for nerve and muscle prep-
arations indicates a fundamental functional dichotomy between
hibernating and non-hibernating mammals, insofar as the rat and
the hamster are representative of these groups. Hence, in these
systems, other than tension production, the rat has a higher
setting’ at 38°C but due to the greater temperature dependence
(higher Ea’s) the processes are slower and less effective in the
low temperature range than is the case for hibernants. This
dichotomy may be characteristic of those metabolic and fune-
tional systems which must be specifically and phylogenetically
adapted for survival at low body temperatures.

It was noted above that frequently the temperature curves
for certain processes of hibernating and control hamsters were
very similar (e.g., magnitude of the spike potential and rate of
conduction) throughout the temperature range. In other in-
stances (e.g., threshold and contraction rate) they were very close
down to a temperature ca 10°, whereupon a divergence occurred
in which the curve for the control hamster fell off more sharply
than that for the hibernating hamster. The plots of the relaxation
rates revealed the possibility of vet another factor, that is, a
difference between hibernating and non-hibernating hamsters
similar to that between rats and hamsters. Such observations
led one to suspect that something more than mere interspecific
differences are involved here. In other words, the hamster may
undergo an acclimatization process prior to hibernation which
involves cellular processes at a very fundamental level.

It is not enough, however, that only the kinetics of muscular
contraction or neural conduction be adjusted for function at
reduced temperatures. These are only mechanical or physical-
chemical results of many metabolic processes which function to
“serve up’? energy in sufficient quantities and in usable form
(e.g. ATP). Consequently, any self-contained system which is
eritieal for survival durine hibernation must be modified in
three ways with respect to temperature, That is, it must be able
to (1) transform the energy content of general substrates (e.g.
glucose) to an immediately useful form (e.g. ATP) at a sufficient
rate to support the needs of the overall process. (2) Intermediate
coupling (e.g. ATP to myosin) must be accomplished at a suf-
ficient rate. (3) The ‘‘effector’’ portion of the system (e.g. con-
tractile protein, metabolic ‘‘pumps,”’ ete.) must be able to
transfer this energy to its environment at a sufficient rate and
quantity to meet the needs of other systems. Therefore, a hypo-
thetical tissue might supply energy at a high rate, but it would
he of no consequence if the effector should be unable to use it.

482 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The heart represents a system which has received a fair amount
of attention from those interested in mammalian hibernation.
We are well aware of its central position of importance in hiber-
nation, functioning not only as the circulatory pump but also
as an important source of heat during arousal (Lyman and
Chatfield, 1950; Lyman and Ledue, 1953). A number of investi-
gators have examined heart rates and EKG’s of hibernating,
awakening and non-hibernating mammals (cf. Kayser, 1953;
Lyman and Chatfield, 1955, for discussion and bibliography ).
One particularly interesting aspect of the EKG studies relates
to the repolarization time (S-T). Since repolarization time is
fairly long it dominates the intervals variously reported as Q-T
and RS-T. Evidence indicates that of all intervals of the EKG,
the Q-T is most profoundly affected as the heart temperature of
non-hibernators declines and aberrancies of the S-T segment
(Osborn wave) often occur (Nardone ef al., 1955; Ruhe and
Horn, 1955; Biorek and Johansson, 1955; Tysinger et al., 1956).
In contrast, the repolarization time of hibernators is usually less
affected by declining temperatures than the other intervals and
seldom shows aberrancies of the type described by Osborn (1953 )
(ef. Dawe and Morrison, 1955; Bidrek and Johansson, 1955;
Nardone, 1955). Since repolarization is dependent upon meta-
bolic processes (Garb and Chenowith, 1953), it is quite apparent
that the comparatively short S-T interval seen for the hearts of
hibernating mammals represents the resultant of phylogenetic
adaptation of cardiae metabolism.

The question remains as to modifications in the mechanisms
of energy supply. Several solutions are possible: (1) the in-
herent rate of supply might be sufficient so that only the ‘‘ef-
fector ’’ is modified; (2) the inherent rate may not be sufficient,
but increases in the concentrations of rate-limiting enzymes
and primary substrates might occur such that enough enerey
could be supplied to the effector (alternate pathways would also
be included here); or (3) the properties of the enzyme system,
or of a rate-limiting enzyme within that system, might be altered
in such a way that at lower temperatures it would operate more
efficiently in reducing the ‘‘energy barrier’” over which the
reactants must pass.

As a first step in answering these questions, the initial rates of
oxygen constunption of heart shees obtained from rats, control
hamsters, hibernating hamsters, and torpid bats (J/. lucifugus )
were determined over a temperature range of 5° to 43°C (Fig. 4)
(South, 1958). Again the now familiar effect of temperature on

1960 MAMMALIAN HIBERNATION 483

rat tissue is seen — the rapid fall of rate from an initially high
value. In a fashion similar to that observed for the rate of
relaxation of diaphragm muscle (Fig. 2), the ventricular QO»
of the control hamsters declines less rapidly than for the rat and
more rapidly than for the hibernating hamster and bat. If one
‘*foree fits’? a straight line to the curves between the tempera-
tures of 17° and 43°C for comparative purposes, the calculated
Ka’s for the rat, control hamster, hibernating hamster and torpid
bat, respectively, are 9.8, 6.0, 4.5 and 4.6 keal. It may be econ-
cluded that in certain systems the hamster does ‘‘prepare’’ for
the hibernal situation on a cellular level. From all evidence

e Rat, ventricle Qo,

4 Hibernating hamster, ventricle Qo,

o Torpid bat, ventricle Qo,

Normal hamster, ventricle Q 0,

36
oO.
4g° 43° 38° 3? 24° bra 10° oi
104
7

Fig. 4. Logarithm of the initial QO» of ventricle slices (kr) as a fune-
tion of the reciprocal of the absolute temperature of incubation.

presented so far, this is not accomplished by merely increasing
concentrations of enzymes and by storing substrates as would be
indicated if the rate-temperature plots were parallel with dif-
ferent intercepts. On an operational level, the lower Ea’s for
the hibernators suggest an adaptation of enzyme properties.
Whether this means that the curves which have been obtained
are due to a metabolic shift such that they are characteristic of
different limiting enzymes, alternate pathways, or to adaptations
of the properties of given enzymes which make them less suseep-
tible to changes in configuration over a range of temperature (as
might be suggested by Kavanaugh’s hypothesis (1950) ), is not at
all certain.

484 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

That these differences in rate temperature plots are probably
not ubiquitous among the tissues was shown by similar experi-
ments with brain shces. In this case, the curves for brain QO»
were essentially parallel with only shg¢ht differences in potential
intercepts (Fig. 5). The Ea’s were approximately 11.0 keal,
except that for bat brain, which was slightly lower at 10 keal.

Rot brain, Qo,

+ Normal Hamster Brain Qo,

a Hiberinating Hamster Brain Qo;

0.3
Torpid Bat Brain Qo,

0.1

31 32 33 34 3:5 36

Fig. 5. Logarithm of the initial QOs of brain slices (kr) as a function
of the reciprocal of the absolute temperature of incubation,

Similar results were obtained for the rates of anaerobic glycolysis
of brain slices, with a slight suggestion of lower Ea’s for hiber-
nating animals (South, 1958). This result was rather unexpected
in view of the experiments of Peiss and Field (1950) im which
brain minees of the Arctic Cod and Golden Orfe were studied.
‘hese animals lived at ambient temperatures of —1.5° to +2.0°C,
and +25°C, respectively. The Ea values between 10° and 25°C, as
calculated from their published data, were 12.3 keal for the
Arctic Cod and 16.0 keal for the Golden Orfe. Between 10° and

1960 MAMMALIAN HIBERNATION 485
0°C the Ea for the former fish did not change significantly while

that of the Orfe rose to about 25 keal — a pronounced decrement
in rate.

Heart Sarcosomes
6
P

ae
> 20-
é
io
E to_
a Ea(kcal)
5-24°C |
fs] ° Rat 28.3 =
<q -| + Control af My
hamster 20.8 ae
‘| Q Hibernating
,| homster 20.4 A
+
17° wp 5°
35 36 (037
lot
no

Fig. 6. Rate of oxygen consumption by heart mitochondria as a fune-
tion of the reciprocal of the absolute temperature of incubation, Semi-
logarithmic plot. Each point represents the mean of 6 to 10 experiments.
Pyruvate (malate) substrate.

Just as not all tissues or systems evidence the same apparent
adaptive response of cardiac tissue, so it is to be expected that
not all the reaction of energy metabolism will mirror these altera-
tions. This implies that only those enzymatically-controlled re-
actions which may be rate-limiting under these conditions need
be altered. Furthermore, the phylogenetic adaptation which dis-
criminates between the hamster which can hibernate and the rat

486 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

whieh cannot may occur at a different loeus in the metabolie
ehain than does the change which apparently distinguishes the
hibernating hamster at 5°C from the hamster dwelling at a com-
fortable 25°C. It must be confessed that these statements were
made utilizing the pecuhar prognostic powers obtained from a
finished experiment.

50
024.156 0.647 X
7) anal
@
E
O 20
Oo
= Y= 7825-0. 467 x
5 10
wy yan
Ve Y 717.3987 0.449X

5 \
fo} \
@m 5
So
=. ny

. oA
4 XY 4
SS
2 |

re Ea(kcal) KN
= 5°- 24°C
X feat 29.5

5
~ Control
= * hamster 21.4
Hibernating
4 hamster 20. 6 s

104
——

Fig. 7. Rate of esterification of inorganic phosphate by heart mito-
chondria as a function of the reciprocal of the absolute temperature of
incubation. Semilogarithmie plot. Each point represents the mean of 6 to
10 experiments. Pyruvate (malate) substrate.

As part of the continuing effort to characterize and isolate
those metabolie factors involved in hibernation, heart mitochon-
dria of rats, control hamsters, and hibernating hamsters were

1960 MAMMALIAN HIBERNATION 487

isolated. The rates of oxygen uptake and phosphate esterifica-
tion and ‘‘P/O ratios’’ were determined (cf. Maley and Plaut.
1953, for general methodology, which was modified for use here).
Pyruvate, with malate as a ‘‘sparker,’’ was the substrate. Ineu-
lation temperatures ranged from 0°C to 88°C.

Reference to Figure 6 reveals that even at the mitochondrial
level the rat heart remains very different, the slope of the line of
rate of oxygen uptake is again steeper and crosses those of the
hamsters as before. An additional fact is also immediately ap-
parent in that the slopes of the rates of oxygen consumption by
hibernating and control hamsters are parallel. This ean be in-
terpreted only as an increased enzyme concentration, accompany-
ing the hibernating state, at some point alone the portion of

===2 ‘Rat

+ Normal hamster
——4 Hibernating hamster

Os 5 10 17 24 =) 38
Temperature (°C)

Fig, 8. P/O ratio of heart mitochondria as a function of temperature.
Rectangular coordinates. Pyruvate (malate) substrate.

the metabohe pathway responsible for oxidative metabolism of
pyruvate. These observations are reinforced by the almost identi-
cal graphic form of the rate of phosphate uptake (Fig. 7) and the
closeness of the values of Ea. These plots and that of Figure
8 indicate that while the efficiency of phosphorylation is essen-
tially unaffected by temperature and not related to hibernation
as such, the rate at which it is accomplished is of central im-
portance.

It appears, then, that a specific enzymologie difference exists
between rats and hamsters along the pyruvie oxidase, dehydro-
genase and terminal electron acceptor systems. Precisely where
this hes, it is not yet possible to say. The factors responsible for

488 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

the change in slope correlated with the assumption of hiberna-
tion by hamsters (I*ig. 4) have been dissected out of the oxida-
tive phosphorylation system as studied here. The change in econ-
centration is, in all probability, an ancillary change.

sefore proceeding, it would be well to emphasize that while
the various values of Ka have been diligently calculated, direct
comparison with values obtained in other experiments and by
other authors studying systems of various complexities has been
avoided. For those desiring such data, the articles of Sizer
(1943), Kavanaugh (1950), Johnson ef al. (1954), and Feigen
et al. (1958) may be consulted.

The reasons for this avoidance are several. Too many variables
exist, such as pI, ionic milieu and strength, the question of pos-
sible inactivation at temperature extremes, presence of various
inhibitors, and the subjective factor of just how and where the
curves or slopes are fitted. Unless it can be shown that a particu-
lar catenary series of reactions depends upon a specific rate-
limiting reaction as characterized by the Ea it very well may
be misleading to assume such a relationship. An example of this
night be to conclude, since the rate of contraction of diaphragm
muscle of hibernating hamsters has an Ea = 20 keal and bacterial
dehydrogenase has an Ea = 19.4 keal, that the rate of contraction
of the muscle is limited by the bacterial dehydrogenase reaction.
Such a procedure must be approached with caution.

Since the approach of listing various reactions and comparing
fa’s has been rejected, we are left with the problem of explain-
ing why, in so many systems, the Arrhenius plots are less steep
for hamsters than for rats, and for hibernating than for control
hamsters. Several possibilities, some of which have been dis-
cussed above, can be cited, but few definitive answers given.

In view of the slower reaction rates at higher temperatures for
hibernants relative to the non-hibernants, and the crossing over
to a comparatively high rate as the temperature declines, the
suggestion of Brown (1956) may be dismissed as a generic solu-
tion. It does have some appheation as exemplified in the rates
of oxidative phosphorylation of heart sarcosomes of hibernating
and control hamsters and, possibly, to isometric tension produe-
tion as well (Fig. 3).

All other explanations must remain in the limbo of hypotheses
until the crucial experiments have been completed. However,
certain of these possible explanations have an attractiveness
which may be of value in the design of experiments.

1960 MAMMALIAN HIBERNATION 489

The easiest explanation would involve the idea that these
curves do represent the energies of activation of limiting reae-
tions in a catenary series. Hence, the hibernator would have
adapted by, again, imereasing the concentration of a eiven
enzyme to the degree that it no longer would be limitine and,
therefore, the curves represent the Ea of a different enzyme. If
this were so, one would expect that the rate of the reaction at
higher temperatures would be greater than that for the same
reaction of the rats, regardless of the slopes. This does not oceur.
A similar objection would apply to most direct applications
of Crozier’s (1924) theory, although it could obtain in certain
circumstances.

In an attempt to explain the laek of linearity of Arrhenius
plots and the alterations in rates at temperature extremes, Kav-
anaugh (1950) developed) an hypothesis which considered
changes in configurations of the enzymes. Henee, at higher
temperatures reversible denaturation of the enzyme occurs with
a concomitant reduction in its specific configuration (ef. Johnson
et al., 1954). In this partially ‘‘unfolded’’ or ‘‘uneoiled’’ state
the ‘‘active centers’? would no longer be in proper alignment for
maximal activity. At low temperatures, the formation of addi-
tional intramolecular bridges would result in the opposite situa-
tion, that is, the enzyme would be excessively ‘‘folded’’ so that
the **aetive centers’? would again be out of optimal alignment.
By implheation, it was areued that a sinele reaction could be
limiting for a complex system throughout the temperature
range. Such a formation could be adapted for our purposes by
postulating that certain critical enzymes of the hibernator might
be modified through formation of ‘*stabilizing bonds’? in a way
that such deviations from an intermediate configuration would
he minimized. Such an hypothesis is not ineonsistent with the
results as reported here except that it also implies that all Arr-
henius plots of enzymatically controlled reactions must be eurved,
a priori,

On a rather intuitive basis, a more attractive formulation
might be constructed from the experiments of Fraser and Kap-
lan (1955) on veast catalase. They found evidence, on the basis
of their values for Ea and other thermodynamic constants, that
intracellular veast catalase is adsorbed to an interface in a
partially unfolded configuration of low specificity and with a
high ATL of activation. Destruction of this interfacial associa-
tion of the catalase }y various agents decreased the AH®# to that
seen for the reaction when catalyzed by extracted yeast and

490 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124.

crystalline liver catalases, i.e. from an original level of 8.5
keal/mole to about 4 keal. Applyine the interfacial hypothesis
to hibernation it could be postulated that while the enzymes of
the rat usually exist in an analagous association with intracellu-
lar interfaces and structures with a concomitant loss of ordering
and high Ea’s, the eritical enzymes of hibernators exist in less
unfolded, less restrained but more active configurations. The
processes catalyzed by them would possess lower energies of ac-
tion.

While at the present time little additional evidence ean be
brought forth to support this idea, it is receiving considerable
attention in our laboratory at the present time.

REFERENCES

BIORCK, G. AND B, JOHANSSON
1955. Comparative studies on temperature effects upon the eleetro-
ORT

cardiogram in some vertebrates. Acta physiol. seand., 34:257-

272.

3ROWN, D. E.S.
1956. Some considerations of physicochemical factors in hypothermia.
In: The physiology of induced hypothermia. Nat. Acad. Sei.,

Nat. Res. Council, Washington, D. C., Publ. 451, Pn. 1-7.

CHATFIELD, P. O., A. F. Barrista, C. P. LyMAN AND J. P. GARCIA
1948. Effects of cooling on nerve conduction in a hibernator (golden
hamster) and non-hibernator (albino rat). Am. J. Physiol.,
155:179-185.

Crozier, W. J.
1924. On biological oxidations as a funetion of temperature. J. Gen.
Physiol., 7:189-216.

Dawe, A. R. AND P. R. Morrison
1955. Characteristics of the hibernating heart. Am, Heart J., 49:367-
384,

FrIGEN, G. A., D. DEVoR AND S. T. TAKETA
1958. Activation energy of ventricular contraction in anionically moddi-
fied solutions. Science, 128:1436-1437.

FRASER, M. J. AND J. G. KAPLAN
1955. The alteration of intracellular enzymes. III. The effeet of tem-
perature on the kineties of altered and unaltered yeast eatalase.
J. Gen. Physiol., 38:515-547.

GARB, S. AND M. B. CHENOWITH
1950. The T’ deflection of isolated mammalan heart muscle eleetro
gram. Cire. Res., 1:135-144.

1960 MAMMALIAN HIBERNATION 49]

GLASSTONE, S., K. J. LAIDLER AND H. EyriIneG
1941. The theory of rate processes. New York, 611 pp.

Hra@nauer, A. H., W. J. SHRIBER AND H. O. HatreRIUs
1950. Cardiovaseular response of the dog to immersion hypothermia.

Am. J. Physiol., 161:455-465.

JOHNSON, F. H., H. Eyvrina anp M. J. PoLissar
1954. The kinetic basis of moleeular biology. New York, 874 pp.

IKAVANAUGH, J. L.
1950. Enzyme kinetics and the rate ef biologieal processes. J. Gen.
Physiol., 34:193-209.

KAYSER, C.
1953. L’hibernation des mammiféres. Ann. Biol., 29:109-150.

LYMAN, C. P. AND P. O. CHATFIELD
1950, Mechanisms of arousal in the hibernating hamster. J. Exper.
Lool., 114:491-515.
1955. Physiology of hibernation in mammals. Physiol. Rev., 35:403-

25.

LYMAN, C. P. anp E. H. Lepuc
1953. Changes in blood sugar and tissue glycogen in the hamster during
arousal from hibernation. J. Cell. Comp. Physiol., 41:471-491.

Maupy, G. F. ann G. W. E. PLaur
1953. Yields ef oxidative phosphorylation by heart mitochondria, J.
3iol. Chem., 205:297-302.

McQUEEN, J. D.-
1956... Effeets of cold on the nervous system. Ju: The physiology of
induced hypothermia. Nat. Acad. Sei., Nat. Res. Council, Wash-
ington, D. C. Publ. 451, Pp. 243-250.

NARDONE, R. M.
1955. Electrocardiogram of the arctic ground squirrel during hiber-
nation and hypothermia. Am. J. Physiol., 182:364-368.

NaARpDOoNE, R. M., C. G. WILBER AND X. J. MUSACCHIA
1955. Electrocardiogram of the opossum during exposure to cold.
Am. J. Physiol., 181:352-356.

OSBORN, J. J.
19538, Experimental hypothermia. Respiratory and blood pH changes
in relation to cardiae funetion. Am. J. Physiol., 175:389-398.

Priss, C. N. anp J. FIELD
1950. The respiratory metabolism of excised tissues of warm- and cold-
adapted fishes. Biol. Bull., 99:213-224.
RaMsSEy, R. W.
1944. Musele physics. Jn: Medical Physies. Ed., Glasser. Chicago.
Vol. I, Pp. 784-798.

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Rune, C. H. W. ann R. H. Horn
1955. Cireulatory and respiratory effeets of hypothermia induced by
blood refrigeration. Am. J. Physiol., 182:325-330.

Sizer, 1. W.
1943. LEffeets of temperature on enzyme kinetics, Ady. Enzyme, 3:55-

SoutnH, F. i.
1958. Rates of oxygen consumption and glycolysis of ventricle and
brain shees, obtained from hibernating and non-hibernating
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Tal, J.
1922. The heart of hibernating animals. Am. J. Physiol., 59:467.

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DISCUSSION FOLLOWING SOUTH’S PAPER

STRUMWASSER noted that, in frogs, neuromuscular fune-
tioning continued at low temperatures. Choh-Luh Li and P.
Gouras (Am. J. Physiol., 192:464, 1958) showed that spontane-
ous miniature end plate potentials (recorded intracellularly from
the sartorius muscle) and contraction still occurred as low as
—1°C in response to direct and indirect electrical stimulation.
Ile then asked, concerning SOUTH’S experiments on differences
in neuromuscular transmission between hibernating and non-
hibernating mammalian species, whether his 7 vitre experiments
had taken into account natural intercellular parameters which
may be the important factor in the hibernating animal rather
than some fundamental difference in muscle or nerve. SOUTH
replied that he knew of Choh-Luh Li’s paper, but he could only
say that the results described were as he (SOUTH) obtained
them. He indicated he intended to carry the work further using
micro-eleetrodes.

STRUMWASSER then asked if, using the proper frequency,
duration and waveform of electrical stimulus to the nerve
terminals at the neuromuscular junction, one might not be able
to extend neuromuscular transmission in the rat to lower temper-
atures than SOUTH had described. SOUTH rephed that he
used various frequencies, strengths and durations, and no dift-
ferences were obtained. The optima were reported.

XX VI
THE EFFECTS OF IONIZING RADIATION
IN HIBERNATION’

By Douaeuas E. SMITH
Division of Biological and Medical Research
Argonne National Laboratory
Lemont, Illinois

The subject matter of this paper falls into two broad categories.
One concerns the use of hibernating animals as tools in the study
of the development of and protection against damage by ionizing
radiations. The other deals with the effects of ionizing radiations
on the induction and maintenance of the state of hibernation.

I. The Development of Radiation Damage in the
Hibernating Mammal

A. Introduction. Practically nothing is known of the sequence
of events that lead up to the morphological and biochemical
lesions detected hours and days after the irradiation of mam-
mals. Because of its markedly low body temperature and metab-
olism, the hibernating mammal appears to be a promising tool
for the study of the development of radiation damage. Although
one would not expect the changes attending the initial transfer
of energy from incident radiation to be influenced within the
ranze of temperatures found in homeothermice and hibernating
animals, one would anticipate that the subsequent chemical
changes would be temperature-dependent. It is possible in hiber-
nating animals not only that the development of radiation
damage mieht be generally slowed but also that it might be dif-
ferent when compared with that in irradiated homeotherms. Such
differences might be expected on the basis of the complex inter-
relationship of possible alternate metabolic pathways, differences
in activation enerey of enzymes and different radiosensitivities
of cnzymes.

To the present, two main types of study have been carried out
on the effects of ionizing radiations on hibernating mammals. In
one, account has been taken of the mortality of x-irradiated
hibernating animals. In the other, histopathological examina-
tions have been made on the tissues of hibernators similarly

1'This work was performed under the auspices of the U. S. Atomic Energy
Commission,

494 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

treated. Observations have been made also on a few other signs
of radiation injury.

B. Lethal effects. Table I summarizes the mortality data
vathered on several species x-irradiated while hibernating and
kept in hibernation for varying periods of time thereafter. It
is clear trom these data that the expression of radiation damage
as mortality is markedly slowed during hibernation. In the
eround squirrel and dormouse there is no evidence for develop-
ment of damage resulting in death during the period of hiberna-
tion, whereas the data from bats clearly indicate the development
of such injury in the hibernating state. (Multiple hemorrhages
and coagulated blood, gross signs of radiation damage, were
found in the gastrointestinal tract of bats dying during the
period of hibernation after irradiation (Osborn and Kimeldorf,
1957). In bats it is also obvious that the level of mortality during
hibernation is dependent upon radiation dosage. Furthermore,
in the httle brown bat observations beyond the 80-day period
shown in Table I reveal a progressive development of damage
leading to death (cf. the 15,000 r group of Figure 1).

If, after the hibernation periods shown in Table I, the animals
are removed to and kept in a warm environment, death occurs
as in the case of non-hibernating animals irradiated and kept in
the warm environment. Accordine to some workers (Kunkel
ct al., 1957) the mortality pattern is that to be expected if the
animals had not been exposed to x-rays until brought into the
warm environment. Examination of the available detailed mor-
tality data, however, reveals that for ground squirrels (Smith,
1959a) and marmots (Smith and Grenan, 195la) the time to 50
per cent mortality and the average survival time (caleulated
from the time of removal to the warmer room) are shorter in the
eroups exposed to radiation while hibernating than in the groups
exposed and kept in the warm environment. This indicates that
radiation damage leading to mortality is developed during the
period of hibernation. Quite the opposite results are found in
the dormouse (Kiinkel ef al.. 1957). Here the average survival
time and time to 50 per cent mortality calculated after removal
from hibernation is about 3 times as long in the group irradiated
while hibernating as in the group irradiated and kept at 20°C.
Moreover, the mortality at 30 days in the 20°C environment is
67 per cent in the former and 83 per cent in the latter group.
These data indicate the possibility that the 21-day period of
hibernation after exposure to x-rays confers some protection
against damage leading to mortality.

1960 MAMMALIAN HIBERNATION 495

TABLE |
Mortality of X-irradiated Mammals During Hibernation

No. of days in

No. of hibernation after
Treatment Reference animals irradiation % Mortality
Ground squirrel (Citellus tridecemlineatus )

S00 r C1)4* 24 30 0
1000 r (1) 12 30 0
1200 r (1) 2 30 0
LOOO Yr (2) 90 22 0
2000 (2) 4 21 0
Control* (1,2 60 — 80-100

650 Yr

Marmot (Marmota mona )

3) i 28-42 14

SOO r (3) il 21 0
Control® (3) 9 — 67

Dormouse (Glis glis )

700 ¥ (4) 21 Pall 0

Control* (4) 18 — 83
Pallid bat (Antrozous pallidus pacificus )

1500 ¥ (5) 9 42 56
3000 ¥ ¢5;) 21 42, 3
6000 + Cd) 9 42 89
Control* (5) 35 — 100

Yuma bat (Myotis yumanensis saturatus )

500 Yr (5) 8 42 3

Control* (5) 10 — 80
Little brown bat (Myotis lucifugus )

500 r (6) 40 30 30
1000 r (6) 40 30 25
5000 r (6) 40 30 32

15000 + (6) 40 30 52
Control* (6) 160 — 100
*The controls were irradiated and kept at 20-27°C. The mortalities are for

the same irradiation dosages and times after exposure as in the hibernating

x. oups.

** (1) Doull and DuBois
(1951a) ; (4) Kiinkel et al. (

(1959b).

(1
195

993); (2) Smith (1959a) ; (38) Smith and Grenan
7); (5) Osborn and Kimeldorf (1957) ; (6) Smith

496 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

C. Tissue damage. Wistopathological findings have been re-
ported for ground squirrels (Fitch ef al., 1955) and marmots
(Brace, 1952) during hibernation, after exposure to x-rays. Evi-
dence of distinet cellular damage (failure of mitosis, nuclear
changes and cell death) was found in the spleen, lymph nodes
and bone marrow of the hibernating ground squirrel as early
as 6 hours after a dosage of 800 r. Thereafter the rate of develop-
ment of cellular damage was substantially the same in the
hibernating and non-hibernatine animal. Preliminary studies
CS. W. Lesher and D. E. Smith, unpublished observations) have
shown somewhat similar results in the erypts of the duodenum
of the hibernatine ground squirrel exposed to 1000 r. In con-
trast to the findines in the ground squirrel, no signs of cellular
damage were reported (Brace, 1952) for bone marrow, lymph
nodes, spleen, lunes, heart and adrenals of irradiated hibernat-
ine marmots. Loss of ovoevtes, ovogonia, spermatocytes and
spermatogonia were observed. Signs of hematopoiesis were ab-
sent in both the irradiated and non-irradiated marmot through-
out periods of hibernation as lone as 8 weeks. Upon removing
both groups to a warm environment, hematopoiesis was apparent
after two days and igh at 7 days in the non-irradiated animals.
No recovery of hematopoiesis was noted in the irradiated group
even after 7 Cavs in the warm environment. In the ground
squirrel, hematopoiesis was absent durine the 29-day period of
hibernation after irradiation and failed to return after three
days in the warm room. This finding is in contrast to the observa-
tion of significant recovery of hematopoiesis at 8 days after
irradiation of animals always in the warm environment. Here
again is a possible difference between hibernating and non-
hibernating animal with respect to the effects of radiation dam-
age. Thus, not only does it appear that recovery of hematopoiesis
is not possible during hibernation after irradiation but it also
seems that hibernation was attended by changes that do not
allow the return of hematopoiesis when the ground squirrels
are removed to a warm environment.

D. Peripheral blood. During 42 days of hibernation after
exposure to 800 r the marmot is reported (Smith and Grenan,
1951b) to show no changes in the number of circulating red or
white blood cells or in the pattern of the differential blood count.
[pon removal to a warm environment the levels of both the red
and white blood cells fell rapidly. No change in circulating blood
cells has been found (Kiinkel and Schubert, 1959) in the dor-
mouse. Indirect indications that the same is true for the ground

1960 MAMMALIAN HIBERNATION 497

squirrel (Fitch ef al., 1955) come trom the finding of increased
hemosiderin in the red pulp of the spleen in irradiated, non-
hibernating, but not in the irradiated, hibernating animal. The
above data suggest that the life-span of the blood cells’ is
markedly prolonged in the hibernating state.

Li. Chemical ineasurements. Alkaline phosphatase activity in
the spleen of the ground squirrel is mereased after irradiation,
hut the increase is smaller in the hibernatine than in the non-
hibernating animal (Peterson and DuBois, 1952). Changes in
serum proteins are significant in the irradiated, non-hibernating
dormouse but are absent during hibernation after irradiation
(Schubert et al., 1957). The incorporation of P82 into deoxyri-
honucleire acid of the intestine in the irradiated, hibernating dor-
mouse, however, is 90 per cent lower than that in its non-irradi-
ated, hibernatine control (Kiinmkel and Sehubert, 1959).

I’, Conclusions. It seems clear from the existing data that
lethality resulting from x-irradiation is markedly slowed in the
hibernating mammal. Further conelusions must be made with
considerable caution because of the limited and fragmentary
nature of the experiments. A number of other influences of
hibernation on the development of radiation damage are stronely
suggested by the existing information, however, and should be
mentioned. Thus, there appear to be species differences with
respect to the development of cellular damage (ef. the ground
squirrel and marmot) and to the development of damage leading
to lethality (cf. posthibernation mortality of the ground squirrel
and marmot in comparison with that of the dormouse). The
latter is of especial interest, since it Indicates that there is repair
or failure of development of lethal damage in the irradiated,
hibernating, as compared with the irradiated, non-hibernating
dormouse. Tlihernation appears to prevent or slow the recovery
of at least one process inhibited by irradiation (cf. the absence
of recovery of hematopoiesis of the irradiated, hibernatine
eround squirrel or marmot upon removal to a warm environ-
ment). It would seem highly worthwhile that the above phe-
nomena be thoroughly investigated. It is possible that the
explanation (based on preferential metabolic pathways, different
activation enereies of enzymes and different radiosensitivities of
enzymes) sugeested above can account for the qualitative differ-
ences between the hibernating and non-hibernating animal with
respect to the expre sion of radiation damage. These differences
suggest that when more detailed examinations of biochemical
systems are made in studies of hibernation per se, phenomena

498 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

explained by differences in activation energy of enzymes and
alternate metabolic pathways will be detected.

Il. Influence of Hibernation upon Chemical Protection Against
the Lethal Effects of Ionizing Radiations

Significant protection against the lethal effects of ionizing radi-
ation is obtained in various homeothermic mammals when cer-
tain sulfhydryl compounds are administered before irradiation
but not when such treatment is given after irradiation (Patt,
1953). Thus 80 to 100 per cent of rats injected with cysteine
prior to irradiation will survive a 100 per cent lethal dosage of
x-rays. No survivors are found when eysteine is administered
after exposure. It has been postulated that the sulfhydryl com-
pounds exert their protective effect during the act of irradiation
by reacting with the decomposition products of irradiated water,
by causing tissue anoxia, or by forming complexes with im-
portant constituents of tissue. It is of great interest, therefore,
that marked protection by cysteine has been reported (Kiinkel
et al., 1957) in the dormouse irradiated while hibernating at 4°C
but not treated with the sulfhydryl compound until 3 weeks
after irradiation when the animals were removed to a warm
room. The details of the experiments are presented in Table II.
None of the irradiated animals died during the 3-week period in
the cold. During the ensuine 30 days in the warm environment,
however, 67 per cent of the non-cysteine-treated, irradiated dor-
mice died; none of the animals injected with eysteme died during
this time. In the same study, cysteine administered within 3
minutes after irradiation to the 12 non-hibernating dormice
always kept in the 20°C environment was followed by 83 per cent
mortalitv in the following 30 days. It thus appears that the
mechanism of the protective action of cysteine may be the same
in the non-hibernating dormouse as it is in the homeothermic
mammal. In the case of protection by eysteine given 21 days after
irradiation of the hibernating dormouse, however, an entirely
different mechanism of action must be sought, since one would
not expect temperatures as high as 4°C to prolong for 21 days
the lifetime of the immediate products of irradiation postulated
for the site of action of cysteine in the homeotherm. It seems
possible that the low temperature of the hibernating dormouse
may, beeause of different radiosensitivity of enzymes and dif-
ferent activation energies of enzymes leading to alternative meta-
bolie pathways, brine about biochemical changes unlike those

1960 MAMMALIAN HIBERNATION 499

encountered in the homeothermic animal in that they can be re-
paired by cysteine. In the absence of administration of cysteine,
these biochemical changes are ultimately expressed as death.

TABLE II]

Influence of Cysteine upon Mortality after X-irradiation

Y% Mortality during
No. of animals ‘Treatment 30 days in warm room

Dormouse (Glis glis) (Kiinkel et al., 1957)

21 700 r while hibernating at 4°C, O7
transferred to 20°C after 21 days.*

i) 700 rv while hibernating at 4°C, 0
transferred to 20°C after 21 days*

and injected with eysteine, 500 mg/kg, ip.

IS 700 yr while awake at 20°C, 83
10 Cysteine, 500 mg/kg, i.p. before 700 1 0

while awake at 20°C,

Ground squirrel (Citellus tridecemlineatus) (Smith, 1959 a, 1960)

15 1000 r while hibernating at 5°C, 87
transferred to 23°C after 21 days.*

15 1000 r while hibernating at 5°C, SU
transferred to 23°C after 21 days*
and injected with cysteine, 950 mg/kg, iv.

20 L000 v while awake at 238°C. SU

20 Cysteine, 950 mg/kg, iv. before 1000 + 0
while awake at 23°C,

16 L000 ry while hibernating at 5°C, 81
transferred to 23°C after 15-30 min.*

16 1000 r while hibernating at 5°C,
ae

transferred to 23°C after 15-30 min.
and injected with cysteine 950 mg/kg, i.v.

*No animals died during the 21 days or 15-30 minutes of hibernation atter
Irradiation.

Protection by cysteine given after irradiation of the hibernat-
ing ground squirrel does not seem to oecur (Table IL). This is
not surprising when one considers the histopathology of the
irradiated ground squirrel (Fitch et al., 1955). Cellular damage
is evident in tissues of the ground squirrel as early as 6 hours
after exposure to 800 r of x-rays and, as pointed out above, the

500 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

rate of development and the ultimate degree of damage there-
after is about the same in the hibernating and non-hibernating
eround squirrel (Fitch ef al., 1955).

It appears that there is a distinct difference between the
eround squirrel and the dormouse with respect to the possibility
of protection by eysteine given after irradiation. Since no
information is available concerning the histopathology of the
irradiated, hibernating dormouse, no explanation involving dif-
ferences in tissue damage can be attempted. One can only say
that the development of radiation damage in the hibernating
dormouse is different from that in the hibernating ground squir-
rel with respect to the existence in the dormouse of a biochemical
defect that is reparable by cysteine. Such a defect is not present
in the hibernating ground squirrel either at 21 days or one-half
hour after irradiation.

[1]. The Influence of N-irradiation upon the Induction and
Maintenance of Hibernation

To the knowledge of the author, there has been carried out
but one set of experiments (Smith, 1959b) on the effects of radia-
tion upon the induction and maintenance of hibernation. These
experiments cousist of exposing bats (Myotis lucifugus) and
eround squirrels (Citellus tridecemlineatus) to various dosages
of x-rays in the non-hibernatine state (ambient temperature
23°C), plaecine them in a cold room at 5°C immediately after
irradiation, and observine them for hibernation thereafter.
Tables IIL and IV show that irradiation can prevent the indue-
tion and maintenance of hibernation in both species. It is appar-
ent that massive dosages of x-rays are necessary for the effect.
In the case of the bat, the phenomenon is dose-dependent, for as
the amount of radiation is increased, fewer animals enter hiber-
nation (Table II]). Irradiated bats that fail to hibernate within
the first 15 hours in the cold environment do not become dormant
thereafter and die during the ensuing 33 hours (Fie. 1). On the
other hand, bats exposed to 15,000 r, 30,000 r, 40,000 r, or
60,000 + and kept at 23°C survive as lone as 15, 15, 7 and 4
days, respectively (Smith et al., 1955).

The bats exposed to 15,000 1 or more show signs of altered
function of the nervous svstem beeimnine immediately after
irradiation. These signs consist mainly of a high level of spon-
taneous activity (running and flying about in the cage), circus
movements, and polydipsia. These activities are minimal after

1960 MAMMALIAN HIBERNATION 50]

15,000 r, but beeome more marked as the dosage is increased.
To test the possible effeets of polydipsia, groups of 6-10 bats
were completely deprived of water after being exposed to 15,000-
60,000 r and placed ina room at 5°C. These animals showed the
same mortality patterns as irradiated bats which always had
access to water when im the cold.

TABLE II]

The Influence of X-irradiation upon Ability of the Bat to Enter
Dormaney when Exposed to 5°C (January experiment )
(Smith, 1959b)

Number of bats? in dormaney

A-ray a Tae ea as ee
losage day 12 day 2 day 3
D00 r 40 40 40
9,000 r 40 40 40
15,000 r 38 38 38
30,000 ¥ aya) 33 33
45,000 ¥ 0 0 0
Control 40 +0 40)

140 bats per group except for the 60,000 rr group which contained 12> bats.

2 Time after N-irradiation and exposure to 45°C,

TABLE IV

The Influence of N-irradiation upon the Ability of the
Ground Squirrel to Enter Ilibernation upon Exposure to 5°C
(Smith, 1959b)

Number of ground squirrels in hibernation

X-ray Number -_
poss ae sapiens ia 1 1 day 2 ; day 3 day + day 3 day G6 day7
800 r 12 7 ii 10 12 12 12 ale
900 r Le 5 i) ) 10 10 10 10
1,200 r 12 ; 12 12 12 10 12
5,000 r 6 ] a 0 0 0 0 02
10,000 » 6 ] l 0 0 0 0 0
Control 70 30 65 67 67 67 67 67

Time after N-irradiation and exposure to 35°C.

2 Animals die on Sth and 9th day after irradiation.

502 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Signs of heightened irritability and increased spontaneous
activity are also seen in ground squirrels exposed to 5000 r or
10,000 r. These signs are apparent for several days after irradia-
tion both in animals placed at 5°C and 23°C.

100 §

PERCENT SURVIVAL
re)
Oo

\
\
02 4 6 8 10 12 14 16 18 20 22 24 26 28 30 60 62 64 66
TIME (days postirradiation)

Hig. 1. The influence of X-radiation upon the mortality of bats in a 45°C

environment (January experiment) @— —~@, control; O——O, 500 r;
Xx————X,, 1000 r; O——_1], 5000 r; @e----- @, 15,000 r; O-——--O,
30,000 r; N-—-———N, 45,000 r; G---~-C), 60,000 vr, All groups contained

{0 animals except for the one exposed to 60,000 r, which contained 12.

It may be of interest to note that the later in the winter that
bats are collected and irradiated, the greater the percentage of
animals that fail to become dormant within the first two days
of exposure to cold (Fig. 2). Data similar to those shown for
30,000 rin Figure 2 were also obtained in a series of experiments
with 40,000 1+ as the radiation dosage.

In vet another experiment, groups of 12 bats were exposed to
30,000 r in late March and kept at 23°C for 5, 10, or 24 hours
before they were placed in a room at 5°C. All of these animals
survived the stay at the higher temperature but died during the
first 48 hours after being placed at 5°C.

The indications that irradiation can disrupt the maintenance
of hibernation have been confirmed in other experiments on the
bat (Smith and Thomson, 1959) and ground squirrel (Smith,
1959a ). Prior to and early in the course of continuous x-irradia-
tion, bats that are hibernating at 5°C have a rectal temperature

1960 MAMMALIAN HIBERNATION 503

of about 5.5°C and an oxygen consumption of 0.03 ml/em/hr.
When a dosage of about 15,000 r has been reached, the rectal
temperature and oxygen consumption begin to rise and reach
values of 10-12°C and 5.0 ml/gm/hr, respectively. At this time
the bats are awake and active. Ground squirrels that are exposed
to 1000 r during hibernation at 5°C remain dormant during
irradiation but become fully awake and aetive about one hour
later. The animals stay awake for several hours and enter
hibernation once again,

100 §
90
80
10
6O

SURVIVAL

50
40
30
20

PERCENT

ne)

0 2@ 4 6.8 AO 2. 14 16-16 20-22-24
TIME (days postirradiation)

Fig. 2. The influence of time in hibernation upon mortality of irradiated

bats ina 5°C environment. @————®, collected and irradiated in January
(40 bats); O—-——O, collected and irradiated in February (12. bats);
A\——— X, collected and irradiated in Mareh (13 bats). Radiation dosage

30,000 r.

The several experiments described above indicate that only
those dosages of radiation that are high enough to elicit signs of
altered function of the nervous system prevent the bat and the
ground squirrel from entering hibernation. Animals exposed
to such dosages show unusually great spontaneous activity, and
appear unable to become quiet upon exposure to the cold. The
ability to assume a quiescent state in a cold environment seems
to be a primary requisite for entrance into dormancy.

yO4 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

The finding that massive dosages of x-radiation are necessary
to mbibit hibernation in the ground squirrel does not agree
with a previous report (Doull and DuBois, 1953) that sueh
inhibition is elicited by a dosage of 800 r. Since both these
and the above experiments were carried out on the same species
collected in the same region of Illinois, it is difficult to account
for the discrepaney in results except perhaps on the basis of a
difference in nutritional status in the two sets of squirrels.

Acknowledgment

The writer wishes to thank Doctors Valeska Evertsbuseh,
Robert L. Straube and John KF. Thomson for helpful suggestions
during the preparation of this manuseript.

REFERENCES
Brace, Ix.
1952. Histological changes in the tissues of the hibernating marmot

following whole body irradiation. Seience, 116:570-571.

Dou, J. AND Iw. P. DuBots
1953. Influence of hibernation on survival time and weight loss of X-
irradiated ground squirrels. Proce. Soe. Exp. Biol. Med., 84:

367-370.

Firen, FE. W., J. DouLL AND R. W. WISSLER
1955. Ilistopathology of the irradiated hibernating ground squirrel.
A.M. A. Arch, Path., 60:644-650,

KUNKEL, H. A., G. HOHNE And H. Maas
1957.) Der Hinfluss von Cystein und Wintersehlaf auf die Uberlebens-
rate der rontgenbestrahiter Siebenschlifer (Glis glis). Zschr

Naturforsch., 12b:144-147.

KUNKEL, H. A. AND G. SCHUBERT
1959. The influence of total body irradiation on deoxyribonueleic acid
synthesis and the protective action of cysteine (Investigations

on rats and loirs). Radiation Research, 9:141.

OsBorn, G. K. Anp D. J. KIMELDORF
1957. Some radiation responses of two species of bats exposed to warm
and cold temperatures. J. Exp. Zool., 184:159-170.

Part, H. M.
1953. Protective mechanisms in ionizing radiation injury. Physiol.
Rev., 33:35-76,

1960 MAMMALIAN HIBERNATION 505

Prererson, D. F. AND Ik. P. DuBoIS
1952. Effects of lethal doses of X-ray on phosphatases. J. Pharm.

Exp. Ther., 106:410.

ScHUBERT, G., H. A. KUNKEL AND H. MAAS
1957. Elektrophoretische Untersuchungen am Serum rontgenbestrahlter
und hibernisierter Siebenschlifer (Glis glis). Strahlentherapic,
103 368-375.

Smiru, D. E.
1959a. Protection of the irradiated ground squirrel by cysteine. Radin-
tion Research, 10:335-338.
1959b. Influence of X-irradiation upon dormaney in vertebrates, J.
Exp. Zool., 139:85-94.
1960. Failure of cysteine given postirradiation to protect the hiber-
nating ground squirrel, Radiation Research, 12:79-80.

SairnH, D. E., D. R. Russ anp E. M. Jackson
1955. Response of the bat (Myotis lucifugus) to X-irradiation. Radia-
tion Research, 2:330-338,

SMmituH, D. E. anp J. F. THoMson
1959. Physiological and biochemical studies on various species exposed
to massive X-irradiation. Radiation Research, 11:198-205,

SM1rH, F. anp M. M. GRENAN
1951la. Effect of hibernation upon survival time following whole-body
irradiation in the marmot (Marmota monax). Science, 113:
686-688.
1951b. Cireulating blood cells following radiation in hibernating wood-
chuck. Fed. Proe., 10:128.

DISCUSSION FOLLOWING SMITH’S PAPER

WIMSATT asked if adequate consideration had been taken
of hypoplastic tissue conditions or relative state of activity
during irradiation. SMITH replied that it had been considered
and explained further that in the marmot there was no evidence
of mitosis either in the controls or the hibernating animals, but
that in ground squirrels there apparently were indications of
low level mitotic activity in tissues in the hibernating state.
WIMSATT then observed that a difference between recoveries of
hibernating vs. non-hibernating animals after irradiation may
be a function of differences in the states of their blood-forming
tissues. In non-hibernating animals homoplastie differentiation
may predominate in the active marrow, whereas in the hibernat-
ing marrow the blood cell formation could be pushed closer to
the stem cells (heteroplastic differentiation). SMITH replied

506 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

that after irradiation one would expect the dividing cells to be
much more sensitive and more easily destroyed, that in the
active animal irradiation would be more destructive than in the
resting marrow of hibernation; this is to be noted in the ease of
the marmot which shows no indication of cell damage during
hibernation, but when removed from hibernation, radiation dam-
age rapidly develops.

MENAKER asked if the remarkable longevity of bats could
be correlated with an ability for unusual repair in the hibernat-
ine bat after radiation. SMITH replied that he knew of no
evidence for or against this.

POPOVIC then showed a slide bearing on the viability of
hibernating animals in a situation of oxygen poisoning. The data
he gave showed that ground squirrels would live in a pure
oxyeen atmosphere at 6 atmospheres for 0.3, 7, and 13 hours when
in the euthermie, artificially cooled, and hibernating states respec-
tively (ref.: V. Popovie, R. Gerschman and D. Gilbert, unpub-
lished observation). MENAKER asked how the results seen
compared to the rat. POPOVIC rephed they were just the same
as in uncooled ground squirrels. SOUTH then indicated he did
a similar probe experiment comparing rats and hamsters at high
oxygen tensions, and found that the onset of convulsions oe-
curred at the same time in both, but that rats died quickly
thereafter, whereas hamsters survived as lone as 6 hours after
the onset of convulsions. He believed that this effect was prob-
ably due to a hypothermic condition, although he had not meas-
ured body temperature.

FOLK asked if SMITH had any information on the local
effeets of radiation of hibernating animals, such as_ possible
protectiveness of hibernation against cataracts of the eyes fol-
lowing such radiation. SMITH replied that he did not.

STRUMWASSER then asked if a relationship existed between
survival time following irradiation and the metabolic rate for
the same species. SMITH replied that there are correlations in
homoiotherms in experiments employing thyroid extract. Be-
cause of their long post-irradiation survival time, it might be
thought that bats are extremely radio-resistant. If one considers
that bats at rest have dropped their temperatures to the environ-
ment, one can account for the prolonged survival time on the
basis of a lowered metabolism and rate of development of dam-

ace.

XXVIT
PANEL DISCUSSION

ALBERT R. Dawe, Chairman
E. F. ADOLPH

GrorGe H. Brsnop

KENNETH C. FISHER
DonaLp R. GRIFFIN

Rev. Basie J. LUYET

C. Lapp PROSSER

DAWE: There are a number of ways of beginning a discus-
sion such as this. I have chosen the method of going from the
general to the specific. I would like to go right to the heart of
the matter rather than sidestep it. The initial question for the
panel’s consideration is: ‘‘Is ‘hibernation’ a valid term?’’ I
begin this with the understanding that perhaps we are more or
less hke a group of people meeting in the 1500’s and discussing
‘*phlogiston’’ before the discovery of oxygen. Dr. Griffin.

GRIFFIN: I think it is a perfectly valid, useful term. I
use it all the time. I think | know what it means, although I do
think it is one that has to have a little qualifying. Obviously
the hibernation of a bear is something very different from the
hibernation of a turtle, but this mammalian hibernation, I think,
has been operationally defined even in the last day or so and
was probably further operationally defined the first day of the
conference (which I wasn’t able to attend). I think if you get
into terminology and start rejecting well known terms, you will
just have to call hibernation something else and that will make
all the problems more serious.

PROSSER: I was impressed by Dr. Bartholomew’s plea the
other day. He indicated that, physiologically, hibernation and
aestivation are not greatly different. His proposal that these
terms be restricted to the changes which oceur under natural
conditions rather appeals to me. I’m not convineed that his sue-
gestion of *‘facultative hypothermism’’ is really an adequate
substitute, however. In a sense, it is ‘‘facultative poikilotherm-
ism’? rather than hypothermism. I wonder whether the phenom-
ena that we are discussing, which are certainly all-inclusive with
respect to physiological processes, don’t fall into something of
a spectrum. Perhaps someone else will comment on this. Not

508 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

really having worked with hibernation myself, it is my impression
that the differences among different hibernators and certainly
the differences in the degree of torpidity are quantitative rather
than qualitative. According to the time involved in the torpid
reaction, the differences will become more marked. I wonder
whether the overall syndrome isn’t perhaps similar qualitatively
for all these patterns but when one starts quantitating, differ-
ences become more marked.

DAWE: It would be unique, of course, for this group to an-
nounce to the scientific world that hibernation was not a valid
term. We have had the question raised by Dr. Bartholomew, and
| think all of us have been stumbling over it ever since that
moment,

BISHOP: | think that the term ‘*hibernation’’ limits your
field if you take it strictly. If vou take this thing as a means of
saving enerey, varying metabolism, then it is a very general term
biologically. Thousands of plants do it. Many more animals take
some precaution to conserve energy. For instance, an injured
animal crawls off in the bushes and lies quietly. He cannot eat,
so he stops activating himself; he is conserving energy again.
There are all kinds of expedients that animals and plants take.
This is quite a general biological proposition — if you take as a
central problem of hibernation the ‘‘turning off of the heat,’’ of
the energy. This mammal is a special case because he has to
have some particular kind of stimulus to do it and some particu-
lar expedient for getting out of it when he is in too long. |
suppose those two things make it ‘‘hibernation’’ rather than just
saving enerey.

FISHER: By the same token then, Mr. Chairman, it would
he necessary to start making some subdivision within the term.
The enormous variety of manifestations included in the word
‘hibernation’’ has struck me most forcibly during these three
days. If ‘‘hibernation’’ is retained as an inclusive term, con-
fusion will result, it seems to me, if we do not clearly differenti-
ate the various forms it may take.

PROSSER: Certainly ‘‘hibernation’’ applies—the word has
been used for poikilotherms fully as extensively as for homoio-
therms. We have not discussed it here as the broad general term.
[ still feel that Bartholomew has a very strong ease for using
this as a very general term and then limiting the specific.

1960 MAMMALIAN HIBERNATION 5O9

BISHOP: Literally ‘‘hiber’? means winter, and ‘‘hiberna-
tion’”’ means passing the winter; this is the way the animal gets
through a cold period. The term as we use it apples to a certain
little corner of the study of the way animals and plants get
around all kinds of stressful situations. You are lmiting your-
self by this definition to a certain narrow field, and if it led
vou to exclusive interest in that field I think it would be a pity.

DAWE: I gather that this group believes the word ‘‘hiberna-
tion’’ (the ‘‘winter sleep’’ or ‘‘winterschlafen’’) no longer
covers all the things we talked about in the last 24% days.

LUYET: The question: ‘‘Is hibernation a valid term?’’
calls, first, for a definition of the term ‘‘valid.’’ Of the several
meanings which may be attributed to ‘‘validity,’’ I would like
to examine shortly here the followme three: (1) Has the term
‘hibernation’? been ‘‘scientifically serutinized’’ in such a man-
ner that its introduction in a biological dictionary as an ade-
quate designation of a phenomenon of nature can be recom-
mended? (2) Is the term ‘‘hibernation’’ obviously misleading
so that a substitution should be proposed? We have an example
of such a situation in the terms ‘‘warm-blooded’’ and ‘‘cold-
blooded?’ animals which were replaced, respectively, by ‘* homoio-
therms’’ and ‘‘ poikilotherms.’’ (3) Is the term free enough from
evident ambiguity or obvious inexactitude that it may, without
serious inconvemence, be retained for practical use? One may
notice that improper terms are often used in common conversa-
tion, e.g. the ‘‘rising’’ of the sun. The answer to (1) is, obvi-
ously, ‘‘No.’’ Even if we would like to scrutinize the nature of
hibernation, our knowledge of facts about it does not seem to be
advanced enough to permit a thorough analysis of that phenome-
non. The answers to (2) and (3) would depend on the actual
disclosure of evident ambiguities, obvious inexactitudes, or mis-
leading connotations of the term ‘‘hibernation.’’

DAWE: The second question for the panel: ‘‘Did you gain
the impression that there was sufficient distinction between
hypothermia and hibernation to clearly separate the two phe-
nomena ?”’

GRIFFIN: Let me have another try and see if I can make a
more congenial comment. As [ look back I see that this program
as a whole is entitled ‘‘Natural Mammalian Hibernation’’ and
T still think IT know what that means, and [ think operationally

510 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

(again) that I learned a lot about natural mammalian hiberna-
tion, although some of the papers have certainly dealt with
rather ‘‘unnatural’’ situations. Just as a basis for discussion,
I will stick my neck out here and say that what impressed me
in the last few days was the difference between a natural hiber-
nating state of an animal, in which it seems to be very cleverly
regulating its physiological functions at several levels of tempera-
ture and several levels of activity, and some of these cases of
unnatural cooling, in which the most striking difference, to me,
has been the absence or the much lesser degree of this very
regulation. So, as one who knows less and less about the more
and more he has heard in the last few days, that stands out.

DAWE: You feel then that there is a distinction between these
two phenomena. One should not confuse them; they are indeed
distinet ?

GRIFFIN: They are obviously going to merge and be a con-
tinumm but the distinction remains very real.

PROSSER: That’s why I would raise the point of quantitative
rather than qualitative differences. We have a whole spectrum of
degrees of the hypothermic state. One comment I would like to
add to a discussion of the day before yesterday. We talked about
this matter of ‘‘turnine the thermostat off.’? Some of us have
used the term ‘‘settine the thermostat down’’ rather than ‘‘turn-
ing it off ;’’ it seems to me that this is a little more appropriate,
although I am not sure that either one is very strictly accurate.

ADOLPH: I would like to emphasize further the regulatory
aspects involved in both hibernation and hypothermia. So often
we think of hypothermia as a way of paralyzing an organism. It
seems to me it is not just that; perhaps we are blocking a few
processes but at the same time we are uncovering a host of other
processes and regulations which weren’t apparent before. This
is the aspect of the subject which interests me particularly. As
an example of the things we uncovered — who would predict
that it is possible for the heart to continue beating to a certain
temperature, for nerves to keep on conducting to another certaim
temperature and then come to a eutoff or ‘‘biological zero?”’
Now the significance of a ‘biological zero’’ is the same, perhaps,
as the threshold for some activity. This is one of the character-
istics of this activity. If we then go above the ‘‘biological zero”’
we find some sort of relation between an activity and that tem-
perature, but the temperature coefficients (or whatever you wish

1960 MAMMALIAN HIBERNATION D1]

to call them) for the biological activity which have been meas-
ured in isolated tissues have in no case agreed with the tempera-
ture coefficients in the same activity in the intaet organism. This,
to me, illustrates the fact that there are factors at work influenc-
ing this activity, such as rate of heartbeat or rate of oxygen
consumption, which were not apparent until we used hypo-
thermia to uncover them. Hibernation, however, is another
combination of regulatory relationships, and I don’t expect to
find very much in common between hibernation and hypothermia,
because hibernation includes so mueh more than hypothermia.

LUYET: The question of the difference between hibernation
and hypothermia is, of course, immediately related to the ques-
tion of the difference between hibernators and non-hibernators.
The following differences have been mentioned, in the course of
this conference, as characterizing these two groups of animals:
(1) a hibernator immersed in cold water, in the summer, cools
more rapidly than a non-hibernator ; (2) the lowest temperatures
from which a hibernator and a non-hibernator recover are dif-
ferent; (3) the temperatures at which their metabolic activity
stops upon cooling are different; (4) the energy consumption of
a non-hibernator anesthetized by eold does not fall below that of
its basal metabolism; that of a hibernator in hibernation falls to
much lower values; (5) some physiological properties of the
nerves, In particular, conductivity, are different in the two
groups of animals; (6) so are some physiological properties of
muscle; (7) so are some of the functions of the endocrine system ;
(8) the respiratory rates of brain slices indicate a different re-
sponse to the same treatment; (9) the resistance to anoxia is
different. Thus, definite characteristies justify the classification
of mammals into two groups: hibernators and non-hibernators.
But the existence of marked differences between them does not
mean that the two groups should be considered as entirely dis-
tinct and self-exclusive. One may, similarly, enumerate a great
number of marked differences between typical animals and typi-
cal plants; but this would not justify the conelusion that ani-
mality and ‘‘vegetality’’ are essentially different and self-exelu-
Sive entities.

FISHER: Would it not be better, Mr. Chairman, to say that
hypothermia might be a part of hibernation. Hibernation is a
larger term and involves more than what is ordinarily understood
in hypothermia, not that they are entirely distinet but that one
nueht be a lesser thine than the other.

512 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

PROSSER: Here is a quantitative difference again. When
the bat or the hummingbird becomes quiescent the body tempera-
ture drops; this is just quantitatively different from the state of
prolonged quiescence as in true hibernation.

GRIFFIN: Can I add to that? I think I agree with Dr. Pros-
ser although I was using a little different wording. I would say
there is a continuum here and in my favorite “*beasty.”* the bat,
a sort of intermediate situation between the poikilotherms and
the most effective and resistant or stubborn homoiotherms. I sus-
pect that there may be one aspect of the whole relationship of
metabolism and temperature that needs further investigation.
and I understand that the panel is supposed to think about
directions or courses of action which may be taken in future
research. I think of bats quite commonly as spending a great deal
of their time in a rather poikilothermic state where their temper-
ature falls to that of the surroundings, and yet I suspect they
are still a part of the continuum with a very real homoiothermic
phase of their lives, too. Now, to my knowledge, this hasnt been
studied quantitatively. But when they are awake and flying
they seem to regulate, as far as I can tell from casual observation.
So you might think of bats ranging all the way from some of
the more labile ones that are almost poikilothermic (but not quite
because even they, when they are awake, may fivy out in sub-
freezing temperatures and, I am sure, keep their body tempera-
tures above 30°, probably above 35°, because bats don’t seem
to be able to fiy below about 30°), to those which fiy in a very
hot place. in which case there is abundant evidence (H. Mislin,
Helv. Physiol. et Pharmakol. Acta, 5:C1S,. 1947; Rev. Suisse
Zool., 48 :563, 1941; W. G. Reeder and R. B. Cowles. J. Mammal..
32 :389, 1951) that their wing membranes undergo considerable
vasodilatation and vasoconstriction, so that I suspect they do
regulate and are at least some sort of homoiotherm. Then, as
various people have pointed out here and elsewhere, they can
fight back against the cold and sometimes do to a limited extent.
Dr. Wimsatt tells me that his favorite pets, the vampires, don’t
seem to fall to low temperatures but resist cooling. Perhaps he
commented on that before I was here, so that I would just lke
to place these particular animals in Dr. Prosser’s continuum, if
T could. :

PROSSER: May I ask a question, Dr. Griffin, in that con-
nection? Do you consider that the hypothermia which a bat
undergoes at rest diurnally is similar to or different from the
state in the winter when it is more easily called ‘‘hibernating’’?

1960 MAMMALIAN HIBERNATION 513

GRIFFIN: I think the two states have a lot in common but
there are some differences, too, and | would borrow your qualita-
tive term and say I suspect some of the main differences between
bats, when they do this either in winter or summer, and other
mammals are in the time constants, Le., they ‘Sturn this thermo-
stat up and down”’ very quickly and flexibly. Superficially, they
are much the same, in my limited experience, in summer and
winter. If you put them in the cold they are quite likely to
cool in either season. But, | have also noticed differences, and
one of the most startling ones in my limited ‘‘philosopher’s ex-
perience’’ is that if you do try to do this in summer, the bat
usually dies in about a week. [I do not think this is just lack
of fat, because it occurs even when they are quite cool and
they can’t have burned up much fat or carbohydrate. On the
other hand, of course, everyone knows that in the winter Inber-
nation can go on for months. [ suspect there are several adjust-
ments to be made, and these are easier to make during the winter.
Dr. Mayer deseribed some of the histological changes.

PROSSER: Thank you.

DAWE: The next question for the panel is: ‘‘ Were the lines
of investigation deseribed all-inclusive or were there elaring
deficiencies in the research efforts represented ?”’

BISHOP: Would [I be extreme in saying that about three-
quarters of the work I’ve heard has dealt with the way the animal
eets out of hibernation instead of how he gets mm, or what ‘‘get-
ting in’’ involves? This change of the musele and so on, that
enables the animal to function at a low temperature, and the
arousal reaction which has been studied so much here are all
secondary sequelae to the fact that he first has to ‘‘go down”?
and has to have some incentive for ‘‘going down.’” That’s the
first point.

DAWE: The technical problem of obtaining data on the
animal goine into hibernation is extremely difficult. In this
respect, | think Dr. Lyman’s paper was somewhat classical be-
eause he has obtained some data on induction. There was a
smattering of other such information. Lack of data on induction
represents more a question of technique and inability for tech-
nique to cateh up with desire. I think all people working in hiber
nation would like to have data on induction.

514 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

GRIFFIN: Might [ put in a plea for my friends the bats. If
you insist on using stubborn animals like hamsters, of course
it is difficult; but if you are working with bats it is extremely
easy to study them while going into hibernation. They won’t do
it absolutely every time but they do it at least half the time
that you want them to.

KISHER: May I then put in a plea, since this is a large
problem, for the golden-mantled ground squirrel which im our
experience can be subjected to high and low temperatures, to
starvation and to platters of food, to water or a lack of it, to
shortening days or lengthening days, or constant days or constant
light or constant dark and still goes into hibernation when it 1s
ready to do so in the fall, and similarly comes out in the spring.
My point is that if both the bat and the squirrel are to be con-
sidered ‘‘hibernators,’’ then we must be careful to realize that
neither the ease with which the bat can be induced to enter
hibernation nor, conversely, the striking difficulty of inducing
hibernation in the squirrel during the summer months can be
regarded as characteristic of hibernation in general.

BISHOP: Nov can aestivation be induced by subjection to a
hot room,

FISHER: Exactly.

DAWE: Were there any other deficiencies in coverage of the
hibernation problem noted at this symposium ?

GRIFFIN: [I don’t see any deficiencies. I think it is a splen-
did coverage but I can see some thines that I hope somebody
will study in the future. [ don’t think these constitute a glaring
deficiency but just real opportunities that perhaps are now
ripe. | was very impressed by Dr. Strumwasser’s work, and by
the obvious importance of the nervous system in regulating
affairs, and hope that he or someone will use similar approaches
to a shehtly different aspect, which TI don’t think his particular
eround squirrel perhaps demonstrates as well as other hiber-
nators — that is, this resistance to aetual freezing. This phe-
nomenon has been deseribed a number of times — a hibernating
manual, as its body temperature gets too near the freezing point,
fiehts back or generates a little more heat and actually regulates
at this very low temperature level. I would like to know what
the brain is ‘‘sayine’’ in a bat or hamster or any animal that
demonstrates this phenomenon at 0.5 degree, instead of 6.1

1960 MAMMALIAN HIBERNATION 515

degrees. | would like further to reiterate the obvious that, if
one is able to study the nervous system with implanted electrodes,
as Strumwasser has demonstrated, one has tremendous advan-
tages. He has certainly stimulated me even further than I had
been stimulated by others before to learn how to do this. I ean
see a number of lines of work that I hope are going to come out
of this.

LUYET: On the question of ‘‘lines for future investigation,’
| would suggest that the program of a future conference in-
clude: (1) Phenomena related to hibernation in lower animals
(lower vertebrates and invertebrates), and in plants (in par-
ticular, dormancy and cold hardiness). (2) The development of
hibernation in the course of evolution, and especially under the
influence of changes in climatic conditions. A problem of great
interest would be that of the origin and development of aestiva-
tion.

DAWE: In other words, a study of comparative hibernation
in the first case and historical hibernation in the second.

ADOLPIL: I think I would like to eall attention to the desira-
bility of listing the thines that hibernators do not do, as well
as listing the things that they do do. This is another way of
describing what happens in a very profitable manner. For in-
stance, I would make the generalization that no hibernator is so
anxious to be cool that he uses evaporative cooling in order to
zet cool. Now this, to me, is a very significant thing. From an
energetic point of view it might be cheaper to expend some
water to get cooler than to have to spend more calories to stay
warmer, and yet we don’t know of any animal which uses this
sort of refrigeration or gets cooler than his environment in this
process of hibernation. However, behavior takes care of some of
this, because animals (to some extent) seem to choose cool places
in which to hibernate so that behavior is one of the elements
in hibernation, no doubt. This, also, we haven’t attempted to do
much with. Of course, the whole matter of induction of hiberna-
tion is bound up with behavior as well as with physiological
processes in the narrower sense. No doubt we all are looking
for triggers which will induce hibernation and the faet that in
200 vears we haven’t found any could be discouraging to us, but
if we now start to list all of the thines that have been tried, really
tried, and have failed, we might be farther alone than if we sim-
ply wait for new ideas on things that haven’t been tried before.

516 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

PROSSER: I have one comment on future lines of research.
We're usually looking for cellular approaches and perhaps we
could comment on that later, but | would like to recommend in
this listing a little more use of the clinical method. It seems to
me, from what I have heard here, that the hibernation phenome-
non is a syndrome, something like the stress syndrome of Selye.
There has been good use made of the clinical technique in ap-
proaching the stress svndrome by making as many different kinds
physiological and biochemical
measurements and, of course, histological — on the same organ-
ism. I can see great advantages in having a nearly complete
clinical description of single organisms. I think that this has
not been done as much as it meht be. We attempt to make one
kind of measurement and then hope to correlate that with another
kind of measurement made by someone else.

of measurements as possible

GRIFFIN: When you say single organisms, you mean

single species?

PROSSER: Yes, of course. But I do think that this clinical
type description of a mammal might be useful. I see advantages
in the cellular approach, also.

GRIFFIN: This is essentially what Benedict was trying to do
in his day. In fact, this analogy occurred to me during the
morning — that here we have this sort of ‘‘super-organism of
investigators of hibernation’? who bring in a whole variety of
methods today and are not concentrating on one species of ani-
mal. But | must say that my own common failings make me
quail at the thought of encompassing this enevclopedic battery
of data.

PROSSER: Most of us ike to work alone, but there is some-
thine to be said for group research. I have seen it succeed in
this kind of problem, specifically with respect to the radiation
syndrome. Sometimes, if you have a whole laboratory of people
who are using different techniques on the same organisms they
vet a complete picture.

DAWE: Thank you. The next question: ‘‘What are vour
thoughts with respect to the ‘thermostat’ ?’’

BISHOP: You would expect this ‘‘thermostat’’ to be espec-
ially illustrated in plant reactions, if you remember how small
a trigger is necessary to set off a plant hormone reaction. For

|
—_"
~)

1960 MAMMALIAN HIBERNATION f

instance, a plant that has a day and night eyele which induces
eoine to seed — that’s a survival trait. Now a flash of Heht in
the middle of the dark period — just one flash — breaks the
cycle. Just a few seconds of light in a 12-hour period will change
the whole pattern of that behavior in plants. I suppose there
are thines lke that in hibernation, such as a cue to set this
thermostat off, which may be so sheht that you have not de-
tected them as yet. Such a sheht cue might, for instance, set up
a hormonal reaction (as in some plants) which in turn could
change the animal’s behavior drastically. Now if you assume, as
happens everywhere else, that these are hormonal reactions which
control metabohe rates, then the essential thing here is a redue-
tion of the energy. If you reduce the energy that an animal ean
put out, it doesn’t matter whether or not he has a thermostat, he
can’t use it. Once vou get the animal with the energy to come
down below where he can keep himself warm, the thermostat is
out. From there on, as | understand, most of the animals or
many of them, follow the environment down.

GRIFFIN: No, I think the evidence presented is quite to the
contrary. Before | came to this conference or heard otherwise
of some of the evidence to be presented here, | would have agreed
with you completely. But | think the data of Dr. Strumwasser
and others have shown that there is some sort of thermostat,
using the term loosely, that is, some kind of regulator.

BISHOP: Only at the beginning of induction when the animal
hasn’t gotten into the full hibernating state. Strumwasser’s
repeated warmings seem to be the persisting though sluggish
functions of a normal or non-hibernating reaction. The only ease
where anybody demonstrated and insisted on regulation at a low
temperature was in the bear and promptly the protest was made
that he was not hibernating — that this is not hibernation.

PROSSER: Didn’t Strumwasser find activity in the brain
even when the animal was quiet and very cold?

BISHOP: Yes, sure.

GRIFFIN: Furthermore, I don’t know whether he said this
or whether it was in his paper that brain temperature seems to
stay quite constant within 1/10 of a degree, at 6 degrees.

BISHOP: It does if the environment stays constant.

d18 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

GRIFFIN: No, I gather with fluetuatine environment. (Dr.
Dawe then called on Dr. Strumwasser, in order to answer this
point. )

STRUMWASSER: Well, | think vou have missed one point
and perhaps I can clarify this. If you remember, when these ani-
mals were dropping their temperature to certain critical points
(the ‘‘test drops’’), although the environment was relatively
stable, plus or mimus one-half a degree, each animal was regulat-
ing in the sense that it shivered and maintained a certain high
level of brain temperature. The particular level of brain tem-
perature was dependent on the number of days it had been in
the cold. It was ‘‘seeking out a preferred level,’’ let us say. I
interpret these findings to mean that the biological svstem was
choosing a level and regulating at that level.

BISHOP: [I can make a different interpretation. I would
say the animal is still sensitive to cold, and responds, as nor-
mally, by shiverine but that his sensitivity to cold was changing,
as my hand would adapt if I kept it in cold water. IT wouldn’t
Feel a cold stimulus as acutely. During hibernation the animal
loses the reaction of shivering or other processes of heat produe-
tion, and thereby cools off. At other times, cooling sets. off
enerey-yielding reactions and thus prevents cooling, acts lke ¢
thermostat if you wish; the thermostat turns on the heat. Shiver-
ine is, In part, a reflex response to a sensory stimulus of cold,
and probably, in part, to internal stimuli from low body tempera-
ture not resulting specifically in sensation. Strumwasser’s evi-
dence of repeated recovery of temperature, but each successive
time from a colder state, appears to me to indicate that his
animal is adapting. If the sense organs adapt, then shivering
occurs, if at all, only to a more severe stimulation than normal.
His animals appear to be cooling more rapidly than their cold
receptors can become adapted. Perhaps if cooling were more
eradual, the receptors would adapt progressively without these
periodie recoveries. The question then would be: what permits
the adaptation in the first place as compared to the non-hiber-
nating state. Perhaps something happens internally, in the
nervous system, let us say, which reduces the effect of cold stimu-
lation but which can do so only slowly and in Strumwasser’s case
cooling as a stimulus overpowers cooling as a central depressant,
and the temperature oscillates as a result. The ‘‘thermostat’’
then has at least two sections, a sensing apparatus and a means
of increasing the heat, both processes being depressed by cold.

1960 MAMMALIAN IIBERNATION 519

| wouldn’t call that a thermostat, where the ‘‘stat’’ implies con-
stancey. | am not defining a thermostat. I am defining a state of
so little enerey the animal never can get up to where the thermo-
stat regulates, unless he can rouse out of hibernation completely.

GRIFEIN: There is regulation. I don’t know whether the
evidence was discussed here but there are several of these cases
where down right near freezing, as an animal is cooled from 1
degree to 0.5 degree its metabolic rate increases, but it doesn’t
wake up .

BISHOP: You haven’t got a thermostat actually but you
have a reaction, perhaps to a further decrease of temperature, as
a shehtly effective stimulus.

GRIFFIN: Well, your cold reaction tends to regulate the body
temperature at this very low level and this is what I mean by
a thermostat.

STRUMWASSER: What is the thermostat? Can we come
to a deseription of it? Is it not something which we put in a box
for purposes of communication but which consists of detectors,
tracts carrying information, integrations going on? We put all
these parameters into a box and we eall it a thermostat. We’re
not selecting any one area of the brain necessarily or just picking
on a few neurons; it is a system. Don’t you agree with that, Dr.
Bishop ?

BISHOP: Sure.

STRUMWASSER: Well then, this is brought into operation
at these critical levels at which the animal’s system chooses to
regulate. If the external environmental temperature begins drop-
ping, then the animal produces a little more heat and if the
environment drops too fast, the animal tries to arouse after the
thermostat has been activated, but the thermostat is never turned

off.

BISHOP: No. That’s what I was saying a while ago; the
thermostat is there all the time but if the animal isn’t producing
enough heat, he can’t use it. If you had a heater in a bath, a little
heater, just enough to warm the bath a couple of degrees and if
you set a thermostat at 10° above this, it would never act at all,
but the bath would warm up a little above its surroundings.

D0 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

DAWE: Next question: ‘‘Do you feel there was good evidence
of an internal ‘clock’ mechanism in operation in hibernation in
the mammal ?’’

FISHER: As I have already stated, the onset of hibernation
in autumn and its cessation in spring in the golden-mantled
eround squirrel are, in our experiments, independent of external
conditions ; this implies that they be controlled by such a ‘‘clock’’
mechanism. This question in relation to the other animals dis-
cussed here is still open, it seems to me. The arousals from
hibernation which occur periodically every 4-5 days in the ham-
ster, every 2 weeks in the squirrel, and so on, also suggest an
internal ‘‘cloek.”’

BISHOP: It seems to me you are getting into another seman-
tie difficulty when you label these things ‘‘clocks,’’ or when
you call a reflex a ‘‘feedback’’— using these mechanical terms
to describe a biological proeess. Now [I would say that it is a
‘fevele’’ .

FISHER: Agreed.

BISHOP: ...a eyele of biological activity which recurs every
so often and gives you a time interval. But you are getting a
little bit — well, it’s like an engineer talking about biology —
he usually gets off on the wrong track.

GRIFFIN: You want to include the thought of the cycle being
endogenous — whether vou turn it off or not —the fact that it
is not just regulated from the outside. That’s the important
thine.

BISHOP: There are plenty of recurrent phenomena in biol-
ogy after all; they were invented before clocks were.

DAWE: Next question: ‘‘Did you feel there was good evi-
dence for the presence of a hibernating gland?’’ (The panel all
agreed that the influence of brown fat on hibernation had not
been established. )

BISHOP: If you change the question a little bit and say, is
there any evidence against a glandular control, I would say that
there is no evidence against the pituitary. Nobody has ‘‘hung
it on it’’ yet, but nobody has given it very thorough investiga-
tion.

1960 MAMMALIAN HIBERNATION 52]

PROSSER: It seems that the adrenal eortex is more involved
than the pituitary. (Discussion of the adrenal cortex was post-
poned by Dr. Dawe.)

DAWE: Next question: ‘‘Should this conference go on ree-
ord as advoeating the point of view that further research of a
erash-type into hibernation may make possible human hiberna-
tion which in turn may make man’s space flight for lone periods
of time feasible ?”’

ADOLPH: I think the less said about space the better re-
searchers we'll be.

DAWE: Next question: ‘‘Is it possible that, with prolonged
hibernation, starvation ultimately results and the awakening sig-
nal is related to the depletion of food reserves, ete.?”’

BISHOP: LT asked somebody the question, suppose you ear-
ried an animal for an indefinite time at a cold temperature,
bevond the normal winter. It would presumably wake up peri-
odically and raise its temperature. Where would it finally die,
at the cold level or the high level? This man thought it would
die at the high level —it would come out sometime before it
died at the cold level and get up to normal temperature and
‘‘burn itself out’’ there. Now, does anybody know?

FISHER: I don’t think anybody, Mr. Chairman, during the
two and a half days of this conference imphed that there was a
lack of either stored fat or carbohydrate at the end of hiberna-
tion.

BISHOP: But suppose you carried it out. Suppose you really
put the animal under a continuous strain beyond that time.
Where would it be?

GRIFFIN: I have oceasionally left bats in a hibernating
chamber that was reasonably good, and left them right on into
the summer and they do eventually die, I think either of too
little energy reserve, or possibly of desiccation, but I have no
idea whether they awake first. Yet I sometimes think it is
clear they die right in hibernation without waking up.

BISHOP: I imagine they might get exhausted and couldn’t
wake up, or might wake up and ‘‘burn themselves out’’ before
they went back again.

522 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

GRIFFIN: You occasionally find them frozen into icicles in
eaves and I can’t beheve they let that happen if they were able
to wake up at the last minute because it would only take a few
inches of crawling to get away from the ice.

DAWKE: It has been my experience that the animal arouses
from hibernation periodically during the winter, irrespective of
its fat stores. Of course, if fat stores are entirely depleted, it
won't arouse. It dies in hibernation.

ADOLPH: I don’t think we ought to limit this question to
ihe matter of nutritional depletion, because there are other
thines which can give out besides the number of available eal-
ories. Presumably these deaths occur, as they do without any
rewarming of the animal, from a large number of things, any one
of which goes wrone or any number of which go wrong. So |
don’t think we can credit the end of endurance to any single
sort of phenomenon in the animal.

BISHOP: That raises the question of what does really make
him come out. Does he come out because he gets a little too cold
or perhaps a degree below where he is safe? Is this a safety
factor, coming out, or does he come out because of some eyechec
activity whieh occurs and stimulates him or does he get some
kind of external stimulus ?

ADOLPIL: Again I would form the opimion from what. |
have read and heard that there are a number of thines that
can figure in warming’.

BISHOP: That is certainly one of them, isn’t it: external
stimulus. Poke him enough and he will come out, isn’t that right?
Another one, [ understand, is abrupt cooling. Didn’t somebody
say that if you cool him rapidly when he is already down, he’ll
come out. I wonder if vou cool him very slowly — sneaked up
on him 2s.

DAWE: He might not react.

BISHOP: He might not react because he was too cold to react
to a weak stimulus.

DAWE: Yes, that’s right. In other words, the Law of Dubois-
Reymond may be operative. It’s quite obvious, I think, that the
deeply hibernating animal has certain sensitivities in this respect
which are not too well understood. Next question: ‘‘ Will the

1960 MAMMALIAN HIBERNATION

|
i)
eu)

panel compare the results they have heard from the various
laboratories ?”’

PROSSER: They’re all good.

DAWE: Next question: ‘‘Does the evidence suggest that the
heart during hibernation is ‘independent’ of the rest of the
body ?”’

FISHER: How could the pressure be maintained, as it ap-
parently is, if the heart were ‘‘independent?”’

PROSSER: Perhaps Dr. Adolph ean speak on that.

ADOLPIL: Well, our evidence was that the pulse rate in the
intact hypothermic or hibernating individual has a_ different
relation to temperature from that in the isolated heart. I don’t
know whether this is the sort of thine that is meant by ‘‘inde-
pendent.’’? This is a very extreme form of independence, to have
the heart cut out of the body and set up to beat by itself. There
are various degrees of independence but I must say that very
few of them have been studied in relation to hypothermia and
hibernation. Why can’t we test various degrees of ‘‘independ-
ence,’ since there are identifiable connections between the heart
and its environment whieh ean be defined in terms of individual
connectors. However, I don’t think that this sort of research is
as penetrating as lots of other forms beeause we'll probably
find multinle wavs in which the heart is related to its surround-
ings and the organism in which it is working. But, on the
other hand, we have an opportunity in hypothermia, and perhaps
in Iibernation, to further define the relationships between the
heart and the organism in which it works, which we don’t have so
obviously in the euthermie organism,

BISHOP: You meht say that in many organisms, when the
metabolism is under strain, the heart has a capacity of getting
energy when other organs fail; the heart is one of the ‘‘tough’’
organs. Is that correct?

ADOLPIL: We could define part of the relation between the
heart and body in terms of energy-yielding materials. The
mobilization of these materials must be of a reciprocal nature.

DAWE: Next question: ‘A hibernator chilled to 6°C shortly
dies, while an animal at 6°C in natural hibernation does not.
What seems to be the most hopeful avenues of approach for solvy-
ing this problem?”

»24 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

ADOLPH: IL have thought about this item a great deal, but
[ haven’t any sovereign avenues of approach. It seems to me
that the fact that a hibernator does periodically wake up, or
Warm up, means that there is some process which is in abeyance
Which has to be turned on or resumed periodically. I have
tried to search for some examples of this in terms of electrolyte
changes in tissues, but when you isolate the tissue to identify the
change you have destroyed most of the situation in which the
tissue worked. | think that it is true there is a longer survival
of the hypothermic individual without rewarming in natural
hibernation than there is after artificial cooling. I think this
much has been established, and I think this gives us an oppor-
tunity to try to analyze what the factors are in natural hiberna-
tion which make the organism more tolerant of the low tempera-
ture. I think the answer is going to be a multiple one. I think
it is going to come out in terms of the composition of the internal
medium, in terms of the activities of the nervous system, in terms
of the activities of the endocrine system, and a number of other

factors.

GRIFFIN: That line of thought suggests to me an interesting
point. While it is quite true that natural hibernators do wake
up from time to time [ think there are important exceptions.
| think there are cases in bats at least, though they may be rare,
where the same animal pretty clearly really did ‘‘stay put’’ for
some months at a time probably without waking — though with-
out having had a recording thermocouple, you can never be
entirely sure. But this might be an important point for future
investigation, to try to find and study more critically those cases,
if they exist (and I think they do), where an animal really does
remain for many days without any waking and to discover what
is different about these animals and the conditions where this
happens from the case where there is this periodic awakening.

BISHOP: It seems to me we have had several papers here
definine this, more or less, which you could summarize under
‘‘oeneral adaptation.’’ For instance, nerve fibers are able to
conduct at lower temperatures after they have been adapted in
the body in the hibernating state. The heart, I presume, will
work at a lower temperature and perhaps I would interpret
Strumwasser’s results as indicating a gradual adaptation to cold,
so that the stimulating value of cold changes during the process
of this cooling off. A ‘‘summer frog’’ and a ‘‘winter frog’’ are
quite different. It is the same thine that was described here in

1960 MAMMALIAN HIBERNATION D20

more detail in the measurement of nerve fibers. A ‘‘summer
frog’” nerve can be raised to a higher temperature before it is
killed than a ‘‘winter frog’s’’ can.

ADOLPH: If think [ would lke to qualify this idea about
adaptation (with which I agree quite thoroughly in principle )
in this respect: I think it is yet to be shown that you cannot
vet as much adaptation in the artificially cooled tissue as in the
naturally cooled hibernating tissue, but I am not sure about this.
A. difference hasn’t been demonstrated and its demonstration
is limited by the fact that the artificially cooled animal doesn’t
last long enough at the low temperature to imitate the naturally
hibernating one.

BISHOP: If you cooled him gradually enough and gave him
time to adapt? Usually vou plunge an animal into cold water and
say, ‘‘ Now he is cool.’’ Well, the hibernating animal has a long
preparation here and the preparation may not be due only to
cooling, it may be a process going on here changing things in
preparation for being cool.

ADOLPH: I think this factor of natural cooling alone has
been imitated, so that the graded cooling was the same as in
hibernation.

DAWE: I have only one more question for this panel:
‘Would the panel discuss the stress svndrome and the adrenal
cortex in hibernation ?”’

BISHOP: You might ask in what sense hibernation is a stress
or cooling is a stress. One might argue quite the opposite in a
sense, since it reheves the animal of certain stresses. It becomes
stressful only when the animal comes to the danger point of
freezine to death.

DAWE: It doesn’t freeze to death. Of course, in that sense
it is not a stress.

GRIFFIN: Would it be too naive to put it the other way
around and say the degree of cooling that is a stress for a non-
hibernator is not a stress for a hibernator because of his adjust-
ment and regulation.

DAWE: Would you expect this to affeet the adrenal gland ?

GRIFFIN: No, naively, I would guess that there would be
less of the stress effect on adrenals on a hibernator going into

526 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

hibernation, in a normal situation. I don’t know whether or not
that is the case.

DAWE: Dr. Suomalainen, of course, has gone over this pretty
carefully and I think he found that the stress syndrome was in
effect. Is that correct Dr. Suomalainen ?

SUOMALAINEN: Yes.

FISHER: It seems to me that there is a difficulty though,
Mr. Chairman. Stress is an ill-defined word. When a rat that
has been kept at 30°C is put at 5°C there is a ‘‘stress,’’ if you like,
that he cannot withstand and he dies. But if the animal is
cooled gradually over a period of weeks, there is no longer the
kind of stress that kills him. Do these two situations differ
merely quantitatively? In the first instance the demand is be-
vond the physiological capabilities of the animal. In the second,
the demand is adequately met.

GRIFFIN: Could I try to clarify that — you don’t mean cool-
ing a rat to a body temperature of 5°C, you mean environmental
temperature ?

FISHER: I’m sorry — in an environment of 5°C — yes, quite.

GRIFFIN: But supposing you do speak in terms of deep
tissue temperatures. [I never heard of anyone, except R. K. And-
jus (J. Physiol., 128:547, 1954), cooling rats to 5°C and I
suspect that was a pretty stressful situation and this is just a
more valid distinction.

BISHOP: That’s the point —if you take an animal that in-
sists on staying at 37°C and push him down, then he fights
against it — that’s a stress. I think anybody will agree that that
is a stress. If this animal doesn’t resist and manages to go down
(slips down without effort), I don’t see any stress about it at all.
For instance, Strumwasser’s animals may be reacting ‘‘stress-
fully’? at first, and not finally.

FISHER: So you would rather not call this ‘‘stress’’ in a
hibernator ?

BISHOP: L would say the same process of cooling might be a
stress in a homoiotherm and not in a hibernating animal.

PROSSER: And yet there are some e¢linical similarities.

1960 MAMMALIAN HIBERNATION SLM

KISHER: It depends upon one’s definition of ‘‘stress,’’ how-
ever. If any change that can be recognized in the adrenal is
defined as the result of ‘‘stress’’ then any question is settled :
hibernation is clearly a stress.

ADOLPH: Let’s leave the adrenal out of the definition —
why not? There must be many ways in which stress is expressed
other than in the adrenal change.

BISHOP: You might say the process of hibernation is to
avoid stress. After all, if an animal is going to starve to death
or hustle for a living if he stays up at a normal temperature, he
is avoiding that stressful existence by going into hibernation.

DAWE: I have no other questions for the panel. The panel
now has an opportunity, individually, to express thoughts which
they feel need expression. Dr. Fisher.

KISHER: This is probably out of order, Mr. Chairman, but |
feel someone should express appreciation to the institutions and
individuals who organized this meeting and made it possible to
hold it under such pleasant circumstances. If it is appropriate
| would like first to move a vote of thanks to them for these
arrangements —and then we can get back to science.

DAWE: Thank you, Dr. Fisher.

KISHER: [1 like the point about the hypophysis mentioned
hy Dr. Bishop. Upon several occasions it has been noted that,
as the body temperature of the hibernating animal goes down,
its rate of metabolism falls somewhat more than would be ex-
pected on the basis of usual values of the Q,9. Now, removal of
the hypophysis, in some mammals at least, lowers the metabolism
of the whole animal and of its parts, if they be isolated for
examination, by about 50 per cent. Could it be that the produe-
tion of the hormone concerned with metabolic rate ceases during
hibernation, thus causing a greater drop in metabolism than
would be expected from the temperature change per se?

GRIFFIN: Has anyone tried to prevent animals that other-
wise would go into hibernation from doing so by adding pituitary
hormones — that would be the obvious experiment.

PROSSER: Didn’t Dr. R. K. Meyer try that? (M. A. Foster,
R. C. Foster and R. K. Meyer, Endoerinol., 24:603, 1939).

GRIFFIN: Did it work?

528 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

PROSSER: As I recall, their controls weren’t adequate.

BISHOP: There was a report — if you take out the pituitary,
the animal still does go into hibernation but dies. Of course, they
die younger anyway.

GRIFFIN: Well, that’s hypophysectomy. Does this include
the other — the addition of hormone?

RISHER: My remarks applied not to the mechanism causing
onset and cessation of hibernation but instead only to the drop in
metabolism which occurs as hibernation ensues. This drop
seems greater than would be expected from the drop in body
temperature assuming common values of Q,o. The situation to
which | was drawing attention is exemplified by the aestivator.
If IT remember correctly, Crtellus mohavensis allowed its body
temperature to fall from 37° to 24° but its metabolism at
24° was only 1/20 of that at 37°. If the change in metabolism
resulted solely from the drop in temperature, the unusually high
(J;9 of more than 10 would have to be assumed. By suspending
all muscular activity, however, the metabolism of the animal
in aestivation might be reduced by 1%. If, in addition, the animal
in aestivation is in this connection functionally hypophysectom-
ized, its metabolism might be dropped because of this by another
factor of 44. The drop remaining to be accounted for by temper-
ature is now only 1/5 (not 1/20) and the Q;9 required is only
3.5, a much more common value. Has anyone else given thought
to the drop in metabolism in aestivation or hibernation?

GRIFFIN: Let’s go back specifically to some measurements
made by Hock (R. J. Hock, Biol. Bull., 101:289, 1951) some
vears ago on metabolic rates of bats at different temperatures
(all the way from 0.5 to 42°C). You could pick your Qo, as I
remember, anywhere from 2 to 5. But at different parts of the
temperature range for this particular animal under these par-
ticular conditions you have a wide variety of Q,os. So this in a
sense reinforces what you were saying. But I extrapolated from
your remarks to the working hypothesis that an animal goes into
hibernation because of insufficiency in some one of the pituitary
hormones. If this is so, you should get some confirmation by
adding the hormone.

PROSSER: It seems to me that the alternative hypothesis
would be to forget the pitnitary and assume that this greater
rate of reduction is due just to complete flaccidity and loss of
all musele tone. The active metabolism, in other words, is gone.

1960 MAMMALIAN ITIBERNATION 529

FISHER: Well, | think a lowering of half the metabolism
might occur this way but no more.

GRIFFIN: At what temperature range?

FISHER: At 87° [ would guess that the loss of all muscular
tone would result in a loss of half the respiratory metabolism.

PROSSER: Somebody ought to try some of these things with
animals that have been made inactive by curarizing agents.

BISHOP: Well, now if you are willing to go beyond the group
of mammals which hibernate, there are plenty of cases of hor-
monal inhibition in metabolism: for instance, the grasshopper
ege which demands a period of cold to remove that imbibition
before it can go on erowine. There is also a certain kind of a
peach which grows until about June and then stops — about as
big as your thumb —and waits for a month during the hot
weather and then goes on growing again. But there is another
peach, just like it otherwise, which keeps on growing and ripens
about a month earlier. This is an aestivation phenomenon in the
plant. What the rule is, I don’t know — but something inhibits
growth for about a month and then growth proceeds normally.
When you get into the other reactions of plants, it is easy to see
that there are all kinds of changes in metabolism factors which
are probably. hormonal, if you could deteet the hormone and
identify it, Involving response to light and dark periods or to
temperature changes and so on. One of the easiest thines with
which to change metabolism is a hormone, in plants certainly. It
looks awfully suspicious here also. Now I suggest that it might
be profitable to look not only at the hypophysis but also at the
hypothalamus which is certainly a center where the cues of
sensation might be reeistered — the reactions for heat, thirst,
hunger, ete. as if the animal had sense organs, neural sense
organs, there that recorded in the blood the presence of certain
critical materials. This would make it a center from which you
could get all kinds of influences over metabolic and = other
processes of the body. It would be a tricky job to do in a little
eround squirrel — to take out some specific part of the thalamus
or hypothalamus — but it might be worth doing.

DAWE: Dr. Adolph, do you have anything further?

ADOLPH: Could I add something to what has just been
discussed about energy metabolism? Popovie has some evidence

530 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

that a hibernating animal has a very low energy metabolism and
within five minutes can change to its expected metabolism with-
out changing its body temperature (V. Popovie, Arch. Sei.
Physiol., 11:29, 1957). In other words, this change is too sudden
to eet rid of a hormone, perhaps. It doesn’t matter to me whether
this agent is a hormone or not, but it is a very fast switch. It is
comparable, in my estimation, to the switch from a resting to an
active muscle: here vou can step up the metabolism 50- or
100-fold with excitation. Hibernation acts in the reverse direc-
tion to step down the metabolism (perhaps not instantaneously
but over an hour or less) after which high metabolism can again
switch on within minutes. This, it appears to me, is the sort of
control we have to look for; one which determines the total
metabolism of an animal — not just the metabolism of one spot
in the animal. It’s not just the presence of more or less eyto-
chrome © or something like that, but it’s an activation of some-
thine that is all ready to be activated by some excitant which
presumably spreads throughout the organism — that’s the way
I picture it.

FISHER: Muscle isolated from an hypophyseetomized rat
respires at about one-half the rate characteristic of musele from
normal rats. In some recent experiments we have found that
the addition of lactate (and probably stimulation to contraction )
raises the rate of each to the same value. Thus, hypophysectomy
appears to lower the resting rate of metabolism without affecting
the maximum rate possible. This situation fits what is known
about respiratory metabolism during hibernation. Part of the
drop in metabolism during hibernation must be accounted for by
a process lke hypophysectomy, which lowers metabolism inde-
pendently of a temperature change and vet permits the maximum
metabolism to be established very quickly as in an arousal.

DAWE: Anything further, Dr. Griffin ?

GRIFFIN: [ would like to add two thoughts. I will just call
attention again to this extreme longevity in bats which puts them
way out of line with other mammals of their size. It just hap-
pens that bats are numerous and various people started banding
them enough years ago so that spectacular longevity records (lke
21 years for a 7 @m species) have turned up not only in this
country but in Europe. Dr. Eisentraut tells me that his bats are
still being caught, just as mine are, and I think that it is as much
a question of the longevity of the investigator as the longevity

1960 MAMMALIAN HIBERNATION 53]

of the animals. | wonder whether this may be shown also in
other hibernating mammals. It would be much harder to get the
data perhaps — but do ground squirrels have any greater longev-
ity than closely related non-hibernating mammals? The obvious
thought is, of course, that they are ‘‘burning their candles’’
only at one end or only at one corner and spreading out their
quota of heart beats or units of metabolic energy. This might
have some vague interest for people concerned with longevity.
That’s one thought. Thought two (suggested by the whole sym-
posium, or as much of it as I have been present at, and particu-
larly by Dr. Strumwasser’s experiments) is one that I have
already expressed this afternoon — that regulation, in part at
least, by the central nervous system is, perhaps more than I
ever realized before, the key to suecessful hibernation. And just
to ** freewheel’? for a moment, how about somebody trying an
experiment in which he takes an animal with a fairly plastic
central nervous system that is not a hibernator, such as a rat, and
tries to train that nervous system to regulate the internal affairs
at lower and lower temperatures? Conceivably, this is the way
to approach human hibernation, not by trying to find some
“maeie pill’? but by trying to find out what it is that has to be
readjusted and to approach this through the one way that in a
gveneral sense is well known — to readjust central nervous sys-
tems, namely learning. And with that final thought, [ will pass.

DAWE: Dr. Bishop, do you have any final statement?

BISHOP: Nothing to add except my own experience, coming
here as a complete ‘‘ignoramus,’’ as to what hibernation is
about. | have had to sort out 50 papers here and try to make
some consecutive sense out of them, and I come up with some-
thing like this: This process of true hibernation in the mammal
(that permits temperature to follow the environment) has three
essential parts. The first is the inciting stimulus which is quite
incidental, it seems to me, because it can be heat or dryness, or
temperature lowering —any appropriate stimulus which the
animal needs apparently sets off the same process. Second, the
specific thing is the process of reducing metabolism. The third
process is recovery from this situation. It will kill the animal if
he can’t recover; the animal is helpless unless he can come out of
this state. Now a lot of the papers presented here described the
various processes by which he comes out — which again however
are more or less incidental. They are not the same for an animal

532 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

that hibernates at 30° as they are for an animal that hibernates
at 5°. They may be different for other reasons, but again these
are more or less adaptations to the environment. I, in my present
view of the whole subject as a result of this conference, see the
central problem here as the problem not of how he lowers his
temperature but of how he lowers his metabolism. On one hand
are the cues which any animal may get and which may be variable
with the environment in which he lives, and on the other hand
are the processes by which he recovers from a dangerous condi-
tion which he biologically prefers, rather than going on and
starving to death at the lower temperature. Of these three parts,
the central problem still is how the metabolism is reduced.

DAWE: Dr. Kayser may wish to speak of that later. Dr.
Prosser, do you have anything further to add?

PROSSER: I would like to comment very briefly about one
viewpoint which seems to me to have been omitted, although I
missed the biochemical papers yesterday. No one has suggested
that some of these metabolic changes may be analogous to enzyme
induction phenomena. Granted that the phenomenon of hiberna-
tion involves many organs and that certamly in the final
analysis one must study the organism as a whole, | would make
a plea for more intensive fractionation — that is, a study of
specific enzyme systems. I think that measuring oxygen con-
sumption is just about as crude a method of studyine biochem-
istry as counting the number of vertebrae and saying that this is
studying morphology. Yet, we are basing most of our conclu-
sions about energy on oxygen consumption of the whole organ-
ism. The important thine as far as energy is concerned (or an
Important measure of it) might be the P/O ratios of the mito-
chondria from certain critical tissues, and it seems to me that we
night well do a little thinking about the possibility of altera-
tions in metabolic pathways. We know that there are many
pathways from substrates out to oxygen and some of these are
cross-linked at intermediate steps. We also know that in many
kinds of organisms (I am most familiar with poikilotherms in
this respect) we ean shift the pattern quantitatively so that one
pathway is predominant over another in a given state of, let’s
say, temperature adaptation. The working hypothesis seems to
he reasonably well supported that we can shift enzymatic path-
ways by slowing one pathway, by causing pile-up of the substrate
of this pathway, and then these intermediates may serve as in-
ducers for an alternate pathway. And we lnow that this kind

1960

I NO ADAPTATION

LOG RATE

TEMPERATURE

Zl ROTATION

4 Clochmise

8 Counter-
clockwise

LOG RATE

TEMPERATURE

LOG RATE

IT TRANSLATION
AND ROTATION

C Translation to
left, rotation
counter-clockwisa

Translation to
right, rotation
counter-clochmise

TEMPERATURE

MAMMALIAN HIBERNATION

D TRANSLATION

A To left or up
8 To right or down

LOG RATE

TEMPERATURE

I TRANSLATION
AND ROTATION

A Translation to left,
a rotation clockwise

8 Tronslation to right,
io rotation clockwise

LOG RATE

TEMPERATURE

Fic. 1. Patterns of acclimation of rate
functions to temperature. In each figure C =
cold, W = warm. Broken lines and numbers
indicate Precht’s types of acclimation. All
patterns are indicated for cold acclimation.
Curves are plotted with decreasing Qin at
higher temperatures. Clockwise rotation
means reduced Qw: counterclockwise, in-
creased Qio. Patterns I—no acclimation; II 4
translation of curve to left or upward on
cold acclimation; II B to right or downward:
HI rotation, 4 clockwise on cold acclimation.
B counterclockwise; IV A translation of
curve to left and rotation clockwise on cold
acclimation, IV B translation to right and
rotation clockwise; IVC translation to. left
and rotation counterclockwise, IV D transla-
tion to right and rotation counterclockwise.

(Reprinted by permission from Physiological Adaptation, C. L. Prosser,

ed. ;

Washington, 1958, p. 173.)

534 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

of enzyme induction can go on not just with substrate (as micro-
Iologists have shown) but with physical factors of the environ-
ment. We can bring this about im poikilotherms by changes in
temperatures ; we can bring this about by restricted oxygen. In
hibernators we have both: reduced oxygen transport, and a
lower temperature. It seems to me entirely possible that some of
the enzyme systems are quantitatively changing in amount. I[
would like to see this general approach of more detailed en-
zymatic analysis, using inhibitors and purified enzyme systems,
rather than just measurement of gross metabolism. Another bit
of evidence is the one I just raised about the difference between
active and resting metabolism. We know that in nerve and many
other tissues the resting and active metabolism go by different
pathways, at least in part, and this may very well play a role in
the reduction of metabolism in hibernation. Now just one other
point — I saw the curves that some of you had of oxygen con-
sumption in relation to temperature, and there was some indica-
tion that there might be differences in the shape of these Q16
functions for non-hibernators and hibernators. [| would suggest
that one technique which might be used, in addition to the en-
zyimatic one, might be to look a little more closely at the interpre-
tation of these curves. In many temperature functions in poikilo-
therms we find that the curve can be shifted in either of two
ways or in both of them. See Figure 1. In many instances there
may be simple translation so that in an animal which is cold
adapted the curve is shifted to the left of that of an animal
which is warm adapted, and when measured at a given tempera-
ture the metabolism in cold adaptation is Iigher. Very fre-
quently we may have a shift, in the sense of a rotation rather
than a translation, and whether or not the rate of the cold
adapted form is higher than that of the warm depends upon
the temperature of measurement in relation to the point of
intersection. The first shift can be interpreted in terms of a
change in total enzyme activity. The second must be interpreted
in terms of a qualitative change, perhaps a change in activation
energy. This type of analysis can really be of use in trying to
eet down to the molecular level in tissue changes. Apparently
some of you with whom I have spoken have not read the very
heautiful work of Precht in Germany along this general line
(U1. Precht, J. Christophersen and H. Hensel, Temperatur und
Leben, W. Berlin, 1955). I hope that another conference may
emphasize the molecular approach to temperature adaptation
including hibernation.

1960 MAMMALIAN HIBERNATION 58D

DAWKE: Thank you Dr. Prosser. Do you have any final re-
marks, Dr. Luyet ?

LUYET: Before attempting to answer questions such as that
of the difference between hibernation and hypothermia, let us
briefly glance at the development of our concepts, in general,
taking the concept of hibernation as an example. One may dis-
tinguish three stages in the history of concepts.

1. The ‘‘name giving’’ stage. Primitive man, lost in a
chaos of sense observations, and pressed for time by the necessi-
ties of material life, made distinctions hastily, often on a super-
ficial basis, and assigned names to the things and actions that he
had distinguished. Thus he developed the concepts of plants and
animals, warm-blooded and cold-blooded animals, sleep and
awakeness, winter and summer, daily sleep and prolonged lethar-
vic sleep, ete. In the course of this observation of the world, he
occasionally noticed that some animals remained asleep during
the cold season; he called that phenomenon ‘‘winter-sleep’’ and
did not think that there was much more in it to be concerned
about. This development of concepts was the work of the budding
scientist 1m primitive man, and coining words was his means of
publishing the results of his observations.

2. The inquiry stage. Later, man, havine more leisure, ex-
amined things more carefully. He developed instruments. to
improve and extend his observations: the microscope, the watch,
the thermometer, electric meters, ete. He now measured the
temperature in the bodies of animals and in their environment :
he determined the time taken by an impulse to travel along a
nerve, ete. Then he tried to fit his new observations in the
old frames of concepts which were a part of his mental equip-
ment. Numerous questions of the following type arose: Is a par-
ticular cell observed under the microscope an animal or a plant?
Is a particular animal a warm-blooded or a cold-blooded one?
Does it belong to the category of true hibernators or not? ITs
winter sleep a true sleep? (The night sleep in man is apparently
considered as the true sleep.) Is the sleep produced by a drug
a true sleep? Is the anesthesia caused by cold a true sleep? One
should notice too that the inquiry stage, also, led to an enormous
number of new frames, that is, of new distinctions and new
concepts expressed in new words, such as homoiothermy, poikilo-
thermy, heterothermy, stenothermy, high and low homoiothermy ;
obligatory, stubborn, indifferent and morbid homoiothermy, to
mention a few.

536 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

3. The semantic stage. Man finally realized that when he had
assigned names to things and actions — either in the primitive
stage of his intellectual life or, later, in the inquiry stage —
he had proceeded too hastily. He started classifying before hav-
ing gathered the necessary information. As a result, his elassi-
fications do not hold. The philosophy expressed in common
language, which is primitive man’s philosophy, and that ex-
pressed in some of today’s scientific terminology are both super-
ficial. The picture of the world supplied by thorough and criti-
cal observations does not fit either in the old or in the new frame
of words. Questions such as: ‘‘Is a particular organism an ani-
mal ora plant ?’’ become senseless, since the frame ‘animal-plant’
has collapsed. In a similar manner, the term hypothermia —
which has been introduced hurriedly because of the urgent need
of a new term for practical purposes in scientific communication

-is now left in our hands with the request that we kindly see
what we can do with it.

Conclusions. It seems to me that the logical conclusions of
what has been said are that: (1) When new terms, such as
hypothermia, are proposed, for designatine phenomena which
differ in some manner from phenomena designated by old terms,
such as hibernation, one could set as the primary criteria for
accepting or maintaining these terms on a temporary basis,
that they be free from obvious contradiction or ambiguity, and
that they be practically useful in communication. (We should,
of course, be aware of the limitations of these terms and of
their temporary status.) (2) Questions imposed upon us by the
accepted terminology, for instance, as to whether a particular
phenomenon is hibernation or hypothermia, then, lose most of
their significance. (3) The real problem, of course, is to learn the
facts beyond the words. In the case at issue, it is to find out
how the various physiological functions are interdigitated in the
various animals, and by which process they became so entangled.
The findines will probably be that typical hibernators and non-
hibernators represent two extremes in a series of complexly com-
bined biological activities. When the gigantic task of establishing
the facts is sufficiently advanced to permit their coordination,
the question of where to place the labels (the words) will be a
relatively simple and a relatively unimportant one. This con-
ference, it seems to me, has contributed greatly to the task of
establishing and coordinating the facts and, therefore, to the
progress of the science of hibernation, in its inquiry stage.

1960 MAMMALIAN HIBERNATION De

DAWE: Thank you, Dr. Luyet. Dr. Fisher.

FISHER: Dr. Prosser has noted that the data of several
participants in this symposium indicate differences in Q,9 fune-
tions between hibernators and non-hibernators, suggesting differ-
enees in the limiting metabolic pathways. This is reminiscent of
the work on an isolated succinate oxidizing system studied by
Z. Hadidian and H. Hoagland 20 vears or more ago (J. Gen.
Physiol., 23:81, 1940). In those experiments the Q,q was
changed by appropriate additions of eyanide and malonate, re-
spectively, owing presumably to the ability of cyanide to make
the eytochrome systems limiting and to the ability of malonate
to make dehydrogenase activity limiting.

PROSSER: I think that you people who are working with
hibernating animals ought not to hide your light under a bushel
but to sell these tissues to the biochemists. It seems to me it
would provide wonderful experimental material quite apart
from understanding hibernation.

DAWE: Now I would like to open the diseussion to the
entire group.

XXVITI
GENERAL DISCUSSION

PENGELLEY: Perhaps [| have some information on what
Dr. Bishop was asking about, that is, what happens if you keep
animals in the cold continuously after the hibernation period is
over? [ have done this with Citellus lateralis and there comes a
time in the spring when they arouse from hibernation on a
permanent basis; this does not seem to be correlated with the
fact that they are running out of fat. This fits very nicely with
Dr. Hoek’s work when he observed the exaet time at which Arctic
ground squirrels come out in spring and the time at whieh
they go in, in the fall. If you do deny them food and water
and still leave them in the cold, the period for which they hiber-
nate will be prolonged, but they seem to decline rather quickly
and eventually they die, and they die in hibernation in the
sense that they don’t come out and run around the eage fran-
tically to get more food. They simply die in a hibernating state.

BRATTSTROAM: | think Beer in Minnesota has studied this
in the big brown bat (.J. R. Beer and R. G. Richards, J. Mammal.,
37:31, 1956). If the winter in Minnesota is sufficiently long the
hats will die in hibernation. Durine mild winters the bats have
enough stored fat to last the winter.

GRIFFIN: Is he sure they didn’t wake up at all; is he just
finding them dead, or did he watch them ?

BRATTSTROM: fle watched them in routine intervals in
caves, and I think in laboratory animals as well. | don’t remem-
ber if he knows whether or not they arouse before death. 1
suspect that they did not.

DAWE: Are there other points to be discussed ?

SMITH: J should like to extend Dr. Prosser’s plea for a more
detailed biochemistry of the hibernating animal to include also
considerations of intra-molecular energy transfer and physical
chemistry. Such considerations seem indicated in the case of data
presented by Dr. South and by myself. With respect to the
im vitro experiments of Dr. South, it should be possible to alter
his systems with agents known to change the configuration of
proteins. Such manipulations might give a elue to the explana-
tion of his results, inasmuch as protein configuration (and thus

y40 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

energy transfer along the protein or from protein to substrate )
might be changed over the range of temperatures employed.

DAWE: Other points?

MENAKER: In the case of the relationship of artificially
induced hypothermia to natural hibernation, it oceurs to me
that it should be possible to use artificially induced hypothermia
as a tool to study hibernation itself, in this way: Dr. Strum-
wasser has suggested that im the Calfornia ground squirrel
the animal mavy be ‘‘testine’’ his state of biochemical preparation
for hibernation. Now it would be interesting, it seems to me,
between these ‘‘test drops’? to subject the animal to conditions
that would induce artificial hypothermia and try to find out in
this way something more about the state of biochemical prepara-
tion and also to do this at different times of the day. For in-
stance, during his nightly natural temperature drop he may be
less sensitive to some of the lethal effects of induced hypothermia.

HOCK: There may be a factor which is being overlooked in
this discussion of hypothermia during hibernation. Hypothermia
as we see it in the laboratory is accidental, but hibernation is
a natural thine for which the animal is prepared or prepares.
This is quite a large distinction and somehow seems to destroy
the hope that we can use hypothermia in all ways that we might
wish to use it to understand hibernation processes.

PROSSER: [n other words, there is a difference between ac-
climatization and acclimation.

DAWE: Other points?

MORRISON: The question was raised, in fitting bats into
our seheme of hibernators, as to whether or not bats regulated
their temperature. Dr. Pearson described at least one example
of a vespertilionid, | remember, that maintained a constant meta-
bolic rate and wasn’t foreed into its natural ciurnal eyele
(throughout at least a 24-hour period). In the bent-winged bat
Miniopterus (whieh was one of the three species I looked at in
Australia), it is possible to show (ander certain conditions when
the animal was not flying) a maintained level. I think the idea
has been advanced that temperature rise in the bat is sort of a
tacit concomitant of muscular exercise — perhaps as in a bumble-
bee or a large fish as it warms up. These animals could main-
tain a fairly high level of temperature up to around 25° without

1960 MAMMALIAN HIBERNA'TION 54]

Aying. Furthermore, under other conditions they seem to main-
tain a temperature around 30° (body temperature 30° and
ambient temperature 20°) and I belheve that when this level of
30° has been reached, either the animal is warming up from
the cold or (in flying animals at a body temperature of 40° )
is going down and regulating again at 380°. So it seems to be
positive regulation and this is a rather significant level because
this 30° figure is one which permits the annnal to fly. Below that
it cannot fly and above that it can fly. It may be a significant
value in Dr. Hoek’s bears also. You saw that his values are
just above 80° and this means the bear is in shape to fight you.

BARTHOLOMEW: The people here who have studied mam-
mals outnumber those who have studied birds with regard to
hibernation in a ratio of 50 to 1. I would like to call to your
attention that some of the problems we are discussing are
amenable to analysis through the use of hummingbirds which
remain active for many hours at a stretch and almost certainly
at high temperatures, and do have prolonged periods of tempera-
ture depression at meht. In addition, one of the most common
birds we see in the United States, the chimney swift, is a natural
for somebody to investigate. At least two kinds of swifts become
torpid, and somebody ought to put a screen over his chimney
and eatch them and see what they can do.

DAWE: T have some questions for general discussion. The
first is: ‘‘ Has anyone tested the threshold of arousal at different
stages of the hibernation cycle for a given type of stimulation ?”’
i certainly naven’t — 1s there anyone here who has?

SMITH: We have irradiated bats collected at various times
during the winter and found different responses as the season
progressed. When we studied arousal from hibernation, we
found that over the period from January through March inereas-
ing numbers of animals awoke from hibernation in response to a
standard exposure to X-rays.

ADOLPH: A number of persons have reported that the in-
terval between the temporary awakenings changes durme the
season; isn’t this a partial answer?

DAWE: I think the person who submitted this question is
looking for a standardized arousal procedure.

WIMSATT: J. DeWilde and P. J. Van Nieuwenhoven pub-
lished a paper 2 or 8 vears ago (Publ. Natuurhist. Gen. Limburg.

j42 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

Reeks VII:51, 1954); they did extensive studies on what will
stimulate arousal in hibernating bats. They were interested in
the micro-climatology of caves. I know they used tickling with
hairs. They finally reached the conclusion that hibernating bats
were particularly sensitive to wind currents, and practically not
at all to sound.

BISHOP: The threshold of arousal might be a measure of
the depth of hibernation.

FOLK: We have observed that some bats remain at a totally
different level of dormancy than others, for long periods.
Within a colony you can characterize the individual animals
staying at different levels. | don’t know just how that ties in
with this except that it was determined by a stimulus.

MORRISON: On this question, | don’t have any quantitative
data on stimuli but I think this is relevant: in experiments with
Muscardinus this past winter, there was a very striking change
in their sensitivity to stimuli. In about 8 to 10 hours, in a
particular case, these animals would wake up with the shghtest
disturbance (in a metabolic chamber monitoring the oxygen
consumption). On the other hand, when we wanted to conclude
the experiment about 3 days later, it was not possible to make
these animals arouse at all, even though we took the chamber
out and banged it up and down on the water bath. We had to
remove the animal and put it in a warm temperature before it
would awake.

LANDAU: Was that ever a two-way change — did you ever
find one that was unarousable one day and two days later was
aroused by just beine looked at?

MORRISON: No. This is the kind of experiment you might
say was not a planned one.

DAWE: Next question: ‘‘What is the first sign of arousal —
temperature rise, heart rate change, shivering, ete.? Is the
arousal initiated by internal metabolic processes or external
stimuli, that is, reaction to cold ?”’

LYMAN: | don’t think [I quite understand the question.
Does this mean natural arousal or disturbed arousal ?

BISHOP: Yes, natural arousal. What brings them back?
Why do they arouse during a steady state of environmental tem-
perature, steady state of the animal’s temperature?) Why do they
occasionally arouse, what sets them. off?

1960 MAMMALIAN HIBERNATION 543

KAYSER: | think that external and internal factors inter-
vene in the arousal: in a refrigerator regulated at *1°C, a
ground squirrel awakes every 6th day in October-November, and
only every 25th day in December ; later on, in January-February
there is a regular decrease of the length of the phases of unin-
terrupted hibernation. The conditions of temperature, illumina-
tion and noise in the laboratory being constant through the whole
hibernation period, the existence of an ‘‘internal clock’’ is
evident. But if the refrigerator is regulated at 0.1 or 0.2°C,
the arousals are generally less frequent; so the temperature
fluctuations also play a part. It may be, also, that the factors
of illumination-darkness alternation intervene, but I have hardly
studied them at all. Another evident factor is the nutritional
one: 1f the animals are given some meat before hibernation, they
enter more reluctantly into hibernation and awake more often,
but the number of accidental deaths is reduced (experiments
done with garden dormice). But the arousals during hibernation
are normal phenomena, probably necessities. In field condi-
tions, many hibernators hibernate in groups in their burrows.
In my experiments (Arch. Sei. Physiol., 6:193, 1952) on two
Kuropean ground squirrels hibernating in two individual cages
placed side by side in the same thermally and acoustically in-
sulated chest, it clearly appeared that the arousal of one induced
the arousal of the other. It is certain that in the field the con-
stancy of external factors is not perfect, and that the long phases
of uninterrupted hibernation which may be observed in the
laboratory are often artificial and harmful. As a conclusion,
internal factors are indisputable, but external factors also play
a role in the determination of the frequeney of arousals.

DAWE: Any other points for discussion ?
STRUMWASSER: [I would like to ask Dr. Bishop a question.
Do vou find it difficult to conceive of a brain at 6°C under con-

stant external environmental conditions (let’s assume that it’s
technically possible) initiating a spontaneous arousal ?

BISHOP: IT don’t know why it shouldn’t be able to.

STRUMWASSER: All right; well, I think that’s responsible
for some of the arousals.

BISHOP: It may be. Hlowever, | wondered just what Dr.
Maver was talking about — what, in natural hibernation, hap-
pened that might cause these periodic arousals. Animals break

n44 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

through the inhibition or depression, or whatever it is, of the
metabolism and start shivering again. Now, shivering is a natural
response to cold. Suppose the temperature went down a little
bit: would the animal shiver like a normal animal shivered
because he got colder at a new level of adjustment, or doesn’t
the change in temperature do it at all—the shivering just
happens trom some other means?

STRUMWASSER: But they do ‘‘spontaneously’’ arouse from
deep hibernation when the environmental temperature remains
unchanged and they arouse in response to some internal alarm.
After all, they do seem to arouse on a rigid kind of schedule at
the start of hibernation, so there are obviously internal factors
which are present and we do not need to always ask for external
factors being necessary for these arousals.

BISHOP: There might be both, of course.

PEARSON: [ am thinking, now, of the daily torpor that ani-
mals like hummingbirds and bats go in for — as I continue to say
‘physiologically’? — in hibernation. Take bats that become
torpid every day and keep them in a roost deep in caves, in
constant darkness, away from sound, and thermostatic, and yet
it is part of their ritual that every evening at the same time
they awaken themselves and come out. This, I am sure, is
spontaneous arousal from hibernation. Hummingbirds, in metab-
olism apparatus under water and at constant temperature,
awaken before daybreak (in darkness) spontaneously. I think
these cases of arousal are clearly spontaneous and Menaker here
has his bats in constant environments giving a little rise of
half a degree each day. All you have to do is magnify that and
it is spontaneous arousal.

LUYET: But then this means only that we do not know,
does it not? You said spontaneous -—— they arouse spontaneously.
Now ‘‘spontaneous’’ means ‘‘ with a cause you can not identify.”’
It may be just a molecular motion some place in the system.

PEARSON: I’m thinking of a cause arising within the
animal —an endogenous event.

FOLK: It is important to add to this picture the fact that
hibernating 13-lined ground squirrels may awaken with intervals
of one day, three days, up to (occasionally) two weeks, at an en-
vironmental temperature of 5 or 6°C.

1960 MAMMALIAN HIBERNATION 545

MENAKER: If we don’t talk about the periodic arousals that
oceur during hibernation, but simply the last arousal — at which
point the animal leaves hibernation for good — it seems to me
that Dr. Hock’s data (obtained from field observations on
ground squirrels) pointing at almost exactly the same date every
year is very suggestive that there is a yearly internal rhythm,
as is Mr. Pengelley’s data on colonies of ground squirrels kept
under constant conditions which awaken spontaneously and then
cease hibernating.

DAWE: Dr. Frank Brown has observed and reported on
annual, monthly, and diurnal rhythms as a general physiological
phenomenon. He has not, however, made these observations on
hibernating animals.

PROSSER: I think that it is entirely possible that there are
‘internal clocks,’’ but | am not yet ready to say that there may
not be extrinsic factors of which we are not aware which are
having influences.

MENAKER: On April 21st every year?
PROSSER: Might very well be.
HOCK: This is 8 feet under the snow, by the way.

DAWE: Next question: ‘‘Would an animal which is ready
to hibernate be prevented from doing so by disturbance or sens-
ory stimulation, or would it finally hibernate anyway? I think
it would finally hibernate anyway. Is there anybody who would
object to that?

SOUTH: Yes, [ think that this has come up a couple of
times but | know that many of us have had a similar experience.
Kor my own part, one of my temperature control rooms broke
down so | moved over to a converted decompression tank which
was much noisier. The thermal insulation probably wasn’t too
much different. It was much noisier, rattled, and conducted
sounds much better. Temperatures were identical to the controls
with excellent regulation in both tanks; but for the whole summer
| didn’t get a single hibernation — for 2% or 3 months, I didn’t

vet a single animal to go into hibernation in the noisy tank.

LYMAN: I’d like to reinforce Dr. South’s observations and |
think a lot depends on the animal, here. But certainly with
hamsters — what were you using, hamsters?

46 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol, 124

SOUTH: Yes, hamsters.

LYMAN: I know that investigators who have come to our
laboratory have asked why our hamsters seem to hibernate better
than theirs do. It almost always turns out that they have some
sort of a compressor that goes on and makes noise or there is a
heat exchange mechanism that blows in. Our hibernaculum is
next to the morgue. This is as quiet, I think, as you can get, and
we get better hibernation.

LANDAU: I think there is another difference here because
we have had some of our ground squirrels in the warm room
where they were with dogs, cats, monkeys, ete. The temperature,
as far as I know, was above 20° all the time and in the fall and
winter we had (I think I observed over 40 times at least) over
18 different animals in a definitely torpid state. Now they cer-
tainly weren't in what you would call ‘‘deep hibernation’’ at a
temperature of 20°, but [| do think that if these animals are
‘ready’? to hibernate they will let their temperatures go. How-
ever, I also think that the more distracting stimuli you have,
the less likely they will be to do this.

SOUTIL: Yes, we have a couple of pet Belding ground squir-
rels in our laboratory which, during this past winter, hiber-
nated very frequently and usually, apparently, hibernate for
about 24 to 36 hours and then wake up due to the distractions,
but they were hibernating on and off all winter lone.

HWOCK: [ think the whole difference here is the fact: that the
eround squirrels make eycle preparation for hibernation and
hamsters do not.

SOUTH: Right.

DAWE: The next question: ‘‘Would a mild anesthetic, a
soporifie only, promote hibernation in an animal ready to hiber-
nate?’

SOUTH: We tried a lot of things. A couple of years ago, Jack
Twente and [I gave some hamsters chlorpromazine (unpublished
observations). We put them in a cold room and about 50 per
cent of them died and the rest became hypothermic; the next
morning the latter were fine.

LANDAU: In this business of anestheties I’d like to ask a
question. We did a great many experiments in which we tried

1960 MAMMALIAN HIBERNATION D47

to anesthetize and cool ground squirrels and we had a terrible
time trying to anesthetize them because they were so inconsistent.
The dose to which one animal would react had no effect on an-
other animal or on the same animal on another day. Therefore,
I don’t know how one is ever going to give them a light anes-
thetic.

BISHOP: IL would suspect that you were injecting the anes-
thetic in shehtly different places in the different animals or in
the same animal. Intrapleural or intravenous injections are
preferable.

ZIMNY: In respect to Dr. South’s findings, at one time I in-
jected Serpasil. Serpasil was new and everybody was doing
something with it. All the 13-lined ground squirrels did was
to lose weight. They wouldn’t eat. But they never did go into
any stage of hibernation, torpor, or reduced body temperature.
If anything, their dispositions became more violent than ever.
That was the end of the Serpasil experiment.

PROSSER: Dr. Luyet has a comment on one of the previous
questions.

LUYET: A question which arose in my mind in hearing this
morning’s report is that of the type of physiological reaction in-
volved in the Auer’s effect, that is, the effect of some magnesium
compounds in bringing about a lethargie state. Is it merely a
form of anesthesia ?

POPOVIC: Dr. Kayser says that magnesium is not necessary.

BISHOP: Magnesium sulfate is a perfectly good anesthetic.
We use it a lot.

LUYET: Is that merely anesthesia then?
BISHOP: As far as I know.

SUOMALAINEN: I have made such experiments. But when
we injected magnesium solution into hedgehogs and put them
into the ice-box, the condition of magnesium anesthesia was en-
tirely unlike the condition of natural hibernation. In the mag-
nesium anesthesia, the heat regulation of the animals was de-
ranged so that the homoiothermic state was changed into a kind
of poikilothermy. Metabolism diminished, the higher functions
of the nervous system were paralyzed and motility disappeared.
On the other hand, sensibility and museular tonus, which are

548 BULLETIN : MUSEUM OF COMPARATIVE ZOOLOGY Vol. 124

preserved in natural hibernation, were greatly diminished. The
animals were very limp. And, finally, the magnesium injection
always produced strong hyperglycemia (magnesium diabetes)
in contrast to the marked hypoglycemia found in hibernating
hedgehogs.

PROSSER: Do the cold and magnesium work in the same
direction ?

SUOMALAINEN: No, they don’t.

KAYSER: It has been known for a long time that magnesium
has narcotizing properties. If magnesium is given during the
summer, then in all hibernators and in all other animals narcosis
and hypothermia obtain. But, like all other suppressions of the
nervous system, the hibernators live 1 to 3 days, perhaps 4 days
in deep hypothermia but without the normal body position char-
acteristic of hibernation. Hamsters are dead after 2 days; ground
squirrels survive 5 ov 6 days in this state.

PROSSER: [| have one more question. I would lke to ask
Dr. Mayer or Dr. Smith: you mentioned the other day that
nutosis is pretty well interrupted, at least in the erypts or the
bone marrow, during hibernation and then as the animals awake
there is an increased burst of mitoses. Is there any evidence that
animals in this situation (where there is a stimulation of mitosis )
might get something that would even become a tumor, or go into
an especially excessive production of white blood cells? I am
interested in it from the point of view of control of mitosis.

SMITH: I know of no evidence concerning this, up to this
point at any rate.

DAWE: Thank you. In my own behalf, in behalf of the
Office of Naval Research, and in behalf of the American Institute
of Biological Sciences, I have certainly appreciated the oppor-
tunity to assist at this symposium. I wish to extend special
thanks to the panel and to the scientists who have come to us
from Europe: Dr. Johansson, Dr. Sarajas, Dr. Kayser, Dr.
Eisentraut and Dr. Suomalainen. I think that this has been an
outstanding opportunity for all of us at last to get together
and to talk over our mutual interests. Now, [ think Dr. Lyman
has some concluding remarks.

LYMAN: I didn’t realize I was supposed to make any con-
cluding remarks but I would like to thank, particularly, Dr.
Dawe whose ‘‘brainchild’’ this conference was.

1960 MAMMALIAN HIBERNATION 549

DAWE: Dr. Morrison was my major professor ‘‘way back
when.’” He is responsible for my basie interest. But the idea for
the conterence itself originated in a discussion you and I had
several years ago, Dr. Lyman, with Drs. Hock, Brace and F.
Smith.

LYMAN: | would also lke to thank Captain Ruebush and
the Office of Naval Research who made the symposium financially
possible and last, but not least, | would like to thank Mrs. Win-
quist and the wonderful staff of Endicott House for taking such
food care of us.

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