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UNIVERSITY OF CHICAGO
_ SCIENCE SERIES

Editorial Committee

_ELIAKIM HASTINGS MOORE, Chairman
JOHN MERLE COULTER
ROBERT ANDREWS MILLIKAN

_ The University of Chicago Science Series,
_ established by the Trustees of the University,
owes its origin to a feeling that there should be
a medium of publication occupying a position
between the technical journals with their short
articles and the- elaborate treatises which
attempt to cover several or all aspects of a wide
field. The volumes of the series will differ’
from the discussions generally appearing in
technical journals in that they will present the
complete results of an riment or series of
investigations which previously have appeared
only in scattered articles, if published at all.
On the other hand, they will differ from detailed
treatises by confining themselves to specific
problems of current interest, and in presenting
the subject in as summary a manner and with
as little technical detail as is consistent with
sound method. —

‘A CHEMICAL SIGN OF LIFE

THE BAKER & TAYLOR COMPANY
NEW YORK

THE CUNNINGHAM, CURTISS & WELCH COMPANY
LOS ANGELES
bs THE CAMBRIDGE UNIVERSITY PRESS

: LONDON AND EDINBURGH

THE MARUZEN-KABUSHIKI-KAISHA

TOKYO, OSAKA, KYOTO, FUKUOKA, SENDAI
THE MISSION BOOK COMPANY

SHANGHAI ‘

KARL W. HIERSEMANN ind
LEIPZIG

“? /

°O

- SHIRO ‘TASHIRO

Instructor in Physiological Chemistry in the University of Chicago

THE UNIVERSITY OF CHICAGO PRESS
CHICAGO, ILLINOIS |

| \
AJICY ,\:

Published March 1917

Composed and Printed By
The University of Chicago Press
Chicago, Illinois, U.S.A.

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PREFACE

- The present work is an attempt to apply facts dis-
Preovered during the study of the physiology of nerves

__ to living processes in general. That mechanism char-

acteristic of all living matter which enables it to respond
to the external world is best developed in the nervous
system. This mechanism may be called the most
characteristic thing in life. The chemical accompani-
ment, or basis, of this mechanism, discovered by the
author in nerve fibers, he has hoped to show exists
also in all forms of living matter, both of plants and of
animals. It gives a chemical method of distinguishing
living from dead tissue, and of measuring the quantity
of life.

This book, therefore, contains somewhat in detail
all the essential facts which he with his students has
discovered from studies of the chemical changes in
nerves accompanying functional change. In the pres-
entation of this work, however, many important refer-
ences and discussions have been omitted in order that
the reader may not lose the main trend of the argument.
The facts themselves are nevertheless given in the form —
of accurate numerical data so that the book may be
useful also to the specialist whose interest lies more
directly in the general physiology of the nervous system.

In an appendix the detailed method for the use of the
biometer is added in response to frequent requests of
many friends and students who wish to use it for various
biological and chemical researches.

The author is deeply indebted to Professor A. P.
Mathews for his criticism, scientific and literary, during

SHIRO TASHTRO
January, 1917
Vii

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III.

IV.

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CONTENTS

ei dari AS A SIGN OF rte lee
‘The Characteristics of Living Matter. Irritability.

Functional Changes as a Sign of Irritability. Functional

Changes in the Nerve Fiber.

CHEMICAL SIGNS OF IRRITABILITY IN THE NERVE

_ FIBER

Signs of Metabolic Activities in a Tissue. Indirect Evi-
dence for Presence of Metabolic Activity in the Nerve
Fiber. Direct Evidence. Experimental Methods with Non-
Medullated Nerve Fiber. Medullated Nerve Fiber. Is
Carbon Dioxide Produced by Living Processes? Discussion.

CHEMICAL SIGNS OF IRRITABILITY IN THE NERVE
FIBER—Continued .

Increased Metabolism on Stimulation. Experimental.
Electrical Stimulation. Other Stimulations. Discussion.

EXCITATION AND CONDUCTION

Excitability and Metabolism. Degree of Excitability ( Com-
pared with Metabolism. Effects of Narcotics. Lower
Concentration. Higher Concentration. Direction of the
Nerve Impulse and Metabolic Gradient. Velocity of the
Nerve Impulse and Carbon Dioxide Production.

. CHEMICAL SIGNS OF LIFE .

Resting Metabolism in Seeds. Increased Metabolism in
Seeds on Injury. Other Tissues. A Chemical Sign of Life.

. CONCLUSIONS

Summary. .Other Criteria of Life. Functions of Resting
Metabolism. Metabolism and Irritability. Chemical Con-
ditions in the Living Processes. Quantity of Life.

7 APPENDIX. THE BIOMETER: How To UsE IT

PAGE

109

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IRRITABILITY AS A SIGN OF LIFE

” | In the pages which follow we shall consider par-

ticularly the question of the chemical processes which

take place in nerves when nerve impulses pass over

them. There is scarcely a subject in the world more
interesting than this one, for the question of what is
the nature of that disturbance in nerve tissue which
shows itself in our thoughts has attracted men from the
earliest days. We must first find out the changes
of a material kind—if there are any such changes—
which occur in the brain when we think before we can
form any probable idea of the relation of these changes
to the psychical changes which accompany them.
Obviously, we must first try to solve the simplest prob- -
lem in this field and discover what are the changes
of a chemical or physical kind when a nerve impulse

flashes over a nerve before we can form any conception

of the relation of the material to the psychic world.
The following pages do not contain, of course, the solu-
tion of this problem of such absorbing interest, but
they do present the first accurate information we have
had of the chemical changes which accompany the
nerve impulse; they have in them, therefore, the
foundation upon which a solid structure of fact can be
based. |

The observations which we have to present are not
confined to nerves, however, for psychic phenomena are

not confined, we believe, to anima!
developed nerves. The course of evolution ‘tron
simplest to the most complex shows us very clearl

the complex psychic life of man and the higher Ree 7

did not suddenly spring fully formed into existence. In
every child, in fact, it can be seen to appear very slowly
and gradually and to increase as the child develops. We
cannot say at what point psychic life begins, for the
simplest organisms show some signs of it. Indeed, as
living originates from lifeless, we are led to conclude
that the simplest rudiments of psychic life must
be found also in the lifeless. And perhaps the universe
as a whole, inert as it appears to us to be, may have a
psychic life of its own. So it is not necessary to con-
fine our studies to nerves, for we find the same phe-
nomena which nerves show, phenomena corresponding to
those of nerve impulses, even in plants, and indeed in
the simplest kinds of plants. The differences between
animals and plants are superficial differences. Plants,
in general, are sessile; they cannot move freely from
place to place as animals do; and they have a green
pigment in them—chlorophyll—while most animals
have a red pigment in their blood. This green pigment
enables plants to make their food from simpler substances
than can be done by animals. But these differences
are superficial, and fundamentally plants and animals
are alike. We must suppose, therefore, that even so
humble a living form as a small plant seed has a psychic
life of its own. Impulses pass through it like nerve
impulses; it may be anesthetized as in the case of
man; it sleeps as does man; and, indeed, many of the
fundamental properties it shows resemble those which

—— _ = 7.” 7. “ee .- P i

IRRITABILITY AS A SIGN OF LIFE nag

we possess. Thus a part of the study which follows has
been made upon such simple things as seeds and garden

peas. It is surprising how closely the results obtained

with these parallel those obtained from nerves.

The really important and peculiar property of living
things is the psychic life they show. And if we actually
had an accurate means of testing the degree or amount
of life, it would be some kind of a reagent or instrument
for testing ‘“‘psychism,”’ as we may call it. But un-
fortunately we cannot at present find any means of test-
ing this property. We do not know what its physical
basis is, and, until we discover that, we cannot make
a psychometer which we can apply to all kinds of
living and non-living things, and thus measure the
amount of psychism, and hence of life, which they
possess. In the absence of any such psychometer we
have to do the best we can, and take as a measure of
this property those physical and chemical changes which
experience or experiment demonstrates to us always
accompany the psychic change. ‘The situation is very
much as it was in the realm of electricity before the
galvanometer was invented; an idea of the quantity
of electricity produced by a battery could be obtained
only indirectly by measuring the amount of chemical
change which the current produced, since Faraday
found that that amount was always a measure of the
amount of electricity.

There are material changes which occur in living .
things as long as they are alive and show psychic life
of any kind. The changes which we may rely upon to
measure the amount of life, and thus indirectly the
amount of psychism, are partly visible changes, but in

4 A CHEMICAL SIGN OF LIFE

part they are invisible and have to be detected by special
apparatus. The visible changes consist in some reaction
of the organism when its surroundings change. If it
moves when it is touched, the degree of movement not
being related to the impulse given it; if it breathes; if
it changes its rate of growth under changing conditions,
we say that it is alive. It is irritable, and it has the.
property of irritability. Another thing noticed in a
living thing is that the impulse which arouses it to
action may cause reaction in a part of the organism
distant from the point of stimulation. In other words,
the change, whatever its nature, set up in the organism
by the stimulus is propagated to a distance from the
point of irritation. We can see the results of this propa-
gation. All things which show this conduction and
response we say are living things. These are physical
processes which apparently always accompany the
psychic process. But sometimes these changes, although
they occur, produce no visible result; consequently we
must have methods which will detect conduction and
irritability, even though there are no visible signs of
them. One cannot see, for example, that anything has
happened to a seed when it is pricked by a pin; it
does not say “ouch!” loud enough for us to hear it, or
in a language we understand; but nevertheless it jumps
when it is pricked as if it did say “ouch!” as we can
show by appropriate methods.

There are two signs, or tests, which all living things
show and which are an index of life. One of these is an
electrical disturbance. ‘This was discovered a very long
time—a hundred years—ago, and its discovery was the
basis of the development of knowledge of electricity. The

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_ IRRITABILITY AS A SIGN OF LIFE 5

— other is a chemical sign, which has just been discovered

and which will be discussed in this book. The electrical
sign of life was discovered by Galvani when he found that
animal tissues are a source of electricity. He discovered
animal electricity. It is now certain that whenever
the response to a stimulus takes place in animals or
plants—the response which is the sign of life—an elec-

’ trical change accompanies it. By placing a galvanome-

ter on the animal or plant we can study this electrical
response. Life and electricity are thus shown to be
related. Electricity and psychism have something in
common, although just what the connection is cannot at
present be said. The English physiologist Waller has
recently introduced as a measure of life a particular kind
of electrical response which he has discovered and which
he calls the “blaze” current, because it is as if the

- electrical display suddenly blazed up when the living

matter was disturbed; this he calls an electrical sign of
life. By it he can tell whether a dry seed is alive or not
without putting it in the ground and letting it sprout.
It is very hard to know whether this electrical dis-
turbance which living things show is due to physical or
to chemical changes in their substances.

It is therefore a matter of very great interest that
I have recently found that there is always and every-
where an accompanying chemical change of a particular
kind which is as sure a sign of life and as invariable an
accompaniment of the vital reaction as the electrical
change. This chemical sign is the sudden outburst of
carbon dioxide which all living things show—plants as
well as animals, dry seeds as well as the nerve tissues of
the highest mammals—when they are stimulated in any

6 A CHEMICAL SIGN OF LIFE

way. The instrument which I have ‘made to detect — :

this carbon dioxide I have called a “‘biometer”’ because,
as will be appreciated from this short discussion, it is
an apparatus for measuring or detecting the amount of
life possessed by different things. I shall show in the
following pages that the increment of carbon dioxide
produced by living things when they are irritated, or
stimulated in any way, is a sure measure of the amount
of life they have; and we may hope that it is to be an |
indirect measure of the amount of psychism they possess,
although of course we cannot be sure of this as yet. It
will be noticed that it is not the absolute amount of
carbon dioxide which is the measure of life, but the
increase above the usual production which occurs when
a definite amount of stimulus is applied to the living
thing, which is the real measure of life. Anesthetized
or sick things do not show the normal increase; those
abounding in life show a remarkable increase.

The first results to be presented will be the proof that
carbon dioxide production is the sign of life of a nerve
fiber.- And it will be well before going into this to say a
few words about the scientific opinion concerning the
nature of the nerve impulse generally prevailing before
the work recorded here was done.

The main function of a nerve fiber is to transmit a
state of excitation from one place to another. It serves
for the conduction of the nerve impulse, which it trans-
mits in the most efficient manner. The nerve is also
excitable at all points, since it can be stimulated by a
variety of methods at any point along the fiber. When
physiologists investigated what takes place in nerve
fibers during the passage of nerve impulses, many

IRRITABILITY AS A SIGN OF LIFE 7

me peculiar results were brought out. In the first place,

if there are no other organs attached to the nerve it is
impossible to determine by casual observation whether
or not the nerve has been stimulated, for there is posi-
tively no visible physical sign of the vitality init. Not
even with a microscope can any structural change in the
tissue be seen. There is, also, no heat change detected
with a method which is sensitive to a millionth of a
degree Centigrade. There was, before this work was
published, no apparent production of carbon dioxide,
or any other chemical change in the tissue. ‘These facts
seemed to indicate that the functional activity of nerve
fibers was in no way associated with any chemical
change. ‘This failure of a nerve to show any chemical
or structural changes similar to those of muscles had a
decisive influence in the formation of ideas concerning,
not only the nature of the nerve impulse, but also the
nature of irritability in general. For nerve fibers

_ not only show the highest type of irritability of proto-

plasm, but they also possess, as stated before, the power
of transmitting the state of excitation in the most perfect
manner. And all attempts to explain the nature of
irritability in general must necessarily account for the
peculiarities of the nerve fiber where we find that prop-
erty in its highest development. If irritability, excita-
tion, and conduction do not involve chemical changes
in nerves, it may be concluded that neither do they in
any other tissues. ‘Thus, on account of the absence
of evidence of any chemical changes accompanying irrita-
bility in nerves, we have gradually drifted away from the
notion that the fundamental condition for protoplasmic
activity is chemical.

‘A CHEMICAL SIGN OF LIFE ——

When it was found that an electrical change occurred

in a nerve when it conducted an impulse, the problem was _ :

considered to be settled. The nerve impulse was sup-
posed to be electrical in nature. This idea was soon
questioned, however, when the speed of the conduction
of a nerve impulse was found to be so slow in comparison
with that of an electrical current. The speediest nerves,
such as those of human beings, conduct impulses only
at the rate of a hundred meters per second, whereas
electricity travels in a wire at a speed of thousands of
kilometers per second. One thing seemed to be certain—
that the nerve impulse can pass through a fiber without
consuming any material. It was found that some
nerves could not be fatigued even on prolonged stimula-
tion. This fact supported the idea that certain quickly
reversible physical conditions must exist in the nerve, and
that the changes in these conditions, rather than chemical
changes, must determine the phenomena of irritability
and conductivity. Ultimately physiologists settled
down to the view that the physical and fundamental
changes concerned in irritability were either a change
of colloidal state, of surface tension, or in the permea-
bility of the nerve to salt, or changes in the distribution
of electrically charged particles in the nerve.

Although such physical changes as these in nerves
have never been demonstrated experimentally, biolo-
gists generally have tried to explain the nature of a
- nerve impulse and the phenomena of excitation purely
on the basis of these hypothetical physical changes;
and they have neglected the chemical changes. ‘They
have also attributed many other important physiological
functions, such as secretion and contractility, to these

f this view will be apparent from what follows, where
B iti is shown that chemical changes are, indeed, an inva-
- 4 -tiable peerere eaent of nervous activity, and of all life.

CHEMICAL SIGNS OF IRRITABILITY. INT
NERVE FIBER oy 7 |

There are various chemical processes which occur in

all forms of living matter and which we might examine a
in order to see whether they are associated with the __

property of irritability, but we naturally seek to make
use of that one which is the easiest to detect. Among
these chemical processes there are, in the first place,
the processes concerned in growth. All living matter
has the power of building up complex proteins, fats,
and carbohydrates as long as it is vigorously, alive.
But it is clear that this process would be very hard to
measure quantitatively without killing the living matter
and determining how much substance it has produced.
And there are also other objections to using growth as a
measure of vitality. Another chemical process found
in all, or nearly all, forms of matter is respiration. By
respiration we mean the gaseous exchange of living
matter with its environment: the taking on of oxygen
and the production of carbon dioxide. This is a very
much more promising line of experiment to follow in
measuring life and metabolism, for, in the first place, it
is universal, as I shall presently show, and, in the second
place, the oxygen may be measured, or the carbon
dioxide given off may be determined, without injuring
the living matter. It was for this reason that the
carbon dioxide was selected for study as probably

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CHEMICAL sic NS OF IRRITABILITY 1

eing thé ‘ope # usitstalive pa Sele and
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vital 1 processes.
The idea that respiration is one of the most funda-
Pricatal of vital phenomena is by no means a novel view.
| _ Even in the earliest times breathing was supposed to
be the process most intimately connected with life.
When a man stopped breathing he died. As early as
the second century Galen had the notion that there must
_ be a pneumatic spirit in the air which kept up life,
and he predicted that some day it would be discovered.
It was after the lapse of fifteen hundred years that this
prediction was verified or fulfilled when Mayow, an
English physician, discovered that there was a gas, or
spirit, in the air which was essential to life and com-
bustion. Later, oxygen was discovered by Priestly, and
it was Lavoisier who first showed that this oxygen after
entering the lungs came out again as carbon dioxide;
and he proved that animal heat was due to the com-
bustion of the materials of the body by the oxygen to
_ form water and carbon dioxide, and that the sole source
_ of energy of living things was this combustive change.
In selecting respiration as the chemical test of life we
are, therefore, selecting that most fundamental reaction
by virtue of which living things get their energy. It
is clear that it is this reaction, rather than any other
chemical reaction, which touches most closely the
phenomena of irritability; for, to move or to think,
we must have energy. It is much better to take this
reaction, rather than those chemical changes which
are related to growth or the repair of waste, as a
criterion of living, for the very essence of a living thing

12 A CHEMICAL SIGN OF LIFE

is that by chemical transformations it sets free energy
and moves itself.

It is much better, too, to take the carbon dioxide pro-
duced, rather than the oxygen consumed, as the measure
of the metabolism associated with irritability, for the
reason that sometimes organisms get their oxygen from
sources other than the air, whereas their carbon dioxide
production is always something positive and universal.

Indirect evidence of the presence of metabolic activity
in the nerve fiber—The search for some kind of metab-
olism, such as the production ,of carbon dioxide, in
nerves had been made by many physiologists on many
occasions, but it was impossible for them to discover
this substance because their methods were not sufficiently
delicate. No carbon dioxide could be found, and for
this and other reasons the conclusion was incorrectly
drawn that there was none produced, or that, if it were
produced, it had no connection with the vital functions
of thenerve. Most physiologists were accordingly of the
opinion that the conduction of the nerve impulse was
a physical process and involved no transformation of
energy and no consumption of material. There was
one exception to this rule. Professor A. D. Waller,
the eminent English physiologist, maintained that,
because of their electrical behavior, nerves certainly
produced carbon dioxide. In 1896 he showed that
carbon dioxide when applied to a nerve produced a very
characteristic change in the electrical response which a
nerve exhibits when it is irritated. It will be remem-
bered that when a nerve or, in fact, any kind of proto-
plasm is irritated in any way, if one applies two electrodes
to the living tissue in such a way that one electrode is on

t rect vity cee the ike on a part which
is less ae ahi be found that there is a current
_ which flows in the tissue from the more active to the less
active part, from the more to the less excited, and outside
in the galvanometer from the less to the more excited
_ part. This electrical current was discovered by Galvani
and is called the current of action, or the action current.
Generally, when an impulse sweeps along a nerve to
which two electrodes are applied, first one electrode
and then the other becomes negative, so that the current
is diphasic, running first in one direction and then in the
other. Now, Waller observed that when a nerve was
. exposed to carbon dioxide this diphasic current showed
a characteristic change, the negative phase being first
increased by small amounts of carbon dioxide and then
diminished. He then discovered that just the same kind
of a change occurred in the electrical response if he stimu-
lated a nerve repeatedly at very short intervals of time.
- He concluded from this that on stimulation of the nerve
carbon dioxide was produced, and that this caused the
characteristic alteration of the electrical response which
occurred in the tetanized or repeatedly stimulated nerve.
| This conclusion was not generally accepted by physiol-
i; ogists for the reason that it was possible that the same
4 change in the electrical response might be produced in
other ways than by carbon dioxide, and while the experi-
ments were regarded as circumstantial evidence of value,

} ;

. showing that a chemical change accompanied the nerve
ie impulse, they were not regarded as conclusive.

+ Waller supported this conclusion by another dis-

covery, namely, that when he stimulated the nerve at
| regular intervals, not too long or too short, by a strong

responses, eas of wphichs is ie prcdteat’ de erve ¢
give at the time, but each of which is a little ore

_ than its predecessor. Such a series of increasing ‘fee

sponses is known as a. staircase, the negative os
increasing steadily while the positive phase decreases.

This Waller explained by supposing that small amounts — |

of carbon dioxide were formed by each nerve activity,
and that this augmented the negative response and
diminished the positive response, just as does carbon
dioxide applied to the outside of the fiber. He considered
that our failure to find the gas was due to the inadequacy
of the chemical methods then in existence. That this
criticism of Waller’s was a just one and that there may
be carbon dioxide produced by nerves, but too small in
amount to be measured by the ordinary chemical method,
is shown by the following calculation: A frog (Rana
temporaria) gives off 0.355 g. of carbon dioxide per
kilogram per hour at 19° to 20° C. A small piece of
the nerve fiber of the same animal, say 1 cm., or three-
eighths of an inch, in length, will weigh, probably, not
more than 1o mg. Now, if this mass of the nerve
fiber respires at the same rate as the whole animal, it
will not give off more than about 0.000,000,7 g. of
carbon dioxide during ten minutes. This calculation
at once suggested that the failure to detect the evolution
of carbon dioxide in nerves was very probably due to the
limitation of the methods for the estimation of the carbon
dioxide, and that it was not at all conclusive evidence
that carbon dioxide was not produced. It was evidently
necessary to devise methods for the detection of very
minute quantities of carbon dioxide.

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roxiee to study the whole of the Papivdtony metab-
a lism of a tissue, we should at least determine the .
Ben consumption as well as the carbon dioxide
no: and also generally the heat production. '
- Inasmuch as the present problem, however, is concerned
only with presenting direct evidence for the existence
of metabolic activity in nerve fibers, we shall attempt
-to measure the carbon dioxide production alone; for
while the lack of consumption of atmospheric oxygen
may not necessarily indicate the absence of chemical
changes, the production of carbon dioxide will surely
prove the presence of metabolism, provided, of course,
that we can prove that such carbon dioxide is formed by
physiological processes. Furthermore, as carbon dioxide
is the only universal expression of the respiratory
activity in almost all anaérobic and aérobic plant and
animal tissues in normal condition, metabolic activity
is probably better represented by carbon dioxide pro-
duction than by oxygen consumption, although we
must restate here, of course, that the study of carbon
dioxide alone will never reveal completely the nature of
the metabolic activity.

Method.—The method which was finally devised to
detect and measure quantitatively the very minute
amounts of carbon dioxide which it might be expected
| would be formed consisted essentially in determining
| the amount of carbon dioxide which was just sufficient
fe to produce a deposit of barium carbonate in a film of
| __half-saturated barium hydroxide solution. Barium car-
& bonate is almost entirely insoluble in such a barium
hydroxide solution, and a very small amount of precipi-
tate can be detected with the aid of a small lens. The

- ra we

16 - A CHEMICAL’SIGN OF LIFE _

method is described in detail in the Appendix. The
special apparatus, the biometer, as I have named it,
which was constructed for the investigation, is shown in
Fig. 1, and its use is detailed in the Appendix. It will —
detect one ten-millionth of a gram of carbon dioxide and
estimate it with accuracy.
As is shown in the figure, the biometer has two
respiratory chambers each provided with a small tube, at»
the top of which the hemispherical drop of barium
hydroxide can be formed. Exceedingly minute amounts
of carbon dioxide produced in the chamber by the small
piece of nerve will be precipitated as barium carbonate
on the surface film of these hemispherical drops and may
beseen withalens. As the apparatus has two chambers,
not only can we detect very small amounts of carbon
dioxide which the nerve may produce, but we can also
compare the output of carbon dioxide of different
tissues under the same conditions, by placing one
tissue in one respiratory chamber and another in the
other.
To discover whether nerve fibers, as distinct from
nerve cells, respire, particular care was taken to select
at first those nerves which are known to be free from
such cells and, as far as possible, free also from connective
tissue. It was necessary to do this because the work of
several investigators seemed to indicate that tissue oxi-
dation was in some way dependent on the cell nucleus.
Certain biologists even went so far as to believe that a
nerve fiber ought not to respire at all, since it contained
no nucleus. The fact that the blood supply to the
brain, where most of the nerve cells are located, is so
copious, whereas the blood supply to the nerve fibers is

CHEMICAL SIGNS OF IRRITABILITY 17

Fic. 1.—The Biometer. One-fourth actual size

we tate studied qunntinivas fie FS of
dioxide from various lengths of nerves which are know:
to be free from nerve cells and which have anor @
connective tissue in them. For this purpose the dave |
nerve from the spider crab was selected. a
Nerve fibers are of two kinds, called rbspeceeen “a
medullated and non-medullated nerves. The essential — a
conducting parts of these are alike, but the medullated
fibers have lying about the conducting core of the fiber a
white, glistening, fatty matter called the medulla, or
myelin sheath. Most of the nerves going to voluntary
muscle in the higher vertebrates are medullated; but
the nerves to the viscera are often non-medullated
and the nerves of the invertebrates are usually non-
medullated. ‘This medullary sheath is evidently some- —
thing which is found in those nerves which it isimportant —
should conduct very quickly and which should not be
fatigued by conduction, and it is clear that the medulla-
tion is an improvement which has not yet been universally
introduced. The function of this sheath is probably
nutritive. But in any case it is important, if we wish to
avoid any complication which it may introduce into the
physiology of the nerve, to examine both medullated and
non-medullated nerves. And that we have done.
Non-medullated nerve fibers—When an isolated claw
nerve of the spider crab is placed in the right chamber —

t Indeed, Bayliss attributes our results, which are soon to be detailed,
to the presence of the connective tissue cells around the fiber, so firmly
convinced does he appear to be that only nucleated parts of cells respire.
For further consideration of this objection see p. 33.

eter ae no nerve in the left, the biometer
y sealed with mercury and filled with air
which is vires from carbon dioxide, and if barium hydrox-
ide is allowed to rise to the top of each tube in such a
‘way as to form hemispherical drops of approximately
~ equal size in both chambers, we observe that the drop
_ in the right chamber, where the nerve is, will soon be
~ coated with a white precipitate of barium carbonate,
but that no precipitate whatever can be seen, even with
a lens, in the left chamber. Carbon dioxide is thus
shown to be produced by this resting nerve of the spider
crab. By interchanging the nerve from the right to the
left chamber, no nerve being now put in the right, we
find that the precipitate is now in the left-hand side of
the biometer, and we have no difficulty in convincing
ourselves that the carbon dioxide has come from the
nerve, for we have thus eliminated any technical error
which might have produced the different results in the
different chambers. ‘The rate at which the precipitate
appears and its quantity depend on the size of the nerve
and the length of time we leave it in the chamber. That
an unstimulated nerve gives off carbon dioxide is a fact
which can thus be demonstrated easily to anyone if the
proper apparatus is at hand. The rate of production
of carbon dioxide by the normal resting nerve of the |
spider crab is found to be proportional to its weight,
other things being equal, and is fairly constant. ‘The
quantitative determination shows that for 10 mg. of
nerve per ten minutes it gives off 6.7xX10 7 g. of carbon
dioxide at 15° to 16° C.
The quantitative determination of this amount is made in
the following manner: The claws of the crab are carefully removed

20 ~~ A CHEMICAL SIGN OF LIFE

from the body, and by gentle cracking the long fiber of the nerve

trunk is easily isolated. After the last drop of water is removed
by a filter paper, the nerve, with the aid of glass chopsticks, is

carefully placed on the glass plate (Fig. 2) and quickly weighed.
The glass plate with the nerve is now hung on the platinum hooks in

the right respiratory chamber, and the chamber is then sealed with —

mercury. The left, or analytic chamber, is now partially filled with
mercury in the manner described elsewhere, and then the appara-
tus is washed as usual by air free from carbon dioxide. The

time at which barium hydroxide is introduced into the top of the -

tube in the left chamber is recorded and the stopcock between
the two chambers is closed. When at the end of ten minutes the
drop on the tube in the left chamber is perfectly clear, having not
a single granule of the precipitate visible to a lens, thus insuring
that the air used for washing is absolutely free from carbon
dioxide, a known amount of the gas from the right respiratory
chamber is introduced into the left chamber in which the clear drop
of barium hydroxide has been exposed, and it is determined
whether or not the amount of the gas taken contains enough
carbon dioxide to give a precipitate in several minutes. Usually
ten minutes will be sufficient for the reaction.t If it does not give
a precipitate in this time, a larger volume should be taken until
the precipitate appears within ten minutes. If it does, the ap-
paratus is washed, dried, and with a fresh nerve the procedure is
repeated, but a less volume of the gas than the amount which
before gave the precipitate is withdrawn into the left chamber
from the right?

In this way, by the use of several fresh nerves, a minimum
volume of the gas for a known weight of the nerve which gives a
precipitate is determined. This minimum volume should con-
tain a definite quantity of carbon dioxide, namely, 1.0X 1077 g.,
the amount carefully determined previously (taking known
amounts of the exceedingly diluted gas) to be just sufficient to
produce a noticeable precipitate. .

* The weight of this plate is known, hence the weight of the nerve
can be determined very quickly (see p. 38).

2 See footnote, p. 126.

ae
i

’ = ——
ve

—)

. P

—
ene

F
|
t

ee =e EF 9 -
A» 24 ~

“a « -

— -
_

Th s gh ee os sEspw the original volume of the chamber in

n the respiration took place and from which this minimum
1 me is withdrawn, and since we know the quantity of carbon

much Sieh didxide’‘s given off by the nerve during the known
_ period. It should be understood that, in determining the mini-
- mum volume of gas taken from the respiratory chamber, a series
of experiments was conducted in order to calculate both the mini-
‘mum volume which just gives the precipitate and the maximum

volume which does not give the precipitate for a known weight of

the nerve for a known period of respiration. In Table I, in the

Appendix (p. 128), columns 8 and 9g refer to these ohahia

calculated from experiments for 10 mg. of the nerve, for ten
- minutes.

Medullated nerve fibers—We have repeated this
experiment with the sciatic nerve of the frog, this nerve
being a typical medullated nerve. The result showed,
not only that medullated nerves also give off carbon
dioxide, but that they give a quantity of about 5.5
1o ’ g. for each 1o mg. of the nerve for the first ten
minutes, which is a little less than was obtained from
the non-medullated nerve.

A large variety of nerves was tested to see whether or
not all resting nerves give off carbon dioxide. As a
result, we found no exception in any of them, although
they vary quite widely in the rate at which they |
produce carbon dioxide. The following nerves were
examined, and it will be noticed that the list includes
all varieties, such as sensory, motor, vertebrate and
invertebrate, medullated and non-medullated nerves.

t. Motor Nerve: Oculomotor nerve of the skate (Raia
ocallata).
2. SENSORY NERVE: Olfactory nerve of the same.

¥ ae
et Ws rc

b&
Rs, J Rea De ‘

oon ts
' ‘ i
d ay
ie ,
eae
‘ os ae ae A.
J > * ‘
.

4. Nott Mivustarkis NERVES:
olfactory nerve of the skate (Raita 0 ocal Il t
5. NERVES OF INVERTEBRATES: Nerves of ‘the s
Limulus, Limax. his ~
6. NERVES OF Vukasonane: I Rertss of frog, dog, n
squiteague (Cynoscion regalis), and skate ean : Raia
ocallata and Raia erinecia). ; ae
7. NERVES OF WARM-BLOODED Anas: Those of dog, rat, S
rabbit, guinea-pig. : .
8. Nerves or Cotp-BLoopep Animats: Those of frog, squi-
teague (Cynoscion regalis), catfish, carp, and skate.
g. SENSORY DENDRITE: Lateral line nerve (ramus lateralis
vagi) of carp and catfish, and ramus lateralis accessorius of
catfish.

This is a partial list of the many nerves examined and ©
it is given only to show that we are justified in making
the generalization that all freshly isolated nerves of
all animals, regardless of the kind of nerve or of the kind
of animal, produce’ carbon dioxide. It is thus certain
that chemical changes of a very vigorous kind are
going on constantly in this tissue without any visible
results. Nerves respire; they are not chemically inert.
It remains now for us to establish the fact that this car-
bon dioxide is a product of normal metabolic activity
and is not due to a disintegration involved in the process
of dying on the part of the tissue, or to a lifeless fermen-
tation, and that it is not simply gas which had happened
to be absorbed by the nerve from the atmosphere or the
blood.

Is this carbon dioxide produced by living processes ?—
Since there are many organic compounds, as well as dead

‘HEMICAL SIGNS S OF IRRITABILITY 23
Bette Shor dioxide either by direct

re x ( Btn, by Seehitation, or by the decomposition of
- carbonates by acids, the possibility that. this carbon
_ dioxide which we have detected is not a product of

vital activity cannot be so easily disproved. Inasmuch
as our apparatus detects such a small amount of the gas
as that which is contained in one-sixth of a cubic centi-
meter of the purest air, we cannot accept the results just
cited as certain proof that the normal nerve undergoes
metabolic changes. We must inquire, therefore, whether
this carbon dioxide is produced by living processes. In
the first place, as the biometer in its present form cannot
examine the carbon dioxide production of a nerve in its
normal position and with its muscle attached to it, we
have to use an isolated nerve. Certain experimental
factors are thus introduced which must be carefully
considered before we interpret our observations. It
is first necessary to be sure that this isolated nerve lives
and remains excitable for a considerable period after it
has been removed from the body. We can be quite
certain that this is the case because of the fortunate
circumstance that the passage of the nerve impulse
through such an isolated nerve produces a characteristic
electrical disturbance, which we may detect by a sensi-
tive galvanometer. As long as this electrical dis-
turbance occurs and the nerve is excited, we may be
perfectly sure that the nerve is living. It is as certain
a sign of the passage of the nerve impulse, and conse-
quently as sure an evidence of the vitality of the nerve,
as would be the contraction of the muscle which the
nerve supplies, had this remained attached, to it. By
thus testing with a galvanometer isolated nerves, such

: the vitality REN, even. as oan as ni
after removal from the body. These facts a:
therefore, that the observations made on the |
dioxide production of isolated nerves are really poles on te:
active living nerves, and they may be regarded as quali.
tatively similar to what would happen in the abe
nerve in situ were we able to measure its carbon dioxide © -
production.

A
fl
bs
-"

7 A.

TABLEI | ie

COMPARISON BETWEEN NORMAL AND KILLED (By STEAM) NERVES OF SPIDER CRAB a

I 2 3 4 5 6 7

Tempera- . entime- | Duration t. of

ture 0 Weight of | stimula- | tets of Gas | .F Respira- BC)

Date Nerve in * Taken *
Room | Milligrams HOR" 1 Gee Rae tion ter Ten
Degrees C. igh piratory Minutes | Minutes
Chamber

November 4.. 73 40 (killed) No 0.5 Io ~
November 4. . Ai 40 (killed) Yes 0.5 5 xe) _
November 5.. scr 16 ae er No I.0 Io +
November 6. . 15 16 (killed) No 1.0 12 -
November 7. . 16 16 (normal) No I.0 b te) +

If the carbon dioxide is produced by vital activity,
its production should be diminished when the nerve is
killed. This we can demonstrate by placing a nerve
killed by steam in one chamber of the biometer and an
equal weight of a normal living nerve in the other
chamber and then comparing simultaneously the output
of carbon dioxide in the living and dead nerves. It is
found that the living nerve continues to give off carbon
dioxide, while the dead gives off extremely little, the
difference between the two becoming more marked as
time goes on. Such a comparison between two nerves
of the spider crab is given in Table I, from which it

CHEMICAL SIGNS OF IRRITABILITY 25

will be seen, if the experiments on November 6 and 7 are
compared, that 1 c.c. of gas taken from the respiratory
chamber in which the dead nerve had been for a certain
length of time contained not enough carbon dioxide to
produce a precipitate, while 1 c.c. of gas from the cham-
ber in which the living nerve had been for the same time
did produce a precipitate and consequently contained
more carbon dioxide. It is clear, then, that a dead
nerve gives off less carbon dioxide than the living.

Comparison of anesthetized and normal nerves.—By
the use of anesthetics we can diminish the irritability,
or, aS we may say, the vitality, of the nerve without
abolishing it altogether, The nerve, although anesthe-
tized, is still alive, but in a condition of suspended
animation. When the anesthetic escapes from it, it
recovers its normal vitality. If the carbon dioxide has
been produced by a vital process and is at all corre-
lated with the state of irritability of the nerve, we
should expect that a diminution of that irritability by
anesthetics would produce a diminution in the carbon
dioxide output. If, on the other hand, this carbon
dioxide is the result, not of a vital process, but of a
fermentation, or of an acid production of some sort, then
we should expect that it would be little, if at all, affected
by the anesthetic. Accordingly, nerves were anesthe-
tized in various ways, for example, by placing them in a
solution of urethane, or they were treated with the
vapors of ether, or the nerve was isolated from a deeply
anesthetized frog, and the quantity of carbon dioxide
produced by such nerves was compared with the quantity
- produced by normal nerves of the same animals. It was
found always that the anesthetized nerve gave off

~~ F - ws t 7" % iar a : 2 = ; 7 -. ,
decidedly less carbon dioxide than the nerves
iad or nerves taken from frogs whose | circ i

between the carbon dioxide phoaubeon and the ae |
of excitability of the nerve. Thus small quantities oo "1
anesthetics have often the effect of increasing at first
the excitability of the nerve, and it was found that such ©
_ quantities also produced at first an increase in the carbon — -
dioxide. A further consideration of the effects of
anesthetics on the metabolism of the claw nerve of the
spider crab will be found in chapter iv. ‘The important
fact is that since these agents are known to affect the
normal uncut nerve im situ and also to modify carbon
dioxide production in an isolated nerve, and in a manner
parallel with their known actions on ‘irritability, it is
certain that at least the larger part of the carbon dioxide
we measure in an isolated resting nerve must have been
produced by a physiological process. |
Carbon dioxide production in a hydrogen atmosphere. — .
Although many nerves remain alive for a long time in an
atmosphere free from oxygen, they generally exhibit a
lowered irritability when compared with nerves in normal
air. It has been found, for example, that if nerves
remain in the body after the circulation of a frog has
ceased, so that they have not been supplied with oxygen
for some time, they are by no means so easily stimulated
by a salt solution as are normal nerves. ‘Their vitality
is reduced. A similar change occurs in nerves taken
out of the body and put in hydrogen gas. In them, also,
irritability is decidedly diminished. If, now, carbon
dioxide is produced in these nerves by a vital process,

pager St

7 i T= ty bay id n
° i’ ; ia
“=.

CHEMICAL SIGNS OF IRRITABILITY 27
ane _ as ro

should in case to find that less carbon dios was
sduced by nerves in an atmosphere of hydrogen than
normal air. On the other hand, if the carbon dioxide
| Baus to some fermentation, or non-vital process, then
a Pi thould not be influenced by the absence of oxygen.
| ‘When, with Dr. Adams, we determined the rate of
carbon dioxide production in nerves placed in an atmos-
phere of hydrogen gas, care having been taken to insure
the gas being perfectly pure, we found that the rate was
only about half that of the normal nerve. It appears
from this determination that in a medium deficient in
oxygen the claw nerve of the spider crab gives off less
carbon dioxide than in an ordinary atmosphere. The
effect cannot be due to the hydrogen, since that gas has
no physiological action, but is quite inert, and we may
conclude that the lowering of the carbon dioxide is due
to the lack or absence of oxygen. ‘This is additional
evidence that the lowering of the gaseous output is a
f physiological phenomenon, and that the carbon dioxide
he measured in normal isolated nerves is a product of normal
metabolism, and is not the mere diffusion outward of the
gas which is present in the tissue, being produced there
by other than living processes.

Carbon dioxide production of the isolated nerve at suc-
cessive time intervals.—If the carbon dioxide production
is due to a vital process, it might be expected to diminish
gradually in the isolated nerve as its vitality diminishes.
On the other hand, there was a possibility that the iso-
lated nerve had become infected with bacteria and that
the carbon dioxide might be due to their action. If

_ this were the case, it would be expected that the carbon
dioxide would gradually increase. Accordingly, experi-

Ah nerve was pe se hoa the Shady.”

sciatic nerves were isolated from several frogs of thes mie te
size and sex and were left for varying periods of time a at
Ringer’s salt solution, in which they live well. The
rate of the gas production was then determined in the -
nerves when removed from the Ringer solution after —
one hour, two hours, and at other intervals up to
twenty-five hours. The interesting results given in
Table II make it clear that the fresh nerve produces the

; TABLE II
SHOWING DeEcrEASED CO: Propuction By LONG STANDING (FRoc’s Scratic)
I oe 3 4
Pron maepas sree ee ;
entimeters Neces- 9) mount o
Wace Time Elapsed | “sary to Give } | COs Produced by
er Isolation ro mg. of Nerve in
Degrees Cs Sv gang 12 = 1o ‘Minutes
Minutes

BAY atv nigh Kom tek Immediately 2.7 Cc. 5.5X10—7 g. CO,
BEL Spe cceing oe others b t hour 7.08 C.c. 2.1X10~7 g. CO:
Ohio ac akawas bos 2 hours 10.8 c.c. ' 1.41077 g. CO,
SES A Rae 5.5 hours 12.8 c.c. 1.1X10~7 g. CO:
oe re PR cay een 7 hours 15.3 C.Cc. 0.9X10~7 g. COz
Pye een ee Ree to.5 hours’ 21.0 C.c. 0.6X10—7 g. CO.
Beeld a teinadh ed 26 hours 9 cc * 1.6X10—7 g. CO;
DA eek ose oveiere Pu a Sates 27.4 hours TO. GC; 8.1X10~7 g. CO:

* The gradual increase at this point should be noted (after 26 hours, it is clear that
bacterial decomposition sets in).

most carbon dioxide and that the amount produced per
unit of time interval decreases rapidly up to about
twenty-three hours and from then on suffers a very
rapid increase. ‘These facts show that the carbon dioxide
output diminishes as the vitality of the nerve diminishes,
and that as bacterial decomposition sets in there is a
sudden and rapid increase. There is, therefore, a

CHEMICAL SIGNS OF IRRITABILITY 29

parallelism between the decrease in metabolism and
decrease of irritability in the nerve. The gas produc-
tion slows up as the nerve approaches death. This
indicates, also, that the carbon dioxide is formed by a
vital process.

Comparison between the metabolism of resting nerves
and other tissues.—While a comparison of the rate of the
metabolism of the nerve with that of other tissues is
subject to a good many limitations, since there are so
many and great variations in conditions which do not —
affect all tissues similarly, it is nevertheless interesting
to note whether the nerve respires relatively more or less
than most other tissues. In order to give a better
numerical picture of the amount of metabolism in the
resting nerve, as compared with other tissues, we have
set down in Table III the figures for carbon dioxide
production in various animals. Since there are no exact
determinations made of the carbon dioxide production of
the spider crab as a whole, or of its tissues, we have used
for comparison various other crustacea where these
data have been determined. It will be noticed from an
inspection of this table that the spider crab nerve pro-
duces, weight for weight, carbon dioxide at a rate three
to four times that of the whole body of crabs, and almost
as much in proportion to weight as a human being at
test. Recently Bayliss, in his admirable book entitled
Principles of General Physiology, expressed a doubt of our
figures. He thinks that the gas we measured must be
due to some cause other-than the metabolic activity of
the nerve, because, he says, the data show that it is
greater than that of an equal weight of muscle. It is
rather difficult for us to understand the force of this

that a any other tissue of the body.

as we know, no physiological reason fri
priori that the nerve has a lower metabolism than. 2
tissues, but, on the contrary, the direct and indirect oo

TABLE III
CO: per T a
Animals Kilogram emperature . Determined by *
per Hour Degrees C.

Crustacea ‘(whole animal).'.33..%.0ecetacn cae tee eee Jolyet and Regnaut _
Crayfish (Astacus)............00- 37:7 C.C. 12/5 = & “* ;
Crab (Cancer pagurus)........... 89.9 c.c. 16 6) ae 7
Lobster (Homarus vulgaris)........ 54.4 C.C. I5 of 4s ¥
Nerve of spider crab (Labinia cana-

YOU AIG) i Bear le Saw ame Ra 212 C.c. or 15-16 Tashiro

0.402 g. oe.

Frog:

(Rana esculenta) (whole animal).. 0.082 g. 17 Schultz

(Rana temporaria) (whole animal) 0.355 g. 19-20 Pott |

(Rana pipiens) (sciatic nerve).... 0.33 g. I5 Tashiro

(Rana temporariat) (isolated) —

muscles, og 0h uy esbaktne we 0.18 g. 2 Fletcher

DOS 62a Ai Rose ear aI Chek Ti3SISR. Wa cenes coe Regnaut and Reiset.
Man at rest........ hive eee Fata re OAD Sap od cette ob Pettenkoffer and Voit

SM EU | Nr 9 oe SIP RRs aN ee GOR Be kalo eis eek

eM Readitie ll ee Eek, Pade 5 =a Bae POPE fant Cast Ae eae le Speck

* All the figures are quoted from Schifer’s Text Book of Physiology, I, 702, 707, and
708, except that of the isolated muscle, which I calculated from Fletcher (04. cit.).
Fletcher fails to state the weight of a leg, but gives the value o.2 c.c. for one-half hour.
er ap aa that if we take each leg as 6 g. in average, the value will not be far from the
trut

t Fletcher fails to state the species of the frog, but it is inferred from Hill’s paper.

evidence shows that it has a more intense metabolism.
It is no doubt true, however, that an isolated nerve, such
as we have used, respires somewhat faster than the
same nerve intact in the body, because the effect of
cutting the nerve is to act as a stimulant. But, even
allowing for this effect, the metabolism still remains
markedly higher than that of most other tissues. We
may add here, however, that the hourly rate of output
of carbon dioxide from the resting nerve of a frog is

Con aon wr ee dioxide output of nerve fliers and
erve ganglia.—From the table already presented it is
clear that the living nerve trunk containing no nerve
“cel ells gives off carbon dioxide at a rapid rate. It is inter-
: esting to see whether nerve tissues containing ganglion

_ cells produce more or less carbon dioxide per gram per

hour than the nerve fibers. For this purpose we studied
the ganglionated nerve cord on the back of the heart
of ‘the king crab (Limulus polyphemus). This is an
elongated automatic ganglion which has been shown
to be the direct cause of the heart-beat. It was isolated
carefully from the heart, the operation taking but a few
minutes, placed in the biometer, and its carbon dioxide
output measured. It was found to give 2.3X10~’ to
4.7X10~7 g. CO, per centigram per ten minutes at 22.8°
to 23° C. The rate was somewhat lower in the larger
individuals, which were usually females. This amount
of carbon dioxide is very small when compared with the
output of the claw nerve of the spider crab, which with-
out stimulation gives off from an equal weight of tissue
6.7X1077 g. If, however, the comparison be made
with the claw nerve or with the optic nerve of Limulus
itsel{—the same animal as that from which the ganglion
was taken—the rate in the ganglion is found to be about
the same as that in the fibers. The claw nerve of
Limulus gives only about 2.61077 g. of carbon dioxide,
while the optic nerve gives somewhat more, namely, 2.6 to
5107’ g., depending on what portion of the optic nerve
is taken (see p. 76). Limulus is a very sluggish, slow-
moving animal, whereas the spider crab is more active.

anything, its rate is a little aver “This j is me in- |
teresting because, as already stated, this ganglion

‘giiaggion gives off about tl
dioxide per gram of its subst ‘
the same animal.

camo
1

‘TABLE IV
SUMMARY OF CARBON DIOXIDE PRopuction FRoM VARIOUS NERVE TISSUES *
rao | Sele te
emper- iven A
Animal ‘| Sex Nerve ature | Off by ro mg. a
DegreesC.| of Nerve in y
to Minutes
f 3 arse cord of heart (30-
SB tec 23-23.5 | 4.7X10~7g.| Tashiro,
ro) Nerve cord of heart (51 i ia
Eats este dk oe 23 2.4X1077g.| Tashiro,
é News cord of heart (52 A
Limulus WD! 6 ss Sot ee ces 23 2.3X10~7g.| Tashiro,
polyphemus Adams
& ft Claw nmeeve. oo test 23 2.6X1077 g. Tae,
Q | Optic nerve, whole ...... 17.8 | 2.6X10~7g.| Tashiro
Q | Optic nerve, proximal Eat 22.5 | 3.0X10~7g.| Tashiro
@ | Optic nerve, distal part . 22 5.0X10~7g.| Tashiro
Claw nerve, whole 15-16 6.7X10—7g.| Tashiro
Claw nerve, whole....... 20.2 | 7.9X10 /g.| Tashiro,
Labinia 4 Claw nerve, proximal part 21.4 | 8.0X10~7g.| Tashiro
canaliculata Claw nerve, distal part. . 23.2 | 3.7X10~7g.| Tashiro
Claw nerve, whole, when
t stimulated ........... 14-16 |16.0X10~—7g.| Tashiro
ae Sciatic, resting .......... I9-20 5.5X10~7g.| Tashiro
Rana pipiens { Sciatic, stimulated ...... 20-22 14.2X10~ 7 g.| Tashiro

automatically active all the time and is constantly dis-
charging nerve impulses. Of course this result may
be due either to an equality of the metabolism in cells
and fibers, or the injury may have raised the rate more
in the nerve than in the ganglion, or in the ganglion the
amount of non-nervous tissue may be somewhat greater
than in the nerve trunk, so that the carbon dioxide pro-

is Mediicedi thereby. But one Leaks
o be certain, ie., that to attribute the carbon
lioxide tpoiucicn | in the nerve fiber to the connective
issue cells surrounding the nerve trunk, as Bayliss does,
is ; rather ridiculous. Nerve cells evidently breathe
& at about the same rate as nerve fibers, and not faster, as
one might suppose. Table IV summarizes the carbon
_ dioxide production by various nervous tissues, some of
_ which contain cells and others only fibers.
Summary.—We have thus far shown, then, that the
living nerve fiber is no exception to the rule that all
living matter undergoes chemical changes. It respires.
_ There is a chemical sign of irritability in the nerve.
By the use of a proper apparatus of sufficient delicacy ©
we can demonstrate experimentally the formation of
carbon dioxide in all nerves; and by estimating the
amount produced under various conditions—conditions
which we know affect the state of irritability of the
normal nerve in the body—we find a very close parallel-
ism between the amount of carbon dioxide produced and
the state of irritability. We are justified, therefore, in
concluding that the gas thus measured is the expression
of the metabolic activity of the nerve. We may now
pass on to discover whether this carbon dioxide is
increased in case of stimulation.

CHAPTER IIT

CHEMICAL SIGNS OF IRRITABILITY IN THE NER
FIBER —Continued mat

Increased metabolism on stimulation pen have

already stated that all living matter, whether it is an
organism or an isolated tissue, normally undergoes
chemical changes and produces carbon dioxide as one
of the final products of its metabolic activity, and that
the nerve fiber is no exception to this rule. In other
words, respiration is one of the unfailing signs of life
and is a necessary condition for living processes. But
carbon dioxide production from a tissue is not by itself
a sufficient sign of life. For there are many chemical
compounds which spontaneously give off carbon dioxide,
among others sea-water, bicarbonate solutions, as well
as organic materials which are unstable. It would
obviously be a mistake to call these compounds living

because of the fact that they give off this gas. This

criterion alone, therefore, cannot be used for detecting
the vitality of the tissues.

Not only is it common for many non-living matters to
give off carbon dioxide spontaneously, but there are
also some whose mode of gaseous exchange is remarkably
similar to that of the living process. Among these
substances there is none in which the parallelism to
vital respiration is more detailed and interesting than
ordinary linseed oil. The many curious resemblances
of the chemical processes involved in painting to proto-

| 34

3 > i ah

‘soe gee“ TRRTTA’ ‘eeu > fou ’

, SIGNS OF I RRITABILITY
‘ my SS .

+ eS ee

‘te Sepapetion and growth nae already. been
Tout It is unnecessary to go into this more
y than to call attention to the facts that linseed oil
es up oxygen, that it gives off carbon dioxide, that it
Ptimulated by light, that it undergoes also other
- shases of metabolism common to living matter, and
- that, very singularly, it exhibits many of the phenomena
of memory, learning, and forgetting. It is striking,
_ too, that this respiration is of an autocatalytic nature—
that is, it becomes more rapid as it progresses and in this
_ respect resembles the psychic phenomena of memory and

Jearning. Thus we see that respiration alone cannot
be taken as a criterion of life; and, furthermore, that
even the characteristic features of protoplasmic respira-
tion itself cannot be said to be peculiar to living things.

A more certain criterion of life is the increase of respira-

tion on stimulation.

It is well known that contracting muscle produces
‘more carbon dioxide than resting muscle. We breathe
faster when we run. We can measure the irritability of
the muscle by its increased metabolism occurring on
stimulation. Is it possible to increase the metabolism
of the nerve also by stimulation? Can the nerve, one
of the most irritable tissues of all, perform its function
without consuming any material? Is the nerve impulse
something similar to an electrical current passing
through a rather imperfect conductor? How is the
electromotive force created in the nerve fiber when the
‘impulse passes through it? Is it simply the equiva-
lent of the energy we put in at the initial stimulation ?
These questions cannot be considered unless we first

t Mathews, Textbook of Physiological Chemistry, 1915, p. 67.

. hese sacs, | pee aa
| dereriaie Soutely cone or not 5 We nctior
involve metabolic change. = oe ba)
As far as the brain—the master nervous tissue oh the
body—is concerned, it is perfectly obvious that its acti ia
involves a very, intense chemical activity. This is ;
shown by various circumstances. It is made evident “fa
in the first place by the fact that the brain has an
extremely abundant blood supply and that the blood
returning from the brain has lost a considerable part
of its oxygen. Direct measurement of the amount of
oxygen actually consumed by the brain shows that it is
greater than that of any other tissue in the body relative
to its weight. The carbon dioxide production is also
greater. Everyone knows, also, that keen intellectual
work depends on a plentiful blood supply to the head.
When one works hard intellectually the face flushes;
often the hands and feet become cold, owing to con-
centration of the blood in the head. If this increase of
blood does not occur, keen intellectual effort is impos-
sible. If the circulation stops, or even if the blood
pressure becomes low, we become unconscious or faint.
These facts are sufficient to prove that the functions of
the brain, at any rate, involve oxygen and are expressed
in its respiration. ‘The attempt to measure the amount
of heat produced in the brain during intellectual effort
has thus far been unsuccessful, owing in part to the fact
that it is impossible ever to get the brain in a state of
rest. It is always in partial activity. In the second
place, the brain makes only a small portion of the total -
weight of the body, so that its heat makes but a small
fraction of that of the whole body, and it is this which
we have to measure. It has been observed, also, that

iA bh,
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oe

Di wie it is stimulated, owing to the consump-
n of oxygen and the resulting decolorization of the

) We may now consider the carbon dioxide ie of
_ nerves on stimulation.

_ Non-medullated nerves ——The biometer is so delicate
that in trying these experiments many precautions had
to be taken to make certain that the experimental con-

_ ditions themselves did not produce an increase of

carbon dioxide independent of the change in the metabo-
lism of the nerve. If we stimulate with an electrical
current, we have to be on our guard lest there should be
direct decomposition of some substances at the elec-
trodes, resulting in more production of carbon dioxide.
But by trying various kinds of stimulation, mechanical
and chemical as well as electrical, we can throw out
these possible sources of error.. We found, in the first
place, that there was no appreciable increase of carbon
dioxide due to any direct electrical decomposition by
stimulating a dead nerve. In all the quantitative
experiments which follow, the current for stimulating
was so small as to be barely perceptible to the tongue.
The heating effect was, therefore, practically negligible.

A nerve of the claw of the spider crab was isolated
as before. A comparative estimate was first made.
Two pieces of the nerve of equal weights and lengths
were placed separately on the glass plates, each nerve
being laid across the electrodes of the plate, in the man-
ner shown in Fig. 2. In this way either nerve can be

stimulated at will. These glass plates are hung upon the

FIc. 2.—Glass weighing plate.
' A, B, platinum wires fused in the

rear of the glass plate, with hooks; —

C, the nerve which is stimulated
at D; G, the plate proper. Another
piece of glass, exactly counter-
balanced with this plate, is used,
so that any wet tissue can be
weighed very quickly.

current, ihe distance between the primary waa

ary coils being more than
to cm., an ordinary ary
battery being used, not —
only does the precipiaeaal

appear sooner in the cham- __

ber containing the stimu-
lated nerve, but the
quantity of the carbonate
is much greater. This
difference in carbon diox-
ide production can be
brought out better in the
quantitative e estimate made

in the manner described above.
As shown in Table V, a stimulated nerve fiber

of the spider crab gave 16X10

7 g. of carbon dioxide for

TABLE V

Nerve

Amount of CO: Pro-
duced by 10 mg. of
Resting Nerve in
to Minutes

Amount of CO: Pro-

duced by ro mg. of

Stimulated Nerve in
ro Minutes

pre aoe aee (spider

e160 -@ > ev eee OE OS

6.7X10~7 g. (15 °-16°) 16. X10~7 g. (14°-16°)| 2.4
5.5 X10 7 g. (19°-20°)|14.2 X10 7 g. (20°-22°)| 2.6 times

1o mg. of the nerve for ten minutes, while a fresh
unstimulated nerve of the same animal gave only

oe same units. In other words, the
oe between roo and 200 per cent by
zation of the nerve.
ical stimulation of medullated nerves.—The fact
tthe increased production of carbon dioxide on
ee tim ilation is not limited to the non-medullated nerve
is shown by our quantitative determination on the
| - sciatic nerve of the frog. Ten milligrams of frog’s nerve
- gave 14.2X10 7 g. of the gas during ten minutes of
stimulation as compared with 5.510 7 g., the amount
produced by the resting nerve of the same animal. Here
again stimulation increased the output from 200 to 300
per cent.

Other stimulationWe have now established the
fact that when a nerve is stimulated by an electrical
current it gives off more carbon dioxide. In order to
test whether this increased production of the gas on
electrical stimulation is due to the direct decomposing
influence of the current or to the state of excitation
produced by the stimulus, many additional facts must
be sought. In the first place, if the increased gas pro-

- duction is not due to a change in rate of metabolism,
but to the current itself, then we should expect that |
the stimulation of a killed nerve ought also to cause more |
gas production, provided, of course, that we may assume
that the conditions under which electrical decomposition
takes place are the same in the living and in the dead.

When we place two nerves killed by steam, one in each
chamber of the biometer, and stimulate one of them, the
stimulated nerve does not give off more carbon dioxide
than the unstimulated when the same strength of current
is employed as was used in the other experiments.

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cceiription that the conden fase which n rical —
decomposition takes place is the same in a living and a

dead nerve may not be strictly true, but that an electrical a

current can in some way drive away carbon dioxide more ne.

_ quickly in the living nerve. Since killing by steam may _ q

also drive out the gas already present in the tissue, the
apparent indifference of the dead nerve toward electrical
stimulation may not prove that the increased carbon
dioxide accompanying stimulation in the living nerve is
really a direct result of a change in its vitality, rather
than an indirect result of the passage of the electrical cur-
rent. If, however, this increased gas production were
due to the mere electrical decomposition, which was not
limited to the point of contact with the electrode, we
ought in the living nerve to get a proportional increase of
carbon dioxide by increasing the length of nerve through
which the current directly passes. The fact is, however,
that we can produce an increase of carbon dioxide by
stimulating with electrodes 2 mm. apart, so that only a
small portion of the nerve is traversed by the current,
as well as by electrodes 15.mm. apart. It makes no
difference how much of the nerve is traversed by the
current. But it does make a difference how much of
the nerve is traversed by the nerve impulse. These
experiments suggest very strongly that the increase of
carbon dioxide on electrical stimulation is due to the
increased metabolic activity during functional activity
in the nerve, and is not due to the influence of the
electrical current as such. With the aid of other
means of stimulation we shall now proceed to prove
that all stimulation is accompanied by an increase of —

a .

a : i ea _ “jen ge-- 24

s shown el the output of carbon

Me ice Nenatiison. —Since the ordinary method
B echanical stimulation cannot be used directly on
the nerve in the biometer in its present form, in view of
the fact that the chamber has to be kept shut, we used —

a different method, namely, that of injuring the nerve

by crushing. That when protoplasm is smashed vigor-
ous chemical changes result is well established. Fletcher
reports that injured muscle gives off more carbon dioxide -
than normal muscle; later he and Hopkins discovered
that muscle under a similar condition is richer in lactic
acid. Mathews observed a similar increase in carbon
dioxide in the crushed eggs of Arbacia. We have

discovered that if a nerve is crushed with a rough edge

of a glass rod it gives off more carbon dioxide than the
normal one; that is, an injury increases the carbon
dioxide output of the nerve. Since this increase of
carbon dioxide cannot be produced by crushing an
unexcitable nerve, we consider this injury to be a form
of mechanical stimulation. (For further consideration
of this subject see p. 91.)

Chemical stimulation.—The study -of the nature of
chemical stimulation has been so thoroughly made that
it might seem ideal to study quantitatively the increased
production of the gas following the stimulation of the
nerve by various salt solutions. But there are com-
plications which seriously interfere with the use of this
method. We found, for instance, that. the presence of
minute quantities of a foreign liquid is a seriously
disturbing factor for a quantitative estimate of carbon
dioxide. Qualitatively, however, we found various

4 2 EES ee exitiuble ‘e ace ri Pp
less carbon dioxide than the normal resting nerve.

_ When the two sciatic nerves of a frog are woes pe "3
one is left in physiological salt solution—o.75 per cent —
sodium chloride—and the other in the body of the frog
for the same length of time, and when they are trans- _
ferred to the two chambers of the apparatus, itisfound,
if the quantities of the carbonate precipitates are com- —

pared, that the nerve which has been in the saline solu- _

tion produces more carbon dioxide than that which has
remained in the body. It is known that such a saline
solution raises irritability and ultimately stimulates the
frog’s sciatic nerve.

The different rates at which carbon dioxide is pro-
duced from different nerves treated by various con-
centrations of potassium chloride are equally instructive.
When a nerve is placed in a molecular solution of
potassium chloride, stimulation takes place for a con-
siderable time. Then finally the nerve becomes inex-
citable. But if the nerve is put in 0.2 mol. solution
of the same salt, nervous excitability is abolished in a
short time without any primary stimulation. The
carbon dioxide production follows exactly analogously to
this. The nerve treated with the stronger solution
gives off more carbon dioxide than the one treated
with the weaker solution. This was true even after
the nerve became inexcitable, showing that the nerve
must still be giving.off more carbon dioxide while being
stimulated. by the stronger solution. Mr. Riggs is.
making an extensive study of the effect of various

olutions of varying concentrations and has
Iready discovered confirmatory evidence for the increase
of metabolism during chemical stimulation. It may
—b e added here in passing that the different solubility of
ea dioxide in these salt solutions cannot alone explain
our results, for there is not enough difference in solu-
bility of this gas in such dilute equimolecular solutions
_ of potassium and sodium chloride whose effects.on carbon
dioxide production are so divergent, the former salt
diminishing, the latter increasing, it.

The fact that during chemical stimulation the nerve
- gives off more carbon dioxide is made evident, also, by
the use of low concentrations of anesthetics. If the
concentration is so low as to give a primary stimulation
to the nerve, the production of this gas is greatly acceler-
ated at the beginning of immersion of the nerve in the
narcotics.. This is an additional evidence that there
is a relation between excitation and metabolic activity.

Stimulation in hydrogen.—The last experiment which
we shall describe in this connection is on the quantitative
estimation of the carbon dioxide production in a nerve
when the latter is in an atmosphere of hydrogen and when
it is being stimulated by an electrical current. We
expected to find here one of two things. First, there
is evidence, to which reference has already been made,
that nerves left in hydrogen gas show diminished irri-
tability and that they give off smaller amounts of carbon
dioxide than do the same nerves in air. This fact led
us to anticipate that these nerves, being thus less irritable
than normal nerves, would produce less than the usual
increment of carbon dioxide on excitation. This would
be the case if the increment were a proper measure of the

a Nien nerve, and if. nerve teh ed not in involv
tion, we expected to find that putting the nery
hydrogen gas would not affect the output on stimulatic ‘ion. q
For this experiment the claw nerve of a spider crab was
used, and stimulation was effected in the usual manner —

by a tetanizing induced current of the same strength as — 4

that which had been used before and found to increase
the output in normal nerves. The results are givenin
Table VI. It will be seen in this table that the pro-
duction of carbon dioxide by this nerve was reduced

“

TABLE VI

CoMPARATIVE RATES OF CO: PRODUCTION IN THE NERVE WITH AND WITHOUT OXYGEN

Amount of CO. Amount of CO:
Produced by Produced by
Nerve Medium ro mg. of Resting ro mg. of Stimu-
Nerve in 10 lated Nerve in ©
Minutes . to Minutes

almost exactly so per cent when the nerve was in hydro-
gen, as compared with when the nerve was in the air;
and still more remarkable is the fact that stimulation in
the hydrogen atmosphere produced practically no
change in the carbon dioxide output. We interpret this
to mean that the excitability of the nerve had been so
reduced by the lack of oxygen that this strength of
stimulus was unable to cause any excitation in the nerve.
We base this conclusion on the known fact that lack of
oxygen lowers considerably the irritability of the nerve

al F also Liles the time during which a current of any
str ength can stimulate the nerve. Exhaustion comes
on much more rapidly in a hydrogen atmosphere.

_ Fréhlich found that when a sciatic nerve of a frog is
__ deprived of atmospheric oxygen its irritability, measured

by the threshold of stimulation for muscular contraction,
decreases more and more, until after the lapse of some
hours the stimulation required is so strong as to approach
the region where electrical currents spreading down the
nerve stimulate the muscle directly. If such is the
case in a frog’s nerve, the claw nerve, too, left in hydrogen
may in reality not be stimulated by such a weak current.
Thérner, also, taking the action current as an index,
found that a nerve continuously stimulated in an atmos-
phere deficient in oxygen was quickly exhausted. It
is remarkable that the action current of a nerve in
nitrogen gas falls to two-thirds of its original value
within the first ten minutes. Fatigue of the nerve by
continuous stimulation during the first few minutes of
our experiments with hydrogen may then have been
brought about.

Whatever interpretation we take—and, as a matter
of fact, both factors doubtless enter here—the fact that
there is no decided increase of carbon dioxide on weak
_ electrical stimulation in hydrogen points inevitably to
the view that oxygen is a primary factor in the excita-
bility of the nerve, as well as in the conduction of the
nerve impulse.

Recently Bayliss has pointed, out what he considers
a probable error in our experiments. ‘To him it seems
that the increased production of carbon dioxide on
electrical stimulation may be due, in consequence of the

cae an

escape sof cahon dioxide which hail been. C
the living cells in the connective tissue around the n
fiber. We have cited several experiments the results HS. a
which exclude this possibility. In addition to these, — a
the apparent lack of any increase of this gas on applica-
tion of induction shocks to a nerve in an oxygen-free
medium like hydrogen should be taken as conclusive evi-
dence that the increased gas production by anerve when
stimulated in the air is due to physiological processes, and
not to experimental errors.
Lack of fatigue.—If the chemical change of the nerve
tissue is as active as the observations just cited indicate,
- one naturally asks how we can explain the fact that the
nerve impulse can pass continuously through the fiber
without any measurable sign of fatigue. There is no
doubt that this apparent lack of fatigue of medullated
nerves is a very remarkable and striking phenomenon.
Nerves can be stimulated for many hours continuously
without marked fatigue. But it does not at all mean
that there is no chemical change in the nerve, for, in
the first place, it must not be forgotten that medullated
nerves have in the medullary sheath a very large supply
of raw material, or food, which is more than sufficient for
their nutrition during the longest experiments which have
been tried. ‘The only surprising feature of the physiology
of the nerve is that in the isolated nerve, where there is
no opportunity for getting rid of the products of decom-
position, accompanying functional activity, by way of
the blood, nevertheless these products do not seem to
act deleteriously on the nerve function. But, after all,
we have only to assume, in order to understand this, that

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e of such a nature that they have very
logical action. It is quite possible that
. taken care of i in the nerve, because it is vitally
cessary to animals that those of their nerves which

a skeletal muscle, at any rate, shall not be easily
fatigued. ‘There are also other tissues in which it is
perfectly certain that there is a rapid metabolism and
-which also show a no less remarkable freedom from
_ fatigue. We may cite, for example, the contracting
_ wings of insects which vibrate at a rate as high as three
_ hundred vibrations per second, and yet these insects can
fly for hours continuously without muscular fatigue.
There is not the least doubt that these muscles which
are undergoing this tremendous activity without fatigue
are at the same time undergoing a very rapid metabolism.
All that is necessary to avoid fatigue is that the tissue
shall return each time after activity to its normal state.
The ordinary induction coil which we use in these experi-
ments only stimulates a nerve about one hundred times
a second, or about one-third as often as the insect’s wing
muscles contract, so that more time is given for recovery
in the nerve than in these muscles. The lack of appar-
___ ent fatigue in nerves is not, then, any proof of the
___ absence of metabolism.

When we examine nerves more closely and by more
| delicate methods, we find unmistakable evidences of
_ fatigueinthem. The only remarkable thing about them
| is that they recover from that fatigue very rapidly.
Thus Gotch and Burch discovered in 1889 that if two
stimuli are successively applied to a nerve within
1/5,000 of a second, only a single nerve impulse is pro-
duced. One cannot generate a second impulse until

vi aa

longed under patie nao Sark as tow temper
high temperature, asphyxiation, various drugs, and
certain anesthetics. Frélich prolonged this refractory —
period by partial anesthesia and succeeded in producing —
fatigue phenomena by repeated electrical stimulations
at shorter intervals iene the prolonged arab co period | =
of the nerve. 4

The idea that all the physiological activities are
composed of at least two opposing metabolic phenomena
was expressed by Claude Barnard and later extended by
Hering. Thus metabolic activities are considered as
consisting of two phases, namely, a breaking down, or
katabolic, and a building up, or anabolic, phase. That
two such phenomena are involved in nervous metabolism
and are closely connected with the phenomena of fatigue
may be shown by the use of certain drugs in connection
with electrical changes and refractory periods. Waller
observed that protoveratrin slows up one of the electrical
changes (positive variation) of the nerve, while the
other (negative variation) is little influenced. He
contended accordingly that this drug does not alter
katabolic changes of the nervous metabolism but re-
tards the anabolic activity to a considerable degree. It
is by its anabolism that the nerve is restored to its nor-
mal state after the passage of the impulse. Since the
pharmacological action of protoveratrin and yohimbin
on muscle are known to be very similar, Tait concludes
from the study of the effect of yohimbin on the refractory
period of the nerve that these drugs must attack nerves
in a similar manner. Yohimbin, in other words, retards

= atlas

bol. cp si esses e BP aattiershiy this prolonging the one
ase e of the refr actory period or increasing thus the
efficic jency of the nerve. From these considerations we
Ly Pbatinde that the nerve may be fatigued by repeated
tin mulations if we can prolong the time interval of either
‘the : excitatory or the repair state.

The general conclusion to which this leads us is that

j what we call fatigue in a tissue of any kind is due to a

failure of the tissue to recover completely its normal

state after it is excited. In some tissues this state of

fatigue is very easily demonstrated, but in medullated
nerves the mechanism of recovery is so perfect that
ordinarily the restoration of the nervous substance to its
original state after the passage of the impulse takes a
very short time—a fraction of a thousandth of a second.
Nevertheless, by the conditions stated, namely, by lack
of oxygen, by partial anesthetization, by the action of

drugs like yohimbin and protoveratrin, the recovery is
__ delayed, and in these cases the nerve exhibits phenomena

which may properly be called fatigue. The failure of a
nerve to show fatigue under ordinary circumstances
should not, therefore, cause us to conclude on this
account that there had been no destruction of nerve
substance by its excitation, but rather that the nerve
had in its medullary sheath an especial supply of a food
particularly formed to serve as a speedy pabulum for
the fibers, and that the means of reconstituting the
nerve tissue after excitation had been so perfected that
the result was accomplished in a very brief time.

Heat formation.—Another evidence which has been

often cited as showing that there was no chemical change
’ accompanying the nerve excitation, is the fact that there

is no heat “poducet ex nerves
-. explain the fact that this relatively tr rt
e\aneh can occur without) iets, ic
There are several explanations which might be iven
of this fact, but before considering them we may see ~
first what the evidence is that there is no heat produced. _
Although there have been in the literature many
contradictory statements as to heat formation in the
active nerve, the original negative results of Helmholz,
Stewart, and Rolleston have been confirmed recently |
by A. V. Hill’s work, which shows that there is no meas-
urable liberation of heat when the nerve is stimulated.
Since his apparatus is exceedingly sensitive, being sus-
ceptible to the change of 1/1,000,000 of a degree Centi-
grade, the lack of observed heat production is not ap-
parently to be explained by any lack of a proper method
of measuring temperatures. His work is remarkably
significant in that according to his calculation not more
than one single oxygen molecule in every cube of a
nerve containing 3.7 cubic uw can be used up by a single
propagated nerve impulse, since more than this amount
would produce a measurable amount of heat. Thus he
is convinced that a nerve impulse is not of an irreversible
chemical nature, but must be of a purely physical nature.
Negative evidence of this kind cannot be taken at
its face value without considering the limitations of the
method. Stewart. calls attention to the fact, that we
should not forget that if the axis cylinder is the only
portion which is conducting a nerve impulse, as we
believe, the measurement in medullated nerves with
which most experiments were made does’ not express
the true state in the axis cylinder. We should consider,

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Betiowed hat : a ‘eiiooth Bikes also, when measured
in the same way, failed to produce heat during its con-
traction. But no one doubts that during muscle work
the metabolic activity is greatly accelerated. He used,
by the way, exactly the same technique as Hill. Either,
then, heat is produced, but owing to some circumstance
it is not detected, or else it is not produced. Now, most
muscles certainly produce heat when they work, and
it is probable that smooth muscle does so also. It is
not to be supposed that the muscles are a perfectly
reversible engine. On the contrary, we all know that
we become warm when we exercise. When, then, it is
reported that smooth muscle produces no heat when it
contracts, we are at once skeptical of the method which
gives such a result. Consequently, therefore, while the
method for the detection of the nerve heat on stimula-
tion appears to be a competent method, we do not feel
certain that this is the case.

But suppose we grant that the results are correct—
that nerves produce no heat when they are excited—
does that mean that there is no chemical change occur-
ring in the nerve? Is this fact conclusive evidence that
these results of a positive kind which we have adduced,
showing that chemical changes do occur in the nerves,
are, after all, due to some secondary cause or to some
undiscovered errors of technique on our part? Cer-
tainly this is not the case, for it is quite possible for
chemical changes to occur without liberating more than

a very small amount of heat, or, indeed, they -
actually be heat-consuming rather than heat-produ

And, indeed, if we had side by side reactions which mon a

duce and reactions which consume heat we might havea

considerable chemical change without the liberation of

much heat. Thus in a Daniels’ cell there is a very __

large transformation of energy with the liberation of
very little heat. The Weston cell has a still smaller
heat coefficient. The energy set free in the cell takes

the form of electrical energy rather than heat. To be
sure, it is ultimately converted into heat, but for the
time being it does not appear as such. There are many
chemical changes also which yield carbon dioxide and
yet liberate very little heat. It is possible that the
carbon dioxide is not produced by an oxidation, but by
a fermentative process which is hardly exothermic.
Many such hydrolyses liberate almost no heat at all.
We might have, for example, the oxidation going on at a
steady rate all the time, independently of the stimulus.
By this means a constant production of heat occurs, but
carbon dioxide is not liberated. ‘That is, the change
has occurred at a steady rate in the oxygen atoms, which
is the essence of the oxidation. A very unstable com-
pound might result, awaiting only the hydrolysis of the
carbon dioxide. This last process might be that which
is accelerated by the stimulation and the passage of the
impulse. This liberates gas, but very little heat. The
reconstitution of the irritable substance might then be
brought about by a second molecule slipping in to take
the place of the first, while the exhausted molecule was
withdrawn to be reoxidized and thus made ready for use
again. This reoxidation perhaps goes on all the time,

, : time, ‘sad yet we “ave a copious production of ele
_ dioxide which is increased on stimulation. The fact, .

therefore, that there is no increased heat production in a
stimulated nerve is by no means contrary to our results,

_although it is certainly surprising. It indicates, perhaps, —

that the act of excitation is not primarily an oxidation,
but that the oxidation is concerned in the processes of
repair. There are several facts which might be cited,
were the space at our command, which would lead to the
same conclusion. There are also other suggestions which
might be made to account for this seeming incompatibil-
ity, but it would be useless to do so without experiments.

We may therefore close this brief discussion with the
statement that the failure to detect heat production in »
nerves during excitation is no evidence of value against
the occurrence there of chemical changes resulting in
carbon dioxide production and correlated with the irri-
tability. ‘The conclusion drawn from it by some authors
that the nerve impulse does not, on this account, involve
any chemical processes is entirely unwarranted.

We may in this connection stop for a moment to
consider what is known of the oxygen consumption of
nerves, for while we have ourselves as yet carried out
no experiments in this line, yet there have been some
observations made which can be correlated with the
carbon dioxide production. In the first place, it may
be noted that there is no immediate dependence of some
nerves, at least, on atmospheric oxygen for their activity.
In this respect the carbon dioxide production and the

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sumption are still rather crude, and the ce i 2
been few. A nerve always gives off more carbon dioxide _

when it is stimulated or active, whereas we know very

little about whether its intake of oxygen is increased
in anything like the same degree. The sciatic nerve
of a frog—a medullated nerve—can remain excitable
for a long time in the absence of atmospheric oxygen,
although its irritability diminishes under these cir-
cumstances, and, as already explained, its langle
increases.

There is a considerable amount of evidence to show
that oxygen is very closely associated with the state of
excitability. ‘To harmonize these two facts, namely,
the independence of atmospheric oxygen and the fact
just stated, the oxygen-storage hypothesis has been
suggested, by which the exhaustion is attributed to
complete consumption of stored oxygen. Excitability _
is restored when atmospheric oxygen is readmitted.
Without committing ourselves to this hypothesis, we may
add that according to Haberlandt’s figure the resting
nerve of 10 mg. weight will consume only 0.0042 C.c.
oxygen in ten hours. If we take our figure of carbon
dioxide output and assume that one volume of oxygen
was necessary to produce one volume. of carbon dioxide
(this assumption is made without any significance
except to give a liberal estimate), the carbon dioxide
production would require a consumption of about
0.015 c.c. of oxygen for ten hours. And if we assume
again that activity will increase oxygen consumption

s that » nerve Ghet sifrnvalztest” bth take up
aly Po: fox c.c. of oxygen during ten hours’ stimulation.
Tt ‘is pie difficult, as everyone who has tried it
knows, to free any gas from such small amounts of
Oxy en as those which are required to keep up irrita-
bility. Our experience in freeing gases from traces of
~ carbon dioxide makes us realize the difficulty of getting
_ the nerve in the first place in a gas quite free from
_ oxygen, and we believe that many experiments have
__. been tried in which there is still some probability that
enough oxygen remained to supply these small amounts
needed. More delicate determinations will have to
be made before we feel certain that nerves have been
found to be irritable for some time in atmospheres which
are free beyond question from all traces of oxygen. How
shall we know when the gas we use is free from oxygen
in these minute amounts? Yet until we know this it is
___ impossible to study accurately the relation of irritability
to oxygen. Meanwhile, however, we may recall the
fact that carbon dioxide production in the spider crab’s
nerve is not only reduced in the absence of oxygen,
but also that we cannot increase its production in such
_ an atmosphere by a stimulation which in the presence
___ of oxygen increased the production of carbon dioxide
over 200 per cent. These facts show conclusively,
negative evidence to the contrary notwithstanding, that
oxygen is in some way involved in the anabolism or
katabolism of nerve fibers:
Summary.—The facts presented in this chapter
prove that all kinds of nerves, medullated and non-
medullated, when stimulated increase their output of

7 ce Ln 4 = _ ek
ype acy

erate

eh? mer ieee

Sats to aes Re, is not a 5 ately price change
state, as it has been represented hitherto as being, b put it
undoubtedly involves a corresponding chemical change. —
Perhaps the excitation is this chemical change itse elf. a
Furthermore, the facts that nerves do not increase
their heat output on stimulation and that they are nee
free from fatigue effects are evidently not incompatible
with the vigorous metabolism discovered to exist in them.

| _ + CHAPTER IV
Ss BXCITATION AND CONDUCTION

a We have shown by this study of nerves that living
_ matter must necessarily undergo metabolic activity and
_ that without an increase of this activity protoplasm
will not function. In short, to be excitable, the
a protoplasm must respire, and to be excited, its meta-
bolic activity must be accelerated. It has also been
demonstrated that the excited state travels along the
fiber with simultaneous increase of the metabolism.
Although our theme in this little volume is not a
consideration of how this state of excitation is trans-
mitted, but is rather an analysis of the conditions
which characterize the irritable tissue, the relation
between these two phenomena is so close that we
shall consider certain facts which are directly con-
cerned with them.

The two phases of protoplasmic irritability are

_ excitability and conductivity, or transmission, of this

excitation. Since it ‘is very difficult experimentally to
produce excitation without conduction, we are accus-
tomed to consider the fundamental processes underlying
these two processes as probably identical. There are
certain facts which are sometimes cited as evidence that
these phenomena are not necessarily interdependent.
In the case of localized and partial narcosis, for instance,
local excitability in the narcotized portion does not
disappear simultaneously with conductivity through
57

part is made use of. Since vi we have no evidence that t
resistance of the surrounding sheath of the fiber and
that of the conducting medium are the same, we cannot 7
assume that in both experiments the same strength
of stimulus was really applied to the conducting portion.
The non-transmissibility of the inhibitory state is A
regarded as another distinction between excitation and _
conduction. We can abolish excitability at one point
without making its neighboring region inexcitable. It
is rather difficult to consider an analogy between depres-
sion and excitation, but the fact is that even if we may
not be able to make other than one point inexcitable
by one depréssing agent, it is doubtful whether we can
produce local inexcitability without affecting the con-
tiguous parts of the nerve. Waller has demonstrated,
in the case of inhibition by heat, that the point of
application of gentle heat became electropositive to the
rest of the nerve instead of negative, as is the case in
ordinary stimulation. According to him, heat does not
stimulate the tissue, but depresses it. If this is the
case, as he seems to have demonstrated in a variety of
tissues, it indicates that although we cannot produce
depression at points other than the point of application,
yet certain conditions along the nerve must surely be
altered through such an inhibition. In any event, we
cannot consider non-conductivity of the inhibitory state
as evidence that excitability and conductivity are
entirely different processes.

Let us now consider in detail the relation between
excitation and conduction.

ease with which fi ei can be ciinsplated: o veloaty of

e nerve impulse, i.e., the speed with which the state
4 ‘excitation travels hs one point to another; (3)
i the direction of the nerve impulse. All nerves are
- classified into two general functional types: efferent
Bia afferent, the former conducting away from the
nerve center (brain, etc.), the latter toward the center.
We shall consider somewhat in detail in this chapter
what relation the metabolic condition bears to these
three phenomena in the nerve.

Degree of excitability—Not all nerves can be stimu-
_ lated equally well by the same strength of stimulus.

_ The threshold value—the minimum strength of stimulus
which can call forth functional activity—is different
in different nerves. Not only have the different nerves
different degrees of excitability, but the same nerve can
be made excitable in different degrees under a variety of

conditions. If we study metabolic activity in nerves
under different conditions which we know affect the
state of excitability, we find that there is a very close
relation between metabolism and excitability.

a) If the sciatic nerve is removed from a frog, it
exhibits electrical phenomena for many hours. Since
electrical changes are characteristic of living nerves
only, we consider that the isolated nerve does not die for
many hours. Such a nerve, although it shows large
electrical responses, is nevertheless less excitable than a
‘fresh one. If measurements are made on an isolated
nerve at successive time intervals for many hours, we
find that the carbon dioxide production steadily dimin-

wy
a

minimum production of the gas C0! Ata pond
mately to the point where an electrical response
(Bee ip..28).0 |
a) Although the nerve remains pcewe for some inet
without oxygen, it is known that the absence of oxygen b
diminishes the excitability of the nerve. This diminu-
tion of the excitability when in hydrogen is accompanied ~
with a lowering of carbon dioxide production in the nerve.

-¢) Further facts showing the relation between
excitability and metabolic activity are brought out by
the study of the effects of narcotics on the nervous
metabolism. ‘There are several compounds which alter
the state of excitability of nerves to a considerable
degree. ‘The discovery of just what happens to réspira- —
tion during anesthesia will throw much light on the
nature of irritability. It is this which we shall now
study in detail.

In recent years many experiments have been per-
formed which are supposed to prove that oxygen con-
sumption can go on uninterruptedly during narcosis, and
’ the consequent conclusion has been that narcosis is not
produced by asphyxiation. This is not the place for us
to weigh the merit of these arguments, nor are we con-
cerned here with the question of how narcotics act on
protoplasm, but it is very important to know whether or
not metabolic activity in nervous tissue can go on undis-
turbed while the tissue is unable to perform its own
function. Are respiration and irritability independent
processes? ‘To show that they are dependent we shall
cite in detail experiments on the effects of anesthetics on

respiration.

1d demon: eta that Sherine the excitability of |
‘is accompanied by a lowering of carbon dioxide
uction. For the quantitative experiments, carried
out in conjunction with Dr. Adams, we used chloral
a 3 _ hydrate and ethyl urethane in preference to the ordinary
y volatile narcotics. If we anesthetize a nerve with the
lowest concentration of ether or chloroform that pro-
# Be daces a reversible loss of irritability, then the anesthe-
ij __ tized nerve regains its excitability during the course of the
experiments, for in order to make the apparatus free
from carbon dioxide after introducing the nerve we have
to wash it with carbon-dioxide-free air several times.
By so doing the most volatile narcotics are removed
from the nerve. On the other hand, if we use higher
concentrations, which, as we know, lower carbon dioxide
production, we may be subject to the criticism that the
lowering of metabolism may be due partly to death or
injury. It is therefore essential that we should investi-
gate the effect of various concentrations, from such as
have apparently no narcotic effect to those from which
i recovery is doubtful or absent. Thus the use of suitable
& narcotics as well as concentrations seems to be of prime
¥ importance. For even those who consider that narcosis
is not due to an asphyxiation admit that the oxygen
5 consumption is greatly depressed if the narcosis is pushed
3 too far, although such depression in the rate of oxidation
E may have nothing to do with the cause of the narcosis.
With a view to studying the effect of various con-—
_ centrations of anesthetics, the claw nerve of a crab was
isolated, its excitability tested by electrical stimulation,
_ and, without being cut off from the claw, it was immersed

EFFECTS OF ETHYL URETHANE ON CLAW NERVE OF SPIDER CRAB, Libinia conittonate

means of filter paper, its see of Tecunes pits TI i
by stimulation, and, with ai: attached, it was plas

TABLE vir*

TREATED BY
EFrects ON| AFTER Pie acral Amount oF CO; Pro-
Concen- Wie EXcItTA- RETURN TO Phelan pe DUCED BY Io MG. OF
dation MET Fie BILITY SEA-WATER Reward NERVE IN 10 MINUTES
Sea-Water | Long
i } 1, Excitable No change | 7.9X10~7 g. at 20°2
oper cent. .|10 min. nerve ;
2, Inexcit- | Excitable No change | 5.7X10 7 g.at 22°
able nerve
I per cent..|/10 min.| Excitable Excitable -| No change |21.7X10~ 7 g.at 23°8
2 per cent. ./10 min.| Narcosis Excitable No change | Not determined
: very slow .
3 percent..}1o min.| Slow, partial) Excitable No change | Not determined
narcosis
4 percent..|10 min.| Practically | Good return} No change |3.3X10~7 g.at21°-21°5
’ narcotized|
5 percent..]10 min.| Completely | Recovery is | No change |} Not determined
+ narcotized| not Sea
goo

* Since our previous determinations of the carbon dioxide production of the spider
crab’s nerve were made at a much lower temperature (15° to 16° C.), the work was
repeated at the higher temperature at which most of the present experiments were made.
In order to make the comparison a rigid one, the normal nerve was subjected to a treat- ©
ment similar to that employed with the narcotized nerve, except that it was not narco
It was isolated, quickly weighed, and immersed in sea-water for ten minutes, after which
the rate of carbon dioxide production was determined in the usual way. As was expected,
the nerve exhibited a somewhat higher rate of metabolism at the higher temperature.
The results are incorporated in the table.

usually required in making a determination of the
carbon dioxide production. After this the nerve was
brought back to fresh sea-water and the return of irrita-
bility was determined, as evidenced by contraction of the
claw or joint in response to the electrical stimulation
of the nerve. Thus the essential conditions obtaining

a ttt art tnt es tt ie a EE
—

oir Beacn the results so obtained the minimum concen-
- tration which produced a reversible loss of irritability was
chosen for our experiments on the carbon dioxide pro-
duction, and we are thus assured that the nerve has been
7 Be icotived, but that, since its excitability returns, no

permanent injury has been caused. The carbon dioxide
production of the nerve thus treated has been determined
and compared with that of a normal nerve. These

results are tabulated with the physiological data and

given in Table VII.

ETHYL URETHANE

As shown by physiological tests, a freshly isolated
claw nerve on immersion in a 4 per cent solution of ethyl
urethane loses its excitability within ten minutes. Such
a nerve, however, if left in a moist chamber for ten or

fifteen minutes and then returned to sea-water, comes

back to a normal condition of excitability with appar-
ently no injurious effects. That the nerve so narcotized
gives off less carbon dioxide than a normal one can be
demonstrated qualitatively as follows:

Two nerves of approximately the same weight are
isolated, and one is immersed in sea-water while the
other is treated with a 4 per cent urethane solution for
ten minutes. At the end of this time their rates of
carbon dioxide production are compared simultaneously
in the biometer by placing the normal nerve, for example,
in the right chamber and the other in the left. Within
ten minutes the difference in carbon dioxide output
will become evident, for not only does the precipitate

chamber pe in the other The nailed i serve
giving off less carbon dioxide than the normalone. a
That the narcotized nerve produces less carbon dioxide a
than the normal is shown more strikingly by quantita- _
tive determinations. The average carbon dioxide output
for the nerve when treated for ten minutes with a 4__ :
‘per cent ethyl urethane solution is less than 50 per cent
of that of the normal nerve. At 20° to 22° C. the narcot-
ized nerve gives 3.3107’ g. per centigram of tissue for
ten minutes’ respiration, while the normal nerve, calcu-
lated for the same units, produces 7.9X1077 g. One
exception may be noted here—an experiment in which
the respiration of the narcotized nerve was 4.9X107~’ g.
—but this is partly explained by the fact that the
particular determination was effected at 25°C. Even
in this case the decrease of carbon dioxide was marked.
Qualitative experiments with a 2 per cent ethyl urethane
solution show that even this concentration produces
a diminution of carbon dioxide output.

CHLORAL HYDRATE |

As indicated in Table VIII, a 2 per cent solution of
chloral hydrate in sea-water partially or wholly para-
lyzes the nerve in ten minutes and recovery is appar-
ently perfect. A 3 per cent concentration produces
complete paralysis and the return of excitability is
good. Treatment with a 4 per cent chloral hydrate
solution for the same period of time also produces
paralysis, but recovery is not always good. In each

G& és <3
me | xt ;
ME ete :

‘Oaee

e decrease of carbon dioxide production is decided. -

Table VIII illustrates quantitatively the difference in
bon dioxide production under these different con-
di ons. The interesting results with 3 per cent and
4 per cent solutions will be considered later.

TABLE VIII

a EFFects oF CHLORAL HyDRATE ON CLAW NERVE OF SPIDER CRAB, Libinia canaliculata

_ TREATED By Amount oF CO. -
: Sat CHANGE
Rrects on AFTER nt Weicet PRODUCED BY
| = Concentra- | For | Excrrasriiry | RETURN TO IN Io 5S. FEE
| ; Gon ia How SEA-WATER Minutes NERVE IN I0
| 3 Sea-Water | Long SLHOTES
| a oper cent...| ro min.} Excitable No change | 7.9X10~7 g.at20°2
| ‘a o per cent...| 10 min.| Inexcitable No change} 5.7X107 7 g. at 22°
fog
| $ : ©.4 per cent.| 10 min. ys a Excitable No change |11.5X10~ 7 g.at20°4
=. irritable
ze I per cent...| ro min.| Slow narcosis | Excitable No change |} Not determined
fal
we 2per cent...| 10 min.| Partial orcom-| Good return} Very slight} 4.2X10~7 g.at22°5
x : plete narcosis : gain
i> 3 per cent...| 10 min.| Completely Fair return | 25 per cent| 2.8X 1077 g.at23°5
ta ; narcotized ; gain
a 4 per cent...| ro min.) Completely Partial or 50 per cent) 3.6X107— 7 g.at23°5
: narcotized doubtful gain
return

Is the decrease of carbon dioxide due to narcosis ?—
The results given above establish beyond a doubt that
during treatment with narcotics, in concentrations which
produce a reversible loss of irritability, the carbon
dioxide output of a nerve is greatly reduced. The
differences thus produced are far beyond the limits
of experimental error and there can be no suspicion that
the phenomenon is the result of faulty observation. The
question might be raised, however, whether this diminu-
tion is directly related to the narcosis, or whether it
might not be due to some factor casually introduced

i B eplict might be sufficient materially to altes ang !

values obtained. This, indeed, is the reason why we
have never been able to investigate the effects of potas-

sium cyanide, since the slight trace of alkalinity thus
introduced seriously modifies the results. This objec-
tion, however, we have been able to refute by direct
experimental means.

If the solution of the narcotic differs in reaction
from sea-water sufficiently to influence the determina-
tion, a similar effect should be observed in the case of a
nerve which has been killed. Two freshly isolated
nerves of approximately the same weight were killed —
simultaneously by means of steam and left for twenty —
minutes, one in a 2 per cent solution of chloral hydrate
and the other in sea-water. A measurement of the
adventitious carbon dioxide production from the two
nerves so treated gave no evidence of any difference.
The diminution of carbon dioxide from nerves subjected
to the action of narcotics cannot, therefore, be referred
to any change in the reaction of the sea-water produced
by the narcotic.

. Another possibility is involved in the fact that certain
narcotics produce phenomena other than those of nar-_
cosis. This is probably the reason why the metabolism
change is never exactly the same in the case of two
nerves in which typical narcosis has been induced by
different means. One of these effects must be a change

:
i
;
P

_ “EXCITATION AND CONDUCTION 67

e in osmotic pressure, but that the lowering of carbon
dioxide output cannot result essentially from the osmotic
effect is evidenced in many indirect ways. We found

that the sciatic nerve of the frog when treated with 2 per
cent ethyl urethane solution gains about 30 per cent in:
weight during ten minutes’ immersion. No change in
weight, however, takes place in the spider crab’s nerve
on a similar treatment with the same concentration of
this narcotic. Loss of irritability ensues in each case,
and in each case the carbon dioxide production is greatly
diminished. A 4 per cent solution of chloral hydrate
causes the spider crab’s nerve to increase 50 per cent
in weight in ten minutes, while 4 per cent ethyl urethane
solution produces no change of weight in the same nerve.
Yet both narcotics depress carbon dioxide output
greatly. ‘That this decrease is independent of osmotic
effect is further shown by our work on the effect of ether
vapor on carbon dioxide production in a frog’s nerve.
In this work with a frog’s nerve it was found that
ethyl urethane will reduce carbon dioxide production,
but that soon after the nerve begins to gain in weight
the tendency is for this production to increase slightly,
though not sufficiently to raise it to its normal value.
Although this point is still under quantitative investi-
gation, it seems certain that this increase of carbon
dioxide is casual, and probably due to a sort of water
rigor. Somewhat similar results are obtained with the
spider crab’s nerve (Table VIII). Thus we find that
with a 3 per cent chloral hydrate solution the carbon
dioxide production is least—about one-third that of the
normal nerve—while with a 4 per cent solution it is a
little less than one-half. Investigation of the effect of

per cent aes has bit little ‘effect for
- ten minutes, though during the course of half. an k
gain of 50 to 100 per cent takes place. A 3 per c a
concentration produces a gain of 25 per cent in ten min- os
utes, while in a 4 per cent solution the nerve gains 50 —
per cent in the same space of time. These results, —
together with those on the frog’s nerve, indicate that, 4
whatever interpretation we put upon this change in.
weight, the result of such a process is temporarily, at
least, to increase slightly the amount of carbon dioxide
evolved, and that, so far as the effects of narcotics are
concerned in our work, its slightly increased production
from the narcotized nerve which gains in weight may be
looked upon as adventitious. ‘The correctness of our
interpretation of the diminution of carbon dioxide output
as an effect primarily connected with narcosis is further
‘supported by a study of the effects of weak concentra-
tions of narcotics for different periods of time.

Effects of weak concentrations of narcotics on carbon
dioxide production in the nerve fiber.—It is well known
that the primary effect of narcotics is to increase irri-
tability, after which the typical depression follows.
This primary effect is well brought out by the use of
rather weak concentrations of narcotics. Although
we have made no quantitative determination of the
degree of irritability, it is evident that a nerve after ten
minutes’ immersion in a 0.4 per cent chloral hydrate
solution has become abnormally irritable. After about
one hour’s treatment, however, the nerve finally becomes
paralyzed. If the carbon dioxide output of a nerve
treated for ten minutes with a o.4 per cent chloral

-
'F
A‘

al nerv: ie cacy d domanatrete that the |
dioxide production of the nerve so treated is
- increased. The quantitative determinations
\ lated above illustrate this perhaps more con-
oy vincingly (see Table IX, horizontal column 3).
That this change in the carbon dioxide production of
i Fai nerve when treated with the lower concentration is
_ closely connected with the physiological state is further
demonstrated in the following experiments. Many claw
nerves were isolated from several spider crabs, each
pair chosen being approximately of the same weight, and
of each pair one was placed in a 0.4 per cent solution of
chloral hydrate and the other in sea-water. A compari-
son was made of the rate of carbon dioxide production
of the first pair in the biometer in the usual manner at
the end of ten minutes; at the end of half an hour a
second pair was compared similarly, and so on. The
result is given in Table IX. This table is of more than
passing interest, for it illustrates an easy source of error
in the study of narcosis. Evidently it is of prime
importance to determine the carbon dioxide output
during a comparatively small time interval, rather
than during one of long duration. If we were to deter-
mine the output of the gas for sixty minutes’ respiration
of the nerve treated with a o.4 per cent chloral hydrate
solution, we might be led to the conclusion that the
_. narcotic has no effect whatever on the metabolic rate.
___ For although we have shown, by taking corresponding
nerves at the beginning and at the end of the narcosis,
‘that the primary effect is to increase carbon dioxide.
production and that later it is greatly diminished, the

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igebr raic sum os ake results svey readily approximate
1e value found for normal respiration.

. ~The general phenomena are the same for ethyl

- x ethane, except that the primary increase of the gas

j in light narcosis is much more marked, being more
_ than twice as great if compared with the value for the

normal nerve at 20°2 C. It was noted that the nerves.
after treatment with a 1 per cent urethane solution were
hyper-irritable, but a part of this large increase must
doubtless be due to the higher temperature at which
the experiment was performed.

Carbon dioxide production from “‘inexcitable” normal
nerve.—During the warm weather we occasionally came
across a claw-nerve preparation in which no sort of stimu-

= lation of the nerve could evoke any response whatever,
although the peripheral organs were perfectly excitable.
Response by the attached muscle to stimulation of the
claw nerve of the spider crab is of three sorts: con-
traction, or relaxation of the claw, and movements of the
lower joint. In general, different strengths of the
stimulating current result in different responses. It was
at first thought possible that in these cases the stimula-
tion might be calling forth opposed responses, so that
one neutralized the other, and thus that no response
resulted. But further investigation showed apparently
4 that the nerve was inexcitable, since after immersion

in the sea-water irritability was often restored. To

whatever cause this may have been due, the interesting
a fact is that such “‘inexcitable”’ nerves invariably showed
an abnormally low rate of carbon dioxide production.
The results of the quantitative estimates on these nerves
are given in Tables VII and VIII, horizontal column 2.

study of narcosis are: (x) carbon dina iction is |
greatly diminished when the nerve is aardtizell ither
by chloral hydrate or by ethyl urethane in concentra- _
tions which produce a reversible loss of excitability;
(2) with a weak concentration of these narcotics, at the |
- beginning, the carbon dioxide production is increased,
but later is diminished. This is in accord with the theta
that these concentrations primarily stimulate, or in-
crease, the irritability of the nerve for a time. The
conclusion drawn from these facts is that metabolism in
the nerve is interfered with by any agency which inter-
feres with the excitability of the nerve. Excitability
and resting respiration go hand in hand.

The direction of the nerve impulse and the metabolic
gradient.—Although it has been established that an
excitation wave travels in both directions from the point
of the stimulus and that this wave is in all probability
identical with the nerve impulse, yet in the normal con-
dition in the body one fiber is supposed to conduct
the impulse in one direction only. Based on this
difference in the direction of the conduction, one set of
the nerve trunks is called efferent and the other afferent,
according as they conduct from or toward the central
nervous system. That there is a very interesting rela-
tion between the direction in which the impulse normally
goes and the rate of metabolism at different parts of the
nerve will be set forth in the following paragraphs.

Efferent fibers—lIf we take the bundle of nerves
between the second and third joints of the claw of the
spider crab and cut it at the middle, the two halves being
of about equal weight, and place each in a chamber of the

a ree ee eS a ey ae) eee
are ie an hae

EXCITATION AND CONDUCTION —S_73

e - biometer and measure its rate of carbon dioxide produc-

_ tion, we find that the part of the nerve nearer the body,
the proximal portion, gives off more gas than the distal

end of the nerve. This nerve is mainly efferent and
normally conducts the impulse down the claw from
proximal to distal direction. A quantitative estimation
shows that the proximal end gives more than twice as
much carbon dioxide as the distal, the former giving
at least 7.9X10~7 g. and the latter 3.7 1077 g. per
centigram per ten minutes.

TABLE X

CARBON DIOxIDE PRODUCTION FROM Two DIFFERENT
PorTIONS OF CLAW NERVE OF SPIDER CRAB,
Libinia canaliculata

A Plime re CO.

: emperature| Given y 10 mg.

Portion of Nerve Degrees C. of the Nerve in

to Minutes

15-16 6.7X10~7 g.

WOON sy oc. esc rn: { nie 7 QXIO77 £..

Goo) ee 2I g. oX10—7g.

3 OE Ee opi Pace 23.2 -3.7X107~7 g.

From a study of the various conditions which mod-
ify this difference in carbon dioxide production in the
various portions of the nerve. we are probably safe in
stating that we are here dealing with a physiological
gradient, experimental error playing no part. There
are three physiological causes which might account for a
different rate of carbon dioxide production: (1) different
degrees of injury; (2) different rapidity of death; or
(3) unequal rates of metabolism. The first alternative
is possible only on the assumption that the proximal
region must be more excitable (greater susceptibility to
an injury), which always causes the production of

; possibility, we must inquire ect one. pobtiol shot
earlier than the other. The fact that an isolated nerve
stays excitable for a considerable period of time makes F
this interpretation quite untenable, although we support
the idea set forth by Child that the death gradient is ig “4
directly associated with the metabolic gradient. It
may be added here that death modifies the carbon
dioxide production gradient.

Whatever interpretation we choose, inasmuch as we
contend that the rate of metabolic activity as measured
by the carbon dioxide output is a function of the irri-
tability, we are assuming that there must be different —
rates of metabolism along the normal nerve fiber.
Not only that such an assumption is correct, but also
that the effect of injury and death are only secondary
and minor factors, can be shown in the light of Child’s
experiments when the same nerve was examined, result-
ing in the confirmation of our results by an entirely
different method.

He has shown that in various concentrations of potas-
sium cyanide, from o.oo1 too.o1 molecular in strength,
the fibrillae of the claw nerve of the spider crab after a
time become irregular in outline and more or less vari-
cose, so that a strand appears more or less granular in-
stead of fibrillar, like a fresh nerve. With this criterion.
he has discovered a gradient similar to our metabolic
gradient, which appears in the structural death changes.
Using a 1 per cent ethyl ether solution in sea-water, or
even a somewhat lower: concentration, he found that
the change from fibrillar to granular appearance begins
at the ends of the nerve very soon after it is brought into

— % Bex,
5 ~

:* ry eh) J
slut oil that it does not progress equally from
two o ends. _ A distinct gradient in this change can be
en extending Satpbeiaily for a few millimeters from the
F nt -end and a shorter distance centrally from the
P . ripheral end. This first change remains limited to
the terminal regions of the nerve and is undoubtedly,
as the interprets it, a temporary metabolic gradient from
- the ends inward, due to stimulation and i injury resulting
from severing the nerve at these points. Later, however,
the fundamental metabolic gradient in the nerve appears,
in that the change begins to progress along the nerve from
the central toward the peripheral end; but the change at
the peripheral end progresses but slowly, or not at all,
in the central direction. From this time on a distinct
gradient in the change is visible until it has progressed
along the whole length of the nerve. Except in the ter-
minal region adjoining the peripheral cut end the death
change always progresses in the peripheral direction.
_ The peripheral third of the length may be entirely
unchanged at a time when the central third or more has
completely lost the fibrillar appearance. Thus the fun-

damental difference of the two ends is made apparent.
Lo Child has further shown that if the nerve is crushed
b or injured at any point similar gradients appear on both
#4 sides of the injury, but do not extend to a great distance
| ‘ before the general change reaches this region in its
| ¢ progress peripherally.

Since it has been demonstrated repeatedly that
susceptibility of other tissues and organisms to reagents
like ether and cyanide is an expression of the rate of
metabolism in the tissue, these results of Child not
only confirm our demonstration that there is a clear

‘this gradient exists in a normal nerve
injury. .
Afferent fibers —The optic nerve. of imulus was”
tried next. It is a non-medullated, long, appar ya :
uniform, nerve. It can be isolated in a oath of four ;
or five inches without cutting it at either end, though
the task is rather laborious. It is important that the ©
peripheral end should be left intact with the eyes. © This _
is accomplished by cutting the shell about two inches
square around the eye. By gently lifting the eye with
the nerve, we can easily trace the nerve centrally up to
the brain without any injury.

TABLE XI

CARBON DIOXIDE PRODUCTION FROM DIFFERENT PORTIONS
oF Optic NERVE OF KING CRaB, Limulus —
polyphemus, FEMALE

Amount of CO,

Portion of Nerve | L&™mperature Given Off by 10 mg.

Degrees C. of the Nerve in
to Minutes
When sco ccivitaks ge 2.6X10~—7 g.
Provimal: oie: 22.5 3.0X10~ 7g,

Distal ....ixwereos 22 5.0X10~ 7g.

When such a long stretch of the nerve is cut at both
ends simultaneously and is then divided at the middle
so as to furnish two parts of approximately the same
weight, and the rate of carbon dioxide production of
these two parts is compared, we find that the centro-
peripheral gradient discovered in the case of the claw
nerve is exactly reversed. In the optic nerve of Limulus
the proximal portion (nearer to the brain ring) gives

na)

ch | s cz Woh dibxide than the distal portion ede
Prcins)) where the impulses normally originate.
on

a Table XI shows the quantitative results.

_ Sensory dendrites —These results. were at first
‘surprising; but they became exceedingly interesting
when we took into consideration the functional or

a - ea difference between the two _ nerves.

_ The claw nerve of the spider crab is believed to be
ee posed mainly of efferent fibers, while the optic
nerve of Limulus is an almost purely afferent nerve.
The direction of the normal nerve impulse in one
of these nerves is, therefore, exactly opposite to its
direction in the other. Developmentally speaking,
however, the distal portion of the optic nerve corre-
sponds to the proximal portion of the claw nerve in that

these portions are in each case nearer the nerve cells

from which the fibers come. ‘Thus our results with the |
two opposing gradients may be subject to two alternative
interpretations. Either the metabolic gradient may
correspond to the developmental gradient, i.e., all the
portion nearer to the mother-cells may have a higher rate
of metabolism, or it may correspond to the functional
gradient, i.e., the nearer the portion is to the stimulus
the higher is the carbon dioxide production. |
This question will be automatically solved if we
study the metabolic gradient of a fiber whose functional
direction is opposite to its developmental direction;
e.g., an afferent nerve fiber lying peripherally to its
nerve cells—i.e., a sensory dendrite—should be studied.
Professor C. Judson Herrick kindly suggested that we
use a lateral line nerve, or an accessory lateral line
nerve, of a fish. In the carp and the catfish both of

are wholly sensory ioe spol of moto1
The fibers of both nerves are ‘dendrites of
cells which lie in the head. | i
When these fibers were subjected to our tests it was —
found that in every case the proximal portion of the —
fresh nerve gave much less carbon dioxide than the distal —
portion, indicating that the gradient is correlated with
function and not with development. And in this case
it should be noted that, although the fibers of both nerves
are dendrites of their respective neurons and conduct
afferent impulses, the function of the two nerves is
widely different, the lateral line nerve (ramus lateralis
vagi) being excited by water vibration and the accessory
lateral line nerve (ramus lateralis accessorius) being
exclusively gustatory in function. The quantitative
estimates on these nerves given in Table XII will illus-
trate the presence of this marked gradient of carbon
dioxide of the sensory dendrites.

@
LP

TABLE XII

CARBON DIOXIDE PRODUCTION FROM DIFFERENT PORTIONS
oF LATERAL LinE NERVE (RAMus LATERALIS
VaGI) OF CARP

Amount of CO:
Portions of | Temperature Given Off by 10 mg.
Nerve Degrees C. of the Nerve in
to Minutes
Proximal...... 24 4.9X10~7— 5.2X10° 7g.
Distak: oU.csxas 24 12.4X107 7-18.5X10~ 7g

That the metabolic gradient is directly associated
with the functional direction is strikingly shown also by
further studies with different fibers in which quantitative
determinations were not made, but in which the output

m dic tide: of two equal weights of proximal and
x portions of the same nerve were compared in the
meter under identical conditions. The portion of

produce the precipitate of barium carbonate in the
shortest time and the greatest abundance evidently
produced the most carbon dioxide.

TABLE XIII

COMPARATIVE STUDIES ON RATE OF CARBON DIOXIDE PRODUCTION FROM Two
PorRTIONS OF VARIOUS NERVES

TEMPERA-
TURE OF NERVE AMOUNTS
DATE Room or CO,
DEGREES COMPARED
S. Name of Kind of | Portion of | Weight of
Aug. 31... 23.2 Optic nerve of | Afferent |/{Proximal | 17 mg.| Less
__ Limulus mainly |\Distal 17 More
Sept. 15... 18 Optic nerve of Proximal | 23 Less
skate | = Distal 23 More
Feb. 15... 22 R. lat. vagi of Proximal 4.4 Less
carp S istal 4 More
Dec. 20... 20 R. lat. vagi of roximal’ 2 Less
catfish e Distal 1.8 More
| Dec. 13... 17 R. lat. acc. of Proximal I.4 Less
\ catfish eS Distal I.4 More
ey Nov. 23... 25.5 Posterior root Proximal 6.5 Less
of dog . Distal 6 More
. Aug. 30... 21.8 Claw nerve of | Efferent |f{Proximal | 32 More
} 7 spider crab mainly |\Distal 44 Less
: ‘3 Feb. 26... 19 Hypoglossal -|{Proximal |\Equal More
& nerve of dog ¢ Distal weights | Less
i Feb. 21... 23 Same nerve of Proximal | |\Equal More
| e carp by Distal weights | Less
j Sept. 15... 17.8 Oculomotor Proximal II More
o . nerve of skate . Distal II Less
Nov. 26... 26 Anterior root Proximal 1.6 More
‘ of dog “ Distal 1.8 Less

y While we have not by any means exhausted the
7 _ various. kinds of nerve fibers available for the study
of this gradient, the results obtained in those nerves
already examined have been very uniform and the
nerves themselves have had varied functions and have
come from several different classes of animals, namely,

rahscetoks that the tees — a Rey tab
in nerve fibers correlated with the functional pe

related to the direction the nerve impulse takes, must k bas
a very general phenomenon. From these facts we come i

to the very simple conclusion that is summarized in the

following statement: The normal nerve impulse passes
from a point of higher toward a point of lower carbon
dioxide production—from the more irritable to the less

irritable parts. There is also 'a decrement in the nerve
impulse. It cannot proceed indefinitely along a nerve;
it will ultimately die out.

Velocity of the nerve impulse and its relation to res pira-
tion.—Ii_ the nerve impulse cannot pass through the
fiber without consuming substance, it is reasonable to
expect that there may be some relation between the
rate of production of carbon dioxide in the resting nerve
and the velocity of the nerve impulse. The reason for.
such a supposition is clear. It has already been indi-
cated that the more irritable a nerve is the more carbon
dioxide does it produce in the resting state. Hence,
the more it respires, the more irritable it is, the faster
should it conduct the impulse. This is actually found,
within limits, to be the case. If one compares corre-
sponding nerves of different animals, or different nerves
of the same animal, it is found, other things being equal,
that there is a relation between the speed of contraction
of the muscles supplied by the nerve and the velocity
with which the nerve supplying the muscle conducts
the impulse. Obviously it would be foolish to have
a very rapidly contracting muscle, or limb, supplied with
a nerve which conducted the impulse to the muscle at a

-e
2
ae
ep ,
r@

7, : a oe ne a ’ 7
_ EXCITATION AND CONDUCTION 81
ay ‘tailgate t sclae

’ ery low rate. And, being foolish, this does not happen.

Je can use the speed of contraction, therefore, as a rough

index of the relative velocities of the nerve impulses in

& nerves supplying the muscles. In this way the rates of the

nerve impulse of the ambulacral nerves of the king crab,

_ the spider crab, and the lobster are in the ratio of 1: 2:4.
This ratio, as far as the king crab and the spider crab are
concerned, is very nearly the same as that found for the
carbon dioxide output of these nerves, as may be seen
in Table IV (p. 32). Low respiration, low irritability,
and low speed of conduction appear to go together.

- We can also test the rate of metabolism in these
nerves indirectly by Child’s method, and this gives us
just the same result. ‘This method consists in determin-
ing the speed with which the excitability of the nerves
is abolished when they are treated with the same con-
centration of anarcotic. The greater the rate of metabo-
lism the more susceptible is the tissue to narcotics. We
found in the case of these three nerves that the excita-
bility of the lobster nerve is most quickly abolished,

- then that of the spider crab, and finally that of the king
crab. ‘This is the same order in which the nerves con-
duct the impulse, the lobster conducting fastest. This
is therefore additional proof of the fact that the speed
of the impulse and the degree of metabolism in the
resting nerve are correlated, at least in these nerves. It
is remarkable to note that the carbon dioxide output

_ of the nerves of Limulus, the king crab, is very low in
comparison with that of other animals. This may be |
correlated with the very sluggish behavior of this animal
and its power of living for a long time without foodsand
with very little air.

oF ren
ie rae a°*

While there seems, ae eect very lee. 0 rre-
lation between the rate of respiration of resting nerves: re
and the velocity with which they conduct a nerve | 4
impulse, the data for the establishment of this generaliza-
tion must necessarily be cumulative, and we are not yet _
able to state positively that all nerves which give off
much carbon dioxide in the resting state will be found _
to conduct the impulse more rapidly than those which
give off less. There are, however, several conditions
which influence the rate of the nerve impulse, and we
have investigated the effect of these conditions on the
production of carbon dioxide. ‘Two of these con-
ditions are: changes in the salts in the nerves, and —
temperature.

a) Changes in salts: Mayer found that the rate of
nervous conduction in the sub-umbrella regions of the
subtropical jelly fish, Medusa cassiopea, increases about
5 per cent.in sea-water diluted with distilled water in the
proportion of 9:1, while it decreases 50 per cent in sea-
water diluted to 50 per cent with fresh water. By sub-
stituting o.g M dextrose for the distilled water he -
demonstrated that the change in rate of the impulse in
diluted sea-water was not due to the decrease of osmotic
pressure, but was due to the change in concentration of
the salt. If under those conditions which decrease
the rate of the conduction a measurement is made of the
amount of carbon dioxide produced from the thin layer
of the regenerating ectoderm tissue, it is found that a
change in the rate of carbon dioxide production goes
parallel with the decrease in the rate of conduction. As
a result of using the regenerating tissue just mentioned,
the nervous tissue regenerates before the muscular, so

‘EXCITATION AND CONDUCTION 83
- that we can in this way eliminate the effect of the salt on
the muscles. |

6) Temperature: It is well known that a change in
temperature affects the speed of the nerve impulse; an
increase of 10° C. increases the velocity of the impulse by
four-fifths, or even more. This is very significant in
view of the fact that for most physical processes the same
increase in temperature increases the velocity of the
process by at most one-fifth. Chemical processes are’
accelerated about 100 per cent. While the magnitude
and variation of the temperature coefficient of velocity
of a physiological process do not necessarily tell us what
kind of reaction is involved in the process, they never-
theless indicate in this instance very clearly that con-
duction by a nerve is not a purely physical process, as
some have imagined it. It is very important, evi-
dently, that we should compare the effect of temperature
on the carbon dioxide output with its effect on speed
of conduction. |

We have made studies of the metabolic rate of the
nerve of the king crab at different temperatures, such as
naturally occur at Woods Hole and at Dry Tortugas, and
we have discovered that the temperature coefficient of the
production of carbon dioxide by the resting nerve is
just about the same as the temperature coefficient of
the speed of conduction. A similar result was obtained
with a sciatic nerve of a frog under experimental changes
of temperature. We thus have this additional point of
parallelism between the rate of conduction and the pro-
duction of carbon dioxide in the resting nerve.

It is extremely interesting and significant that the
fundamental condition for the conduction of a nerve

$s -
ciple, eno
5 .

in ‘the nerve at the time of ddmacanen lt
‘the resting respiration, or metabolism, which. 3et
determine how fast the nerve impulse should travel al 1 ag
the fiber. It is exactly as if, during rest, the nerve s he s
stance was sustained in a very unstable state by thee! ie,
expenditure of energy by processes which set free the —
carbon dioxide. It is as if the irritable or unstable con-
‘dition was like a stone rolled partly up a hill and kept ~
there at the cost of considerable panting by the toiling
demon of life. When the nerve impulse comes along, —
the stone escapes from his grasp and rolls downhill.
During the period of rest or recovery which follows,
this tiny, toiling Sisyphus, with infinite labor and pant- _
ing, pushes the stone uphill. The higher he gets it the
more he gasps, the more unstable it becomes the more
easily it escapes his grasp, the more rapidly does it crash
down, and the more irritable is the nerve the more ~
rapidly does the impulse travel. _

Conclusion.—Basing our conclusions on the foregoing
experimental facts, we may express the relation between
excitation, conduction, and respiration in nerves as
follows:

The maintenance of chemical activity, or metabolism,
is responsible for that unstable condition in the nerve,
whatever its nature, which we call the state of irrita-
bility or excitability. All irritable tissue must respire.
The tissue cannot be:made irritable and then kept so-
without effort. Chemical energy must constantly be
expended to keep the tissue irritable. The amount of
this expenditure of energy is not the same at all points
along the fiber, but it diminishes in one direction or the

“
iz
5

*
W

-_- EXCITATION AND CONDUCTION 85

other, generally toward the central nervous system
x in sensory nerves and toward the periphery in motor
nerves. The nerve impulse generally travels in the
direction of this gradient. When we stimulate a nerve
at any point, the stimulation consists in the local in-
_ crease of metabolic activity at the point of irritation.
_ The irritability is raised and the carbon dioxide output
is increased at that point above the production on either
side of it. This causes a local metabolic gradient in
the nerve in both directions from the point of excitation,
but the difference between this point and its surroundings
will be greater on one side than on the other, owing
to the gradient in the nerve just mentioned. If this
state of excitation is sufficiently great, it upsets the
4 equilibrium and the impulse will be propagated in each
direction from the point of excitation. The possi-
bility exists that it ought to travel more easily in the
direction which the nerve impulse normally takes, and it
ought to be possible, with a proper amount of stimulus,
to start a propagation in only one direction from the
point of stimulus, but we have not yet tested this possi-
bility experimentally. The excitation always travels
from the point where the excitation is greatest to that
‘where it is less. The repair process, or the anabolic
process, is also propagated.

The conditions which affect the rate of nervous
metabolism not only alter the state of excitability of the
nerve, but also change the speed of the conduction of the
state of that excitation. .Although we have no evi-
dence to show that the chemical change itself constitutes
the nerve impulse, the conclusion is almost inevitable
that the nerve impulse is brought about by, or is itself

CHEMICAL SIGNS OF LIFE

; We have endeavored to show that the living nerve,
as long as it is irritable, is chemically active and that
when it functions this metabolism is accelerated. As the
irritability of the nerve varies, there are simultaneous
changes in chemical activity. What characterizes the
living state is respiration and its increase on stimulation.

We have come now to our main inquiry, namely,
whether or not all living matter undergoes respiration
as long as it is alive, and whether stimulation always
increases its respiration. In addition, we have to ask
whether, if this is true, it can be used as a sign of life in
all living matter.

- Seeds—It has hitherto been maintained that since
dry seeds do not respire but are irritable, irritability is
independent of respiration. The work of Horace Brown,
Thistleton Dyer, and others indicates that dry seed can
be kept alive at very low temperatures in conditions
where no ordinary gaseous exchange is possible. It is
argued, therefore, that life is possible without any ©
metabolic activity. Dry seeds, kept for long periods
in a closed vessel, have not been found to give any evi-
dence of this fundamental chemical change occurring
in living matter, namely, the production of carbon
dioxide. Such seeds, it is well known, are not really
dead, for under proper conditions they germinate.
They appear to live without respiration, but this is but

“1 aA o>. y ;
ae ata i Aces aca 5 .
rth 4 ‘eae wo a ie 2) a 7 ‘
4) feds ak 88° $ - eS te Sa CHE Bagh FS ae ae
") * ‘ ° , 7 Cee , “HT \ i ‘ ery y ne 5
* 1) .t J TCA j

an appearance. The | eeds really respire. \

Geleyed that our present chattel inique is no ;
enough to reveal to us the smallest and most infinitesim; al
chemical changes which may be going on in the appar- rd
ently dry and perfectly dormant seed. He based his —
hypothesis on two considerations: First, was the fact —

- that the seeds wear out, as shown by their losing their _ ’
power of germination and growth in proportion to the
length of time they have been kept. The deterioration
is more or less rapid, according to the nature of the
seed and the character of the protective coats, but in
every instance there is deterioration sooner or later.
He attributes this gradual deterioration to chemical
activity in the seed. |

In the second place, there was the fact, which he

showed by his electrical method, that a living seed not
only differs from the dead one in respect to its electrical
response, but that the amount of its response varies
according to its age. Thus, if he took a living seed, a
dead seed killed by heat, and a very old Egyptian seed
from about the Twelfth Dynasty (about 4,400 years

old) and determined their electrical response, he found
a very interesting result. The first, or living, seed gave
a large electromotive force, while the others, the old
as well as the dead, gave none. If he took a group of
seeds from crops of different years, he found that there
was also a gradual decline in the electrical response as
the seed became old. He considered this electrical sign
as the expression of the chemical changes which cannot
otherwise be determined, and such a sign of death,
according to him, is manifested long before microscopic
or chemical changes can be detected.

ee ie it, y= ae ala a

_ ™

CHEMICAL SIGNS OF LIFE 89

He found that this electrical change—the blaze

current, as he called it—which appeared when the living

seed was stimulated by a strong induction current was
not confined to seeds, but occurred also in other varieties
of living matter, such as the eyeball, skin, leaves, petals,
and many other tissues of plants and of animals. ‘This

momentary electrical change produced thus only by

living matter is accordingly a reliable sign of life, since
it does not occur in dead matter.

When we discovered that even a resting nerve gave
off carbon dioxide if we used a sensitive method, we
at once proceeded with some curiosity to determine
whether or not ordinary seed is chemically inactive.
We had in mind thus to test Waller’s conclusion that the
electrical sign in the seed is really the sign of chemical
changes which, however, were not large enough to be
detected by ordinary chemical methods.

Resting metabolism in seeds.—If a few kernels of
wheat are placed,in one chamber of the biometer, there |
is no difficulty in showing that seeds give off carbon
dioxide, since a drop of barium hydroxide in the chamber
containing the seeds becomes covered after a time with
a precipitate of barium carbonate. It is true that the
amount of carbon dioxide given off is exceedingly small,
being many times less than that of the resting nerve,
but that this carbon dioxide is produced by a vital
metabolism is shown by the fact that living seeds
give far larger amounts of the gas than dead ones.
A seed respires, therefore, as long as it is alive; and
we can measure the amount of respiration. Of course
the mere production of this gas from a seed does not
mean necessarily ‘that the seed is alive, for the reason

ht to inquire whether te at is | SEN:

stimulation. Pb 5s ‘as oH)
Increased metabolism in seeds. The auaet interesting a )

thing ascertained was that the living seed, like any other —

living tissue, can be made to give off more carbon dioxide :

on stimulation. It responds to an injury and is, there-
fore, irritable. It has already been stated that a nerve
injured by crushing gives off more carbon dioxide than
a resting nerve, just as if it had been stimulated by an

electrical shock. Since there was no way of telling —

what strength of electrical stimulation was required
in order to arouse the seed, we stimulated it by an
injury, namely, by crushing it. The seed thus stimu-
lated showed a marked acceleration of its respiration.
If two apparently living kernels of wheat are taken and
one of them is crushed and their carbon dioxide produc-
tion is compared in the biometer, the crushed one always
produces more carbon dioxide than the normal one.
That this is a vital response is shown by the fact that only
living seeds behave in this way. If one takes two kernels
of any similar seed, which have been killed in an elec-
trical oven heated to 60° C., and one of them is crushed,
there is no difference in the carbon dioxide output of the
two seeds. The difference in amount of carbon dioxide
produced by crushing cannot be observed in dead seeds
or in anesthetized seeds. In this respect a seed and a
nerve are alike; the chemical signs of irritability are
identical. Both, as long ‘as they are alive, respond to a
mechanical stimulation by producing more carbon
dioxide.

‘CHEMICAL SIGNS OF LIFE : gI

Is an injury a stimulation?—Are we justified in
regarding the increase of carbon dioxide following injury
by crushing as in the same category as the increase of
carbon dioxide production by ordinary stimulation ?
That this conclusion is justified is shown by the fact
that such an acceleration of carbon dioxide production
will not take place in inexcitable tissue. Neither killed
nor narcotized tissue can be made to give off more carbon
dioxide when crushed. Response to an injury is given
by living irritable tissue only. It is impossible to injure
the dead tissue.

Other tissues.—When we discovered that the irrita-
bility of a kernel of wheat and that of the nerve fiber are
identical, so far as their metabolic expressions are con-
cerned—i.e., no irritability without resting metabolism,
increased metabolism on stimulation, and changes in
metabolic condition, according to the state of excita-
bility—we thought it might be possible that this similar-
ity between the nerve and wheat is special, and that
other plant tissues may not behave at all in the same
way asdoseeds. Similar experiments were consequently
tried on several other seeds, including wild oats, Lincoln
oats, Swedish select oats, rice, corn, mustard, and various
others, with the result that, although the amounts of
carbon dioxide given off varied considerably, all living
seeds were found to be metabolically active. All of
them responded to an injury, giving off more carbon
dioxide on crushing. And in no case did we succeed in
producing more carbon dioxide on crushing killed seeds,
or seeds which had lost germinating power. Thus we
made certain that under the experimental conditions in
the biometer it is possible to detect the fundamental

3 creased carbon dioxide REDE nSS HE on stimulation ,
chemical sign of life of seeds and aiden: senerally,
well as of nerves. -
Once this interesting similarity betwee bods ‘and
nerves was well established we made further investi-

gations on other “plant tissues, in which conditions

were somewhat different. It was possible that the
removal of the heavy coat from seeds in crushing them
might have something to do with the increased metabolic

activity, and that this activity, therefore, might not be ©

. manifested by all tissues. In fact, Crocker has shown
that the removal of the coat is one of the factors which
initiates germination in dormant seeds. When we tried
different leaves, however, they all behaved in the same
manner as did seeds and nerves. The leaves selected

for test were necessarily small, with the object in view —

of being able to place the whole leaves in the chamber
with the least injury. They included such as Japanese

ivy, common grass, Australian pine, and various others.

We may add here that the increase of carbon dioxide
output as a result of some other forms of injury in leaves
has been recorded by several investigators.

Some objection might be made against our experi-
ments, however, on the ground that the injury to the
stomata may be responsible for the output of more gas
in the case of leaves. ‘That this is not the sole cause
of the escape of the gas is shown by our experiments on a
plant tissue without stomata. Red algae were tested
at the suggestion of Professor Osterhout and gave
similar responses. |

aa
-) ,
"3 ~

‘CHEMICAL SIGNS OF LIFE 93

Thus we extended our experiments to the best-known

tissues in the plant and animal kingdoms, and found

no exception to the general rule cited. These results

surely justify the generalization that all living tissues

differ from all dead tissues in that they respond to

e. injury, producing more carbon dioxide than the normal |

tissues; and that by measuring this output of the gas
in comparison with the uninjured we can detect the
vitality of the tissue. |

Chemical sign of life-—We have now come to a con-
clusion on all the facts that we have presented so far.
Of all the signs of living processes irritability is one of
the most universal. This phenomenon of irritability is.
expressed in the power of feeling the external world. It
is the inherent power of the living to react against a
stimulation. The necessary condition for this irritability
of tissues is metabolic activity. Although this chemical
condition is necessary for all tissue in order that it shall
be irritable, yet it is not a sufficient criterion for the
detection of vitality init. We must inaugurate a further
test of whether or not it reacts chemically to.a stimula-
tion. In order to test this power, we injure the tissue
and watch the response. If the tissue is alive, me-
chanical crushing will produce a metabolic response;
if it is not alive, there is no response. :

The detail of testing the vitality of a tissue is as
follows:

In order to test that of a seed, take two or more
kernels of the seed in question having about equal
weights. One is placed in the right chamber of the
_ biometer, and the other is crushed, or is cut to pieces,
and placed in the left. The apparatus is filled with

depending on the size, the number, ad the kind of seed —

we are testing. With several seeds, as with the fresh 7 b
nerve of a frog, we can detect vitality in this ie —_ a a
few minutes.

{
i

’ d
$ :
1g

CHAPTER VI
CONCLUSIONS

| Ee ‘Summary.—While we cannot define life in a physical

sense, for the reason that we have no measure of the
Be eychic phenomena shown by living things, and. these
- psychic phenomena are, after all, the most important.
of the characteristics of life, there are nevertheless certain

phenomena associated always with the living processes

which are so characteristic that for the majority of
organisms with which we are familiar we have no diffi-
culty in determining whether they are living or dead.
Irritability is the universal sign of life, and by it living
matter adjusts itself to its environment. The sign
of this irritability is the functional power of the tissues.
Thus by measuring the functional power we can speak
of measuring the amount of irritability. The changes
of a physical or chemical kind which accompany this
functioning are very important for an understanding of
the living process, for when we know them completely we
shall probably understand the nature of irritability itself.

In chapter ii we showed how it happened that, because
of the apparent exception in the case of nerves, it has
been generally concluded that chemical changes could
not be considered to be essential to all living processes.
Some of these changes, it appeared, must be due solely
to physical processes, and for this reason irritability
had come to be regarded as a purely physical phenome-
non. Various hypotheses had been made to explain how

, SF Bars. vs
2 : 5 <a i
. ,, i he ogee as i an

- this could be; ‘the change te

the colloids or the structure of ite cote or, mo a
recently, in the state of its permeability., But ci on
this basis irritability was to be understood was by no
means clear. On examining the irritability of nerves— a ;
the apparent exception which had led to the conclusion
that irritability had a physical and not a chemical —
basis—we found that this apparent exception was
really due to the fact that our methods had not hitherto
been sufficiently delicate to detect the chemical changes
which accompanied the process. By devising a new
method for the study of carbon dioxide—one of the —
terminal products of metabolism everywhere—we found |
that living nerve fibers in reality were undergoing
chemical change at quite a remarkable rate and were pro-
ducing carbon dioxide faster than any other tissue of the
body, if equal weights were compared. And we found,
further, that reagents or physical methods which change
the state of excitability of the nerve changed also the
rate at which it was producing carbon dioxide, so that
the gas production was evidently correlated with its
vitality and not with adventitious processes.

In chapter iii we found that although the chemical
activity is a necessary condition for all living nerves, yet
by itself it is not a demonstrative sign of life; i.e., it is not
a sufficient criterion of living. An additional criterion is
needed in order to be sure that any tissue is living.
In the case of the nerve, we demonstrated that this
additional sign was also present. ‘This sign is the fact
that all living matter, including the nerve, responds to
a stimulus by the production of more carbon dioxide.

|
k
i

sum Apion 1 of ie nervous SRE In tue: nerve,

then, irritability can be measured by the increased
- metabolism which occurs on stimulation. If the irrita-
| - bility i is high, the carbon dioxide increment is also large,
: 5 and vice versa. The response to stimulation is the sign
of irritability. We measure this response by measuring
the simultaneous output of carbon dioxide, which must
be the sign of that metabolic activity in virtue of-which

the function is performed.

In chapter iv we demonstrated further the importance
of the metabolic changes for the functional activities of
the nerve, and we hinted that the real mechanism which
makes the nerve able to perform its function must be

_the chemical changes which go on in the resting con-
dition of the nerve, and which must determine not only

the degree of excitability of the nerve—the direction of
the nerve impulse—but also how fast this transmission
travels. We found, for example, that the part of the
nerve from which the nerve impulse normally comes
always produces more carbon dioxide than the part
toward which it is going. It is well known that nerves
are more excitable in the parts which normally originate.
the impulse and that the excitability decreases down

_the fiber. Thus there is a parallelism between the

degree of excitability and the amount of carbon dioxide
produced in different parts of the same fiber, a parallelism |
the profound importance of which is not easily over-
looked. For nerves are thus shown to have in them a
metabolic gradient. They are, as it were, polarized
metabolically, and thus we have for the first time the
explanation of the electrical current which has been

nerve, up or pri it, as ‘the case me ‘back
electrical current is generated in a nerve between t 1
parts which are unequally irritable, or unequally ° under- —
going chemical change. The part of the nerve wh ch
is respiring most is in a different electrical state from that
which is respiring less, and thus we see the very clear _.

and definite relationship between the chemical and the a .

electrical changes which have been particularly dwelt
upon by Waller. This is one of the most important and
fundamental discoveries which we have noted, for it
means that there must be a decrement in the rate of the
impulse as it flows down the fiber, and that the distance
to which a nerve impulse can be transmitted is not
indefinite, but that that impulse diminishes as it pro-
ceeds, and will ultimately die out. In the medullated
nerves, to be sure, this decrement is not large, for it is
very necessary that it should be as small as possible in
the more highly developed nerves; but it is to be found
everywhere. And in simple undifferentiated proto-
plasm of plants and animals it is easily shown to exist.
The more rapidly nerves respire the faster do they
appear to carry the impulse; irritability and the rate
of production of carbon dioxide in resting nerves thus
appear to be correlated. The more respiration the
more life! If we abolish respiration temporarily, or
reduce it, we find that irritability has been reduced in
‘somewhat the same proportion. Anesthetized nerves
of all kinds show a reduced output of ‘carbon dioxide,
and they recover their irritability when they breathe
again. Anesthetics do not, therefore, affect the physical
state of the protoplasm only, as they have been supposed

“

CONCLUSIONS © “99

ry to do by many, but their action is shown by the change
in respiration in a manner more perfect than in any
other way except by the electrical response. Small
amounts of anesthetics at first increase irritability; and
at first they increase the rate of respiration and coinci-
dently they increase the electrical response. Irritability,
respiration, and electrical response parallel each other so
completely that they are evidently different aspects of
the same thing. 3

In chapter v what we had established as being true
in the case of nerves was shown to be true in the case
of all forms of living matter. ‘Taking the least promising
kind of living matter, that of a dry seed, we demonstrated
that it, too, breathed as long as it lived, that it
produced carbon dioxide, and increased its output
of carbon dioxide when it was mechanically stimu-
lated by being crushed. Seeds, too, it was shown,
could be anesthetized, in which condition they give off
less carbon dioxide and no longer respond by an outburst
of carbon dioxide when injured. Extending our observa-
tions, we found that all kinds of plant and animal tissues,
‘without any exception, respond in a manner similar to
that of the nerve fiber. In all cases stimulation causes
an increase in carbon dioxide. We could never find
any response unaccompanied by an outburst in car-
bon dioxide. Hence the best way to discover whether
a tissue is living is to crush it and see whether it reacts
to the injury by producing more carbon dioxide. It is
not necessary to put seeds in the ground to determine
whether they live; by crushing some of them we may
discover whether they are alive or not. Thus the
chemical test of life in the tissues, a test which parallels

although its Moplieabiieee is “fa pretens ae bg

testing the degree of vitality of a tissue., ar a

We have now to compare for a moment this criterion — f a

of life—the chemical—with other criteria which have —
been proposed, and to see whether it lacks anything of wee

precision of these other methods, and whether life can be |
shown to exist by other methods where we cannot prove _

its existence by ours. There is one criterion other than
the obvious one of growth which has been proposed to
determine whether a seed or other living thing, or piece
of a living thing, is alive or not. That is the criterion
suggested by Waller. It is the electrical sign of life.
Waller discovered a very remarkable electrical sign of 5
life, which may be described as follows: Two electrodes
are placed on opposite sides of a garden pea which is
living, the electrodes being connected on the one hand
with an induction coil and on the other with a sensitive
galvanometer. A single induction shock is then sent
through the pea. If the pea is alive, this shock is fol-
lowed by a remarkable outburst of electromotive force
in the pea. A current suddenly blazes out, as is shown
by the deflection of the galvanometer. It is as if the
pea jumped when stimulated. This current sometimes
travels in the same direction as the induction shock,
and sometimes in the opposite direction. Itis of momen-
tary duration. Waller calls it the blaze current. As

long as the seed lives, you get it; when the seed dies, you

do not get it. The dead or anesthetized seed does not

a.
—s

AWS

ue + these ise! ereanita: as asi as
2 alive. ,and the outburst is not only a sign of life,
n index of the amount of life. Life and electricity
are inextricably bound up together. In the sea algae
~ alone Waller failed to find this blaze current, but he does
not doubt that it exists there. One does not obtain
_ it for the reason, probably, that the salts’of the sea-water
_ close the current through the tissue rather than through
_ the galvanometer. Perhaps a low-resistance galva-
- nometer would detect it here too. This sign of life of
_ Waller is in many ways the most convenient that we
have, if only we have the apparatus for the detection
of these currents ready at hand and set up for use.

But up to this point we were still in the dark regarding
the cause of this electrical response. We could not know
whether it was due to a physical or a chemical change
in the tissues. It might be due to some change of
permeability of the tissues, or it might be due to a chem-
ical change. Waller believed it to be caused by the
latter, and his conclusion was undoubtedly correct.
| Waller also observed that following this electrical
display there was a sudden lowering in the electrical
resistance of the pea or other tissue. This might
also be called a sign of life, but it is not so clear and
striking as the blaze current. Evidently this is by no
means so reliable a sign’ of life as the other. -The de-
creased resistance might be due to a physical change of
state of the protoplasm or of the membranes, so that the
salt solution became more continuous; or it might be
due to the stimulation increasing in some way the ions in ©
the protoplasm. It is impossible to say which.

eho

fans wih,

ies CHEY ICA | oie

changes, then, we seem to be deating with something
more fundamental than when dealing with the electrical, r
although, if we admit that all processes of oxidation are _
in reality electrical, this distinction cannot be sustained.
Wherever Waller has been able to show the electrical
sign of life, we can show the chemical sign, and we can |
show life at some points where he could not, as in the ©
case of the sea algae. These, under our method, respond
in the same manner as do all other forms of living matter.
Moreover, we can use this method where it is impos-
sible to use the electrical; for example, in very minute
forms of living things, like eggs of small size, bacteria,
or infusoria. Our method can make it clear that they
are alive and breathing and responding to changes in
their environment like every other living thing. It
appears, then, that this sign of life has also certain
virtues of its own, although it is not so striking and
elegant as the method of Waller. It is also not so easy,

perhaps, for the ordinary man to set up and work ~

this apparatus as a galvanometer. But what it lacks
in ease it makes up in precision, in the quantitative nature
of its results, and, above all, in its fundamental char-
acter. By it we get as near as we have yet got to life
itself. | 7

In still another way the results which are recorded ~
here are of a most fundamental character, for one of the —

1c living matter irons. It is this: the Fairibiens of
lems, which we wish to have solved. Is that

ess physical or chemical? Is it simply an altera-

m of permeability of membranes, as some have

_ supposed, or is it in reality in the nature of an explosion ?

Z ‘Is. the living thing essentially a bag of jelly with a
Z "wonderful membrane about it, that membrane being so

- wonderful that all the phenomena of life are to be
ascribed to its changes in state? For this is the view

' which some maintain. They lead us to the holy of
holies of cells and tell us to behold a membrane! _Is life

nothing more than a membrane? What kind of a subter-

fuge is this which we encounter? All the riddles of life
are but the peculiar properties of a membrane! Upon
this membrane, as upon a magic carpet of Arabia, we
are invited to mount and travel over that unexplored
country whose mountain peaks shine in the distance.
Are we, then, beings of but two dimensions, nothing
but membranes, of which the magic proportions mock
us derisively, since we can never hope to seize that
which has but two dimensions? That such a view
resembles the membrane it has conjured up, in that it is
surface without depth, is self-evident.

In no such simple and naive a manner can the un-
knowns in the equation of life be determined. For we
have found that everywhere, paralleling the irritability
changes in a perfect degree, as far as we have been able
to determine, go the chemical changes. Carbon dioxide,
that very simple substance, the last term in the katabo-
lism of living matter, rises and falls with irritability.

. Paliere: RespitaGon) or at pie “this. phase ¢ “a:
tion, and irritability are in some way bound up together,
and we many now very briefly ask ourselves how they
may be related. wy

The connection between irritability and metabolism.—
What, then, is the connection between the irritable el y.
the respiratory process? Could we answer this ques-
tion we should have solved one of the most fundamental
of all questions of science. However, we do not hope
to be able to answer it at present. But let us at least
see what facts we can discover. The first of these facts
which strikes us is that living matter, even when it is
not stimulated, continues to give off carbon dioxide
and to respire. What is the significance of this fact?
Why should this constant consumption of material go
on in the absence of outside work to do? The main
function of the resting metabolism is to keep the tissue
irritable. As long as a tissue remains living and irritable
we find it to be the seat of production of carbon dioxide.
To be sure, it continues sometimes to give off carbon
dioxide after death, but it never ceases to do so as long
as itis alive. After death the rate, with possibly a tem-
porary increase, soon diminishes. For nothing is more
certain than that living matter burns up faster than
the same matter after death. Death extinguishes the
torch of life, although it may continue to smoulder for a
time when the spirit of its flame is gone.

Does not this fact mean that life, or rather the living
state, is a dynamic rather than a static phenomenon ?
We might conceive the living matter as a very highly
explosive substance, very unstable and ready to go to

ted. But this, raites aa: is a static view.
re dis bvered this very fundamental fact, that
, e resting metabolism goes faster when the tissue is
‘ ‘more abounding i in life and is more irritable. It is the
i _ burning substance which is irritable, or, perhaps, the
carbon dioxide thus formed conditions in some way
the vital or irritable reaction. It requires the expendi-
_ ture of energy to keep living matter in an irritable state.
The reaction is dynamic and not static. Living matter
has been conceived by many physiologists (of whom I
may mention only one of the leading exponents, Verworn,
for the various modifications of this view of individual
authors are not fundamental modifications) as being
composed of very complex unstable molecules, or aggre-
_ gates of molecules which are very unstable. These
are called biogens. Now this view is essentially static.
There is no reason why a biogen should not be isolated
if our methods were but fine enough. ‘There is no reason
___why a collection of biogens should not exist without any
metabolism; why, in other words, suspended anima-
tion should not be possible. But the facts which we
have discovered of the parallelism of the production
of carbon dioxide and irritability lend support, it would
seem, to the dynamic, rather than to the static, view.
We can picture the process, perhaps, in the following
crude and, of course, indefinite manner: The life-
process may be considered as a bicycle in motion. The
_ living process is an unstable condition. It is like a
-chemical system, the system as a whole having a certain
stability, but being at the same time the seat of intense
chemical change. It is in an unstable equilibrium. The

in the sectiine state. Rie i SO init abilit al
measured by the force necessary to change nee
makes with the ground. Perhaps the amount of life m
be compared to the angle the bicycle makes with he
ground. The metabolic activity is the force which —
moves the wheel. The locomotion of the wheel is the
functional activity. There is, however, this difference—
the faster the bicycle moves the more stable it is, whereas’
the faster the respiration drives the less stable is the
irritability of the tissues. Our simile breaks down here.
And indeed it is but a poor picture, of not much value.
But whatever view we may take of the matter, we may
at least be sure of this much: that chemical change is in-
volved in irritability. The transmission of a nerve
impulse involves material decomposition in the fiber.
The impulse may be nothing else than the increased
metabolism itself. The nerve impulse is a very real
thing, and it has a material basis which we may hope to
discover. So far we have found two facts about it:
first, it liberates carbon dioxide as it passes over the
fiber; and, second, it depends on the nerve fiber having
been previously oxidized or exposed to oxygen. LEvi-
dently combustion is involved in the process somewhere,
but it appears at present more probable that it is involved
in the creation of the irritable substance rather than in
the very act of excitation itself. In other words, the
oxidation is part of the process of repair or the recovery
of the tissue—the process by which the state of irrita-
bility is maintained—and not the process of transmission
of the impulse itself. |

i
a :

1
“

a Concerning the nature of the material basis of the
_ nerve impulse we can only say that it appears to involve

_ that part of the chemical transformations in protoplasm

CONCLUSIONS — 107

which result in the production of carbon dioxide.
Farther than this we cannot go at present. But it is

certain that it has a chemical basis. Whether it has also

a physical basis, such as a change in state of the colloidal
substratum of the nerve, or not, we cannot yet say.
Who shall write the chemical reaction of the future,
embracing, not only the energy exchange, but the change
in psychism as well ? !

Finally, we come to the quantity of life, the point
from which we started. The méasure of this is the
amount of respiration, or the amount of electrical
response shown on stimulation. The question of how
much we are alive must be answered by the determina-
tion of the extent to which we are undergoing energy
transformation. Death and peace, life and struggle—
these are the pairs which go together. ‘The most perfect
young life is that which shows the highest metabolic
rate. We have shown the general correlation between
the carbon dioxide production and the nerve impulse
in its speed of propagation and ease of origin. There
must, then, be a close correspondence between the habit
of the organism and the general metabolic rate. The
simile of the torch is obvious. The faster it burns the
more light and life it has. The most vigorous life is
that with the keenest chemical change. And this is also,
as has been shown in another volume in this series, the
criterion of youth. The most successful life is that in
which the nervous system remains active, youthful, and
alive for the greatest number of years. It is the youth-

vaakt dts pie chemical ‘2 accomp: vant ent. Perhap:
the nerve impulse is something in the mene an Pp
agated explosive wave in a continuous substan ce.
Whether that wave is in the nature of a hydrolysis or an
oxidation we cannot say, but at any rate it results in the.
liberation, in some manner, of carbon dioxide. This ‘ :
substance tells us whether the nerve impulse has passed.
this way or not. The change which liberates it may be
_ the impulse itself. Three kinds of changes occur, then, _
in our brains when the nerve impulses are passing—an
electrical change, a chemical change, and a psychical
change. Which is the fundamental change ?

APPENDIX
THE BIOMETER: HOW TO USE IT

The study of carbon dioxide has been so connected
with various forms of human activity that in spite of
~ natural difficulties methods for its accurate quantitative
determination have been highly developed. Never-
theless, none of the various methods of analysis can be
used for very minute quantities of carbon dioxide. The
greatest difficulty in using any micro-gas analysis is in
securing freedom from the external variations of tempera-
ture and pressure. Particularly is this so in the case of
carbon dioxide, for we need to consider, not only the
effect of temperature and pressure variation, but also
how to free the apparatus from atmospheric carbon
dioxide. After we discovered a new method which
detected exceedingly minute quantities of this gas we
found that the ordinary method of freeing air or any
other gases from carbon dioxide was not sufficiently
accurate, although it should be admitted that our
experience in washing gases was not very extensive.
The biometer is constructed with a view to meeting
these difficulties and has shown itself to be remarkably
convenient for many biological as well as chemical
investigations. |

Uses of biometer——The biometer can detect carbon
dioxide in as small quantities as one ten-millionth of a
gram. ‘This is the amount contained in one-sixth of a
cubic centimeter of the purest air, in which we assume

109

2 eka of dite apparatus can ae ill 1s
of experiments we can use it for, e. Pee
1. The different rates at which carbon dioxide
duced by a single fertilized and a single unfertiliz 3
of a fish (Fundulus hectroclitus) can be distinguished ie
2. The unequal rate of metabolism of two different
species of the little banana flies can be detected within
ten minutes by using a single insect in each chamber. _

3. The vitality of a single kernel of wheat can ac
detected in ten minutes.

4. The daily variation of respiratory activtig ae a 8
single isopod has been determined. 4

5. The carbon dioxide production of the different
parts of a small nerve fiber can be measured, and the
unequal rates of different segments of the nerve detected.

_ 6. The effect on the metabolic rate of the muscular —
contractions of very small animals, like a worm or insect, —
or the effect of light on small pieces of a leaf, can be
demonstrated in the class in a few minutes.

7. By the use of proper reagents small basis of
many other gases can be measured.

Principle of the method.—The principle of the method
was first devised in conjunction with Dr. H. N. McCoy,
and, with some modifications, the biometer is constructed
so as to conform thereto. The principles involved are as
follows:

1. Exceedingly minute quantities of carbon dioxide
can be precipitated as barium carbonate on the surface —
of a small drop of barium hydroxide solution.

2. When the drop of barium hydroxide is exposed to
any sample of a gas free from carbon dioxide it remains _

”

THE BIOMETER: HOW TO USE IT III

clear, but when more than a definite amount of carbon
dioxide is introduced, a precipitate of carbonate appears,
which is detectible by means of a lens.

3. By the use of accurately known quantities of
carbon dioxide of exceedingly high dilution it was found
that the minimum amount of carbon dioxide which gives
a precipitate is 1.0X107” g.

4. By determining, therefore, the minimum volume
of any given sample of the gas necessary to give the
first visible formation of the precipitate its carbon
dioxide content can be estimated accurately, since this
volume must contain just the known detectible amount
of the gas, which we found to be 1.0X1077 g.

5. By having two chambers side by side the different
rates of metabolism from two different tissues can be
estimated by the different speeds of formation of the
precipitate and extent of the precipitate.

Description of the apparaius——The biometer shown
in Figs. 1 and 3 is made of glass. It consists of two
respiratory chambers connected by a three-way stop-
cock L, the other arm of which is connected to one arm
of another three-way stopcock K. (As is shown in
Fig. 1, for an ordinary experiment we can connect it
directly to the nitrometer.) Each of the other two
arms of stopcock K is connected to a nitrometer, W or
X, which is used for removing the final traces of carbon
dioxide from the gas with which the chambers are to
be filled. The nitrometer on the right is connected to a
_carboy F (see Fig. 5, apparatus III), filled with air free
from carbon dioxide; and the other, on the left, to a simi-
lar carboy as a reservoir for any other gases that may be
used as a special medium for different experiments, such

& > ey _ A CHEMICAL SIGN OF LIFE

ss piss ES.

ates

callie Sat

Fic. 1.—The Biometer. One-fourth actual size

‘THE BIOMETER: HOW TO USEIT 133

as oxygen-free air, hydrogen, or volatile anesthetics, and
_ which are exposed to an alkaline solution in order that
every trace of carbon dioxide can be removed from the
gas in question. As stated before, for ordinary metabo-
lism experiments we may use but one nitrometer for
_ ordinary air only, as shown in the photograph (Fig. 1).
Chamber A is drawn to a capillary stopcock C; chamber

Fic. 3.—Biometer. One-third actual size. The shaded portions
of the apparatus indicate the rubber connection, which is first coated by
shellac and then sealed with a special sealing wax. Some parts are
also sealed with mercury.

B is drawn to a similar capillary stopcock C’, one arm
of which is connected to another three-way stopcock G,
one arm of which is connected to a mercury burette T,
which is used for adjusting the pressure in the apparatus.
(The slightly different structures should be noted here
in Figs. 1 and 3, the latter having no capillary stopcock
C’, but being directly connected with the three-way
stopcock G. As the latter apparatus requires consider-
able experience in order to make it perfectly air-tight,
the former type only, as shown in the photograph, is

‘to 25 c.c. and is oravidel viith the me e
n and m for stimulation purposes, and also wi
glass stopper S or R, which can be sealed with mercury.
The air pump is connected through J and the aria z
hydroxide solution is introduced through V to . and | i r
where the drops are to be formed. om
How to set up the biometer—In order to get up this _
apparatus, the following materials will be necessary:
one biometer proper; two ordinary three-way stopcocks;
one nitrometer; one ordinary glass stopcock; one
water pump; one mercury burette, made of four or five
inches of any broken burette; one bottle with a side
neck at the bottom of about 300 c.c. capacity (aspirator
bottles); two large carboys; one capillary T-tube to be
bent to fit the biometer proper at Q and F; three
pinchcocks; one empty acid bottle for a half-saturated
solution of barium hydroxide; two CaCl, tubes or wash-
bottles to protect barium hydroxide, and one carboy;
one 1o-pound can of Greenbank alkali; 500 g. of C.P.
barium hydroxide; one yard of thick-walled pressure
tubing; one yard of good antimony tubing to fit the
ordinary glass tubing; a little sealing wax; 200 c.c. of
redistilled mercury; a few yards of glass tubing of or-
dinary sizes. :

Excepting for the biometer proper, most of the
materials mentioned above can be found in an ordinary
laboratory or can be substituted with homemade appa-
ratus without losing the accuracy of the method.

With these materials on hand, the apparatus can be
set up without any difficulty if one follows the figure very —

oe WwW AN

~ a> ke

‘= ] oe Iie «
iad

lias hae
he be ws igs to mount it per
Ay, in tead o} se eae it with several iron
d stands. ‘The apparatus set up as in Fig. 1
ind frame or in the wooden frame not only looks
a but is subject to less damage.
How to clean the apparatus.—The apparatus is con-
_ structed and is mounted in such a way that washing and
cleaning can be done after each experiment without
taking it apart. Although the procedure of washing
is exceedingly simple, it is better for the beginner to
follow exactly the directions given below, for there are
many stopcocks which have to be turned in a particular
direction in order to avoid unnecessary accidents which
sometimes necessitate the expenditure of considerable
time in bringing the apparatus back into a working con-
dition. An example will illustrate this: If one forgets
to turn the stopcock Z during the washing, and the
space between Z and K becomes wet, it will require
about five or six hours to wash the space and clean and
dry it. The old saying that an ounce of prevention is
worth a pound of cure should be borne in mind here.
Turn the water pump on. Now turn stopcock E
180° to the right, so that the barium hydroxide solution
is entirely out of connection with the other two arms of
the stopcock E. Remove mercury from stoppers S and
R with a pipette, and then remove the stoppers S and R,
and tissues if there are any. Turn stopcock Z in sucha
way that the bore inside will look like this L, thus
severing the connection between the vertical arm of L
and the horizontal arms. Turn on stopcocks J and
Q and F in order. Turn stopcock G go to the left.
Withdraw the mercury from chamber A by opening

| 5 Us a vada oer ra : ie

stopodck. Fill both cheaches A> ae By hy rie = |
-acidulated with nitric acid (not more than 1 per c -ent)
having both stopcocks open, then with distilled wate aul
times, then once with alcohol,and once with alcohol-ether.
Two funnels placed under these two chambers (see Fig.1)
are connected with a sink, to form an outlet for this —
waste water. ‘The alcohol and alcohol-ether drained out
of these chambers should be saved for re-use. Replace
the stoppers S and R. Let the machine remain un-
touched for drying while the suction pump is going and
while the tissue for an experiment is being prepared.
Five or ten minutes will be sufficient for complete drying
if the water pump is in good order and the alcohol used
has not contained too much water.

It is very important that we should leave the appa-
ratus in this condition until we are ready for an experi-
ment, and that no stopcock should be touched, for this
is the only condition under which all parts of the appara-
tus will dry.

How to obtain air free from carbon dioxide.—It is very
difficult to make air completely free from carbon dioxide
by the ordinary method, i.e., by merely passing it through
several alkaline bottles or alkaline towers. A simpler
and surer method is shown together with apparatus III in
Fig. 5 (p. 131). Itis prepared by shaking air with a 20 per
cent solution of sodium hydroxide in a tightly stoppered ©
carboy F, supplied with suitable tubes, one of which is
led to another carboy E, which is filled with about to
to 15 per cent alkali solution. When the air in carboy
F is to be used, it is driven into the nitrometer C (appa-
ratus III) or W (in case of the biometer), which is filled

THE BIOMETER: HOW TO USE IT 117

with a less concentrated alkaline solution (a weaker

solution is necessary so that the chamber may be filled

with air which is not too dry). Driving the air into
the nitrometer is accomplished either by increasing the
pressure in carboy F, by introducing more alkali from the
carboy above £, or by introducing more alkali through
funnel instead of from another carboy. After each
evacuation of the apparatus by a strong suction this
air free from carbon dioxide is introduced from the
nitrometer C or W into the chambers through the stop-
cock J. For ordinary experiments one can keep the
pressure in the carboy F high enough so that air free
from carbon dioxide can be driven into the nitrometer
simply by opening stopcock 9g after each evacuation.

How to test purity of aiy.—In order to test whether
or not the air in carboy F is free from carbon dioxide
the following experiment is necessary. It may be
stated that in all ordinary experiments we use exactly
the same manipulation as the one now to be described.
When the apparatus is perfectly dry, the pump being
at work, open stopcock (or pinchcock) 9 so that the
nitrometer is filled with the air freed of carbon dioxide
from carboy F. (If no bubbles come out by opening this
pinchcock it means that there is not enough pressure in
the carboy. In that case open pinchcock 8 to let more
alkali siphon down from the carboy above £, until
about 200 to 300 c.c. of air can be obtained by opening
pinchcock 9.)

Shut stopcocks C and C’ and J. If the pressure
pump is strong enough*and all the joints are tight,
chambers A and B should be under a strong negative
pressure by now, so that when you open stopcock C

the wercary in the little v essel can be. ‘0 the
mark, thus making the remaining wtledae ae ft
chamber exactly 15 c.c.1_ With a pipette fill the mercury
burette T with mercury to the mark, open stopcock. G a
go to the right, open stopcock C’ very gently till
mercury falls to the second mark in burette J, which is
so marked that by introducing this amount of mercury —
the remaining volume in this chamber B is now 15 c.c.
after the barium hydroxide is introduced to the top of
the barium hydroxide tube in left chamber A. (There-
fore, by introducing mercury to this mark, chamber
B has a capacity less than 15 c.c., but by introducing
barium hydroxide in the left chamber A, some of the
mercury will be pushed back, so that the capacity of the
right chamber B is now finally exactly 15 c.c.) Now
shut stopcock C’ (very important). With a pipette add
mercury to the mercury burette till the level of mercury
in it becomes a little lower than that of the mercury in
chamber B. Now pull out the core of stopcock J, so as

t The exact volume of each chamber should be calibrated once for
all. If this is done, one can always work with a constant volume in
both chambers, so that when a known amount in cubic centimeters of
mercury is introduced so as to bring it up to the marks in the chambers
the remaining volume will always be the same. The advantage in
having both chambers equal in capacity is obvious. One of our appa-
ratuses has a capacity of 21.5 c.c. in chamber A and 22 c.c. in chamber B.
We therefore introduced 6.5 c.c. into A and marked the level of the
mercury, and 7 c.c. into B and did likewise. Thus when mercury is
introduced up-to the marks, both chambers have the same remaining
volume, namely, exactly 15 c.c. A little error in calibrating the chamber
A is not very serious, as this chamber is used for the analytic purpose
only, while the biometer is to be used as a quantitative apparatus; but
the chamber B must be calibrated with extreme care, and each intro-
duction of the known amount of the mercury must be done accurately.
This can be accomplished by means of the mercury burette T, which is
well calibrated and can measure off any known amount of mercury with
a high degree of accuracy.

es ee ee ee eee

sat

THE BIOMETER: HOW TO USE IT 119

to admit air into the apparatus, and shut it. (Remove
_ stoppers S and R; introduce a tissue to the right cham-
ber B if performing an actual experiment, and replace
the stoppers.) Seal the stoppers S and R with mercury.
Turn stopcock L 180° to the right, so that three arms are
now in communication. Shut stopcock J and open
I very carefully and shut 7. (It should never be
opened unless the nitrometer contains more than
40 c.c. of air and stopcock J is shut.) Open J and
shut J; open J and shut J. In this way we evacuate
the chambers by opening J, and fill them up with
pure air by opening J. This process of washing the
apparatus with air freed from carbon dioxide is repeated
at least five times. At the end of the last washing,
having stopcock J shut and J opened, shut stopcocks Q
and F. Without touching stopcock J open stopcock J
and raise the safety bottle D,so that the pressure inside of
the apparatus is now equal to that of the atmosphere, and
then shut J. Open stopcock C’; the mercury in the
burette ZT should not move’if the previous pressure
adjustment with the safety bottle D and nitrometer is
properly done. Shut the stopcock J so as to cut off
suction; turn stopcock £ to right go°, so that the space
between J and £ will be filled with barium hydroxide;
turn it 90° more to the right, so as to fill all the capillary
T-tube below Q and F with the clear solution of barium
hydroxide. Open stopcock Q very gently until a hemi-
spherical drop of half-saturated barium hydroxide is
formed at d. Then shut Q and make a similar drop at /
in the other chamber. Turn stopcock L 45°, so that the
connection between the two chambers is now severed.
Shut stopcock C’. If the air is completely free from a

cits should be pis ee ans at
introduction of the drop at the beinnings A > aft
standing for several hours, having not a single granul
of the precipitate visible with a lens. Tan

Since the main point of accuracy in our appé
depends on having the air free from carbon dioxide
indeed, it is the most difficult part of the manipulation —
of the biometer to have good air—particular care must —

be taken to have every point of junction perfectly air- — 4

tight. The points most susceptible to leaking will be the
stopcocks and the mouth of the carboy where the air is
preserved. A strong suction is essential for a complete
washing of the apparatus with the air free of carbon
dioxide.

Methods for the qualitative detection of carbon dioxide
production in the tissue.—After the apparatus is cleaned
and dried and the air is ascertained to be pure for use,
a prepared tissue is placed on a cover-slide or the glass
plate shown in Fig. 2 (p. 38) and introduced into the
chamber B, no tissue being put in the left chamber A.
The detailed method is exactly the same as the one
just described. After both chambers are closed with the
stoppers S and R and sealed with mercury, the apparatus ©
is washed with the air free of carbon dioxide in the usual
manner. Barium hydroxide solutions are introduced
into d and f, forming hemispherical drops, and the con-
nection between the two chambers is severed by turning
the stopcock between them (Z); then watch the drop
with a lens. If the air is free from carbon dioxide, the
drop in the left chamber ought to be perfectly clear,
while the drop in the right chamber, if the tissue gives

THE BIOMETER: HOW TO USE IT 121

- off carbon dioxide, will not be coated with the precipitate,

but will have on its surface some crystals of barium car-
bonate, which becomes more heavily precipitated as the
respiration goes on. By repeating the same experi-
ments after interchanging the chambers, using the left
for the tissue and the right for a blank, it will be possible
to eliminate any possible error which might come from
some technical fallacy characteristic of one particular
chamber. For a casual observer the initial granule
will not be distinct from a granular spot on the glass.
The granules, however, will soon increase over the surface
of the drop and will gradually collect downward at the
edge of junction of the drop with the glass tubing.
The thick band of white precipitate around the bottom of

_ the drop will gradually extend toward the top of the

drop, so that after the band reaches more than half of the
hemispherical drop of barium hydroxide one can see
with the naked eye, not only from the side, but also
from above, the whole drop, now resembling a contract-
ing iris. When the very top of the drop is filled with the
precipitate, the whole drop of barium hydroxide will
look very opaque, covered with a thin layer of the car-
bonate. If one take a small piece of sciatic nerve of a
frog, say about 20 mg., he can see these different stages
of precipitation very distinctly, but when the amount
of tissue taken is very large it is very difficult to observe
these phenomena on account of the too rapid formation

of the precipitate all over the surface of the drop. It

is therefore best to take a very small piece of the nerve
for the purpose of following these different stages of the
precipitation of carbon dioxide as carbonate, for the
practice of distinguishing these different stages is very

+ al for ek rative estimate of the
samples of tissue. isc f ta
_ Methods for a quick sempafative estimate of ca
dioxide production from two different samples of tissu
By repeating quantitative experiments it was found th that
_ the speed with which the first precipitate appears, thet
sizes of the precipitates, and the shapes of the aggrega- —
tion of the deposits at different stages represent different
quantities of carbon dioxide, if compared simultaneously _
under the same conditions. Thus, with this remarkably —
simple means we can determine quickly the comparative
output of carbon dioxide from two different tissues at
the same time. The method of procedure is best illus-
trated by the following example: .
Two pieces of the sciatic nerve are isolated from
the same frog and weighed into approximately the same
mass. One piece is laid on one glass plate and the other
on the other plate in such a way that one part of the nerve
lies across the electrodes of the glass plates as shown in
Fig. 2 (p. 38). In this way, when the plates are hung on
the electrodes 1 and m either nerve desired can be stimu-
lated with the induction current. These plates are now
hung on the electrodes in each chamber, and the usual
procedure is followed for eliminating carbon dioxide from
the apparatus. After the connection between the two.
chambers is closed by means of stopcock L, having the
drops of barium hydroxide in each chamber as usual,
the nerve in chamber A is stimulated by the current.
Then if one watches over the surface of the drops care-
fully from the start, the deposit of carbonate will be
seen to appear first on the drop in chamber A, in which

THE BIOMETER: HOW TO USE IT 123

the stimulated nerve is placed. Later the total amount
of the precipitate grows much larger on the drop in this
chamber. ‘This clearly shows that the chamber in which
the larger amount of the precipitate is found must have
the higher concentration of carbon dioxide. Since we
had exactly the same kind of air at the beginning, the
conclusion is that the nerve when stimulated must give
off more carbon dioxide than the resting one. This
conclusion can easily be confirmed by exchanging the
nerve in the chambers as usual.

The following figures will illustrate the different stages
of the granulation of barium carbonate and will show

AY -
oS
+ Gis
I 2 3 4 f 5a 6 6a
Fic. 4.—Different stages of the granulation of barium carbonate-on
the surface of the hemispherical drop of barium hydroxide; 5a and 6a

show the top views of the drops at the time when “‘iris effect” is pro-
duced.

definitely how easy it is to compare the amount of carbon
dioxide production at several points. And such com-
parison can be confirmed more exactly by the quantita-
tive determination; the details of the method are given in
the next paragraph. |

The method for quantitative measurement of carbon
dioxide.—While the apparatus is drying, prepare the
tissue and weigh it. If everything is perfectly dry, fill
both chambers with mercury up to the marks, as directed
on p. 118. Remove the stopper from the right chamber
only, which is to be used as a respiratory chamber,
and the tissue is to be left in this; the other chamber

ae -

is for the purpose of -qdeheie toe nirintdot a i Li
stopper need not be removed. The tissue i ref -efully
laid on the glass plate and on the lation el slectroc _
fused into the chamber, or it can be laid on thee
slide and placed on the mercury. Close the =bappae 7 3
and seal both chambers with mercury. Wash the
apparatus with air free of carbon dioxide, as directed
before. At the end of the sixth or seventh washing
stopcocks G and F are closed and the time is recorded,
since it is plain that from this time on we are retaining
any gas given off by the tissue in the chamber. The
apparatus is filled once more with air free of carbon
dioxide by opening stopcock J; the pressure is quickly
adjusted by raising the safety bottle D, while the stop-
cock J is still open, and then J is shut. After opening
stopcock C’, barium hydroxide is introduced into the
tube d of the left chamber A only, but the solution is ©
never introduced into the respiratory chamber B. Turn
the stopcock L in such a way as to sever the connection
between these two chambers. It is imperative, not only -
that the hemispherical drop formed at d in the left
chamber should be perfectly clear at the time of intro-
duction of this solution, but also that no visible granule
of any kind should be produced on standing. No
quantitative experiment can be performed unless the air |
is absolutely free from carbon dioxide. We have thus
a control for each quantitative experiment. If at the
end of the desired period of respiration, say ten minutes,
the drop is perfectly clear, not having any deposit visible
with a lens, a portion of the gas from the respiratory
chamber B is introduced into the left chamber. This is
accomplished by drawing a designated amount of mer-

aber A Gy peed ta stopcock C
l ig the same eiriouitt of | mercury to the mer-
burette T, opening the stopcock L, and quickly
x the stopcock, so that the communication of

por paw een between these chambers is momentary. This
_ process of driving the known amount of the gas from the
respiratory chamber to the analytic chamber must be

done in a few seconds. The volume of mercury with-

drawn from the analytic chamber is easily determined

by drawing it into a small graduated cylinder, or, more
accurately, by weighing it, and this volume corresponds
to the exact amount of the gas we took from the right
chamber to the left, since the pressures in A and B are

_ kept exactly equal to atmospheric pressure during the

transfer of the gas.

One now watches the surface of the drop at d with a
lens to see whether or not any deposit is formed during
ten minutes. The presence or absence of any visible
precipitate will decide whether the amount of gas
taken from the respiratory chamber contained enough |
carbon dioxide to give a visible deposit. With this
apparatus we have repeatedly introduced accurately
known quantities of carbon dioxide of very high dilution
into the left chamber and found with remarkable regu-
larity that 1.0X1077 g. of carbon dioxide is the mini-
mum amount which will cause a formation of detectible
precipitate of barium carbonate during ten minutes.
Smaller amounts of the gas than this will give no pre-
cipitate for a long time, while larger amounts give it more
quickly and it appears in larger quantities. ‘There is a
sharp line of demarkation at 1.0107’ g., no matter how
large a space this amount of gas is occupying with the air. ©

many cubic centimeters a iene . Sab
before we can obtain the precipitate in ten 1 m: mate |
this volume must contain: then 1.0X107~7 g. inne we
know the volume of the original respiratory chambe

from which this known amount of gas is withdrawal ag

we can easily determine how much total carbon dioxide —

is present at the time of analysis. That is to say, the — ;

original capacity of chamber B divided by this mini-
mum quantity of the gas which gave the precipitate, —
multiplied by 1.0X10~’ g., corresponds to the total
amount of the carbon dioxide given by the known weight
of the tissue for the known period of time.

The following example will make the details of the
method and calculation clear: _ |

We took 10 mg. of the sciatic nerve of a frog and
after ten minutes of respiration we drew 1 c.c. from the
respiratory chamber into the left chamber, and found no
precipitate visible within ten to fifteen minutes. Instead
of now taking more gas from the respiratory chamber, we
should take another fresh nerve, and, after it has respired
ten minutes or longer, draw, say, 1.5 c.c. to the analytic
chamber. As will be noticed, we have three variables
which we can choose from, namely, the weight of the
nerve, the time of respiration, or the amount of gas
withdrawn from the respiratory chamber at the time of
analysis. To estimate the carbon dioxide production
from the isolated tissues it is far better to keep the time
constant and vary the other two, for in many cases the
rate of respiration varies as the time elapses. As far
as the weight of the tissue is concerned, we cannot but

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t only aw Pat teak as fey bo eet
8 of the tissue for each experi-
- in many cases such an attempt will lead to
a numb Por physiological errors. Of course there is a
time when we must select the same weights of the tissues
_ for a particular experiment, such, for instance, as when
we are to test the relation of the sizes of the tissue and
rate of the carbon dioxide production.
The quantitative experiments, therefore, consist in
- determining the least volume of the gas necessary to give
the precipitate for a known weight of the tissue for a
known period of time. This can be found by experi-
menting on several tissues of different weights (too much
variation of the weight should be avoided), ie., by
obtaining two sets of results, namely, the one which does
not give the precipitate and the other which gives the
: precipitate.
| ‘These results are calculated on the standard unit, so
; that we can compare them with each other. We have
usually taken 10 mg. and ten minutes as units. An
example will explain: 14 mg. of the nerve for fifteen
minutes of respiration did not give a precipitate when
we took but 1 c.c. from the respiratory chamber. There-
fore this nerve for 1o mg. for ten minutes’ respiration
will not give any precipitate when we take 2.1 c.c. from
the chamber. In another case we took 13 mg. of the
nerve, which after ten minutes’ respiration produced so
much carbon dioxide that 2 c.c. gave a precipitate;
thus 2.6 c.c. will give precipitate for 10 mg. of the nerve
for ten minutes’ respiration. In this way a series of
experiments with several fresh nerves was conducted in
order to approximate both the minimum volume which

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THE BIOMETER: HOW TO USE IT 129

will precipitate and the maximum volume which does
not give a precipitate for a definite time and weight. In
Table I columns 8 and 9 refer to these columns calculated
from the experiments for ten minutes’ respiration by
10 mg. of the nerve. Since we know that the minimum
volume which gave a precipitate must contain a definite
amount of carbon dioxide, i.e., 1.01077 g., and since
we had 15 c.c. of original volume of the respiratory cham-
ber, we can calculate the total amount of the gas given
off by the sciatic nerve of the frog.

APPARATUS III

Although the use of the biometer is perfectly satis-
factory for almost all micro-metabolic analyses, and
sometimes is indispensable for a quick quantitative com-
parison of two different rates of carbon dioxide production
from the different tissues, yet it is extremely inconvenient
for a complete determination of carbon dioxide pro-
duction from a single tissue, the metabolic rate of which
is constantly changing and the availability of which is
not very great. The necessity for a new device to meet
this difficulty was keenly felt when we were studying the
metabolic changes before, during, and after the cleavage
of a single fish egg.

The new feature of this special apparatus is a device
by which the air after a definite period of respiration by
the tissue can be withdrawn into a tube from the respira-
tory chamber for subsequent complete analysis. With
the new arrangement, therefore, one can make not only a
complete analysis with a single tissue, but also several
duplicate determinations.

ery abit analytical chatabers of “the | bior mi
- capacity is about 30 to 4o c.c., but can become smé
by introducing mercury in the same way as we mana

in the other apparatus. The barium hydroxide tube a

is —

is inserted through its wall, and the three-way stopcock . 455

is attached to the bottom of the chamber. Just opposite

the top of the barium hydroxide tube d there is another "4

three-way stopcock 2, one arm of which is connected to
the nitrometer C and the other arm of which is con-
nected to tube B, into which the respired air is to be
drawn for a subsequent analysis. This tube B is

attached to a mercury burette G, by which the pressure ‘:

in the tube and the chamber can be adjusted. The

similar mercury burette H is attached to the chamber |

proper for the same purpose as well as for the means of
driving air into the tube B. The remaining parts of the
apparatus are exactly the same as in the biometer and
are shown in the figure with dimensions.

Method for quantitative determination of carbon dioxide
with apparatus III.—The detailed method is as follows:
Open stopcocks 2 and 3 in such a way as to connect the
chamber A and the tube B only. Fill the tube B with
mercury by raising the mercury burette G. Close stop-
cock 2 when a little excess of mercury is pushed over into
the space in the capillary tube between the chamber A
and the tube B and when the tube B is known to be
absolutely free from any bubble of air. The closing of
the stopcock must be done in such a way that there
is a connection made between the chamber A and

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THE BIOMETER: HOW TO USE IT

nae che. Giallo ing the aly Dit Db mn open
stopcock I. Tn this" way the tcoor of is

in the capillary tube will be pushed over into the |

chamber A and flow through stopcock 4 into a receiv- i:
ing vessel. %

If stopcocks 2 and 3 are absolutely air-tight, there
should be no air bubble present in the tube B on standing.
This being assured, a known amount of mercury is intro- |
duced into the chamber A by means of mercury burette
H, thus making the capacity of the chamber what was
desired. Tissue is introduced into the chamber in the
usual manner, the glass stopper is replaced, the chamber
is sealed with mercury, and the nitrometer is filled with ©
air free from carbon dioxide. After evacuation of the
chamber and introducing pure air several times, stop-
cock 5 is closed and the time is recorded, the pressure is
adjusted, and stopcock 2 is turned 45°. At the end of
the desired period of respiration any portion of the air,
say 10 or 15 c.c., from the chamber can be driven into
tube B. This is accomplished by raising the right-hand
mercury burette H and by simultaneously opening
stopcocks 2 and 4 and gradually lowering the left-hand
‘mercury burette G. Stopcock 2 is now closed and the
pressure of the air in B is made equal to that of the
atmosphere and is kept under this condition, having
the mercury burette G at the proper height.

Remove mercury from the stopper S and unstop the
chamber, take away the tissue, and lower the mercury
burette H so that all the mercury in the chamber will
flow back into the burette. The little excess of mercury

‘THE BIOMETER: HOW TO USE IT 133

now left in the chamber A can be withdrawn through

‘stopcock 4 into a receiving vessel. In order now to
analyze the air in the tube B, it is better to clean the
apparatus once more with water and dry it, as directed
elsewhere.

The chamber is now filled with mercury so that the
remaining volume of it will be as little as possible, say
15 c.c. (the exact volume need not be known here),
the apparatus is sealed with mercury as usual, and then
washed several times with air free of carbon dioxide, and
then clear barium hydroxide is introduced into the usual
tube inside of the chamber, forming a hemispherical
drop at the top of d. If no deposit of barium carbonate
forms on the surface of the drop within ten or fifteen
minutes, we are sure that ordinarily the air we use is
free from carbon dioxide and that the apparatus is in
perfect condition. This point. established, a small
portion of the gas is driven from the tube B into this .
chamber A. This is done by withdrawing a desired
amount of mercury from the chamber A into a receiving
cylinder and adjusting the pressure in the chamber and
tube B by means of mercury burette G. Close stopcock
2 by turning it 45°.

The surface of the drop at d should now be watched
with a lens, as usual, for a deposit of carbonate. If no
deposit appears within ten minutes, we should introduce
more air from the tube, with usual care, until we get the
first visible precipitate detectible with a lens during ten
minutes’ standing. It is very important that we should
give about ten minutes,of time for the reaction after
each withdrawal of the air from the tube B into the
chamber A.

ing the ey, volume. pai BB. Oe
alivans of the tissue are used and are allow
respire in the chamber for ten minutes. Then ab |
1o to 15 c.c. of the gas are withdrawn into the —
tube B; 0.5 c.c. of this gas gave no precipitate
during the first ten minutes; 0.5 c.c. more of the e 4
same sample gave no deposit in another interval of | :
ten minutes. Thereupon o.5 c.c. more, a total of 7
I.5 C.c., was run into the chamber. A marked evi- —
dence af a precipitate appeared in ten minutes. There-
fore 1.5 c.c. of this gas must contain 1.0X107’ g. of
carbon dioxide.

The apparatus is then cleaned and dried and a clear
drop of barium hydroxide is again introduced upon the
top of the tube d; and after making sure that the airis
free from any carbon dioxide by waiting, 1 c.c. of the
sample gas which has been left undisturbed in the tubeB
is introduced into the chamber; no precipitate will be
found to have formed within ten minutes; 0.25 c.c.
more of the sample will not produce any precipitate;
but if 0.25 c.c. more is taken, crystals of barium car-
bonate appear after ten minutes. Itfollowsthat1.5c.c. ~
of the respired gas must contain 1.0X 107’ g. of carbon |
dioxide. a

From these duplicates it becomes certain that 1.5 c.c.
of 25 c.c. capacity of the chamber now contain 1.0X
‘107? g. of carbon dioxide. Therefore the total amount

“

| order to test the accuracy with which our new
method can be used for the estimation of the exceedingly
minute quantities of the carbon dioxide, a series of
determinations was made on the samples whose con-
centrations were unknown to the experimenters at the
time of analysis.
_ The results are given in Table IT:

TABLE II
VoLumE or SAMPLE WEIGHT oF CO: IN I C.c.
F, REQUIRED TO GIVE A
= PRECIPITATE Found. > Taken
COP A oe oe ee te 1.0 X10~7g.| 0.92X10~ 7g.
Oi CRE LS fia exe ces 2.0 X1o~7g.| 2.3: X10 7g.
ROT aa vinta cna 1.82X10~ 7 g.| 1.83 X10 7g.
£8 PCB es cueaaee ded 0.67 X10 7 g.| 0.62X10~7 g.
CON Cinescore cosets 0.45 X10 7g.| 0.45 X107 7g.

One disadvantage of this apparatus III is that we
must take into consideration temperature and pressure
variation, which was entirely unnecessary for the
biometer proper. If the respiration and analysis are
carried out at different temperature and _ pressure,
the ratio between the minimum volume which gives
the first precipitate and the original volume of the
chamber will not be rigid. In that case the minimum
volume should be translated to the volume at the
temperature and pressure at the time of respiration.
Such correction, however, will not be necessary if the

of the chamber, aiid sis 6 mmf pen Con

and 730 mm. of pressure gave the first precipitate. 1 We

shall then obtain the following results: “4 oe:
a) Without correction we get Bo

I.OX10-7 gx 16.6X 107 g.

b) With a correction,

ae 7 25 wicca a
I.OX 10 8-5 5x (ayo 38) 790 17.6X10-7g.

(270+22) X 760

This shows a little over 5 per cent error, which will be
the maximum and almost an impossible variation, con-
sidering the ordinary weather in the laboratory for a
short interval of time. Besides, we are dealing with a.
very small sample of moist tissue, the weight of which
may easily vary within 5 per cent.

- ‘e ‘a * hte, pure, 23.

Algae, ‘red, 92.
_ Alkali, Greenbank, 114.
Anaérobic tissue, 15.

_ Anesthesia: partial, on local exci-
tability, 58; on conductivity,
58. See also Carbon dioxide
production.

Anesthetics, 25, 61; on refractory
period, 48. On carbon dioxide
production see Carbon dioxide.
See also Ether, Urethane,
-Chloral hydrate,

Animal heat: discovery of nature
of, 11; source of, 11, 51.

Apparatus, for carbon dioxide
determination. See Biometer;
Apparatus ITI.

_ Apparatus III, 129; description,

130; diagram, 131; method,
130.

Arachnid, 80.

Arbacia, 41.

Asphyxiation: on refractory

period, 48; as cause of anes-
thesia, 60.
Astacus, 30.

Atmospheric oxygen. See Oxygen.

Automatic ganglion. See Gan-
glion. |
Axis cylinder, 50, 51. _ °

Bacterial decomposition, 27, 28.

Banana fly, 110.

137

y sprouts fe a eggs, 41; . Barium carbonate: detection of,

111; precipitation of, 16, 19;
solubility of, 15; stages of
granulation of, diagrams, 123.

Barium hydroxide, 15, 19, 20, 89,
94, 110, 114,115, 118, 121, 130,
133.

Bayliss, 29, 33, 45, note 1, 18.

Bicycle, compared to life-process,
107.

Biometer, 6, 15, 17, 19; accuracy
of, 135; bubbles in, 123; cal-
culating results, 127, 128; cali-
brating volume ‘of, 118; carbon
dioxide, free air for, 116; clean-
ing of, 115; description of, III;
diagram of, 113; photograph of,
17, 112; principles of, 110;
qualitative use of, 120; quanti-
tative use of, 123; quick quanti-
tative comparison with, 122;
sensitiveness of, 111; setting
up, 114; testing purity of air
in, 117; uses of, 109.

Blaze current, 5, 88, tor. See
also Electrical signs ‘of life.

Blood supply, 16.

Brain, 16, 50; increased metab-
olism i in, 36; ring, 76.

Brown, Horace, 87.

Burch, 47.
Cancer, pagurus. See Carbon
dioxide production.

Carbohydrate, 10.

Carbon dioxide production: by
life process, 22; comparison of,
in nerve and other tissues, 29;
in different animals: crabs,
cancer pagurus, 30; crayfish,
astacus, 30; Crustacea, 30; dog,

3 ? ?

30; lobster, Homarus vulgaris,

30; man at rest, 30; in differ-
ent parts of nerve, 76, 77; in
hydrogen, 26, 43; in “‘inexci-

table” nerves, 65; in killed

nerves, 24; in nerves: carp,
R. lat. vag., and R. lat. acc., 79;
catfish, R. lat. vag., and R. lat.
acc., 79; chloral hydrate, 65;
dog, anterior root, posterior
root, hypoglossal, 79; frog,
Rana pipiens, sciatic, vesting,
and stimulated, 32; guinea
pig, 22; hypoglossal, 79; “in-
excitable,” 62; Limulus, claw
nerve, 32; mouse, 22; optic
nerve (whole) proximal and dis-
tal, 32; rabbit, 22; rat, 22;
6 skate, Raia erinecia, and
Raia ocallata, optical, olfactory,
oculomotor, 22; spider crab,
Libinia canaliculata, claw nerve,
whole, proximal, distal, 32;
squiteague, cynoscion regalis,
22; stimulated, non-stimulated,
under treatment of different
concentrations of ethyl ure-
thane, 62; turtle, 22; under
anesthesia, 25; in resting nerves,
19, 22, 32; in stimulated nerves,
at successive time intervals, 65;
under anesthesia, 25, 61.

Carbon dioxide: as a measure of
metabolism, 12; as end product
of metabolism, 11, 34; gradient,
79; increment of, on stimula-
tion as sign of life, 87, chap. v;
influence on electrical change,
14; method of analysis of, see
Biometer; method of detecting,
in nerve, 16, 20; sources of,
23.

Carbon dioxide free air, 116.

Carbonate, 23.

Carp. See Carbon dioxide pro-
duction.

cal processes,
the living matter, ro.

Chemical sign of life, algae, 93; ia 2
Australian pine, 92; in common ae

glass, 92; in corn, 91; ir
Japanese ivy, 92; in Lincoln oats,
91; in mustard seeds, 91; in
nerve, 34, 55; Mm fice, 91;
in Swedish selected oats, 91; in
wheat, 89.

Chemical stimulation. See Stim-
ulation.

Chemical transformation, 12.

Child, 74, 81. ;

Chloral hydrate, 61. See also
Carbon dioxide production

Chloroform, 61.

Chlorophyll, 2.

Claude Barnard, 48.

Claw nerve. See Nerve.

Cold-blooded animals, 22.

Conducting medium, 18.

Conduction: as phenomenon of
living matter 4; also chap. iv.

Conductivity, 6. See also Con-
duction.

Connective tissue, 16, 18, 46.

Contractility, 8.

Crab. See Cancer pagurus.

Crayfish. See Astacus.

Crocker, 92.

Crustacea. See Carbon dioxide
production.

Current of action. See Action
current.

Cyanide. See Potassium cyanide.

Cynosion regalis. See Carbon di-
oxide production.

Daniel cell, 52.
Death, rapidity of, 73.

Dendrite, 77; metabolic gradient

in sensory, 78.

Dextrose, 82.

Diphasic current, 13. )

Dog, metabolism in. See Carbon
dioxide production.

Drugs, on refractory period, 48.

Dry seed. See Seed.

Dyer, Thistleton, 87.

Efferent fiber, 58; gradient in,
see Metabolic gradient.

Eggs, production of acid in, 41.

Ehrlich, 37. |;

Electrical changes: as functional

_ change, 4; discovery of, 4;
as sign of passage of nerve im-
pulse, 23; influence of carbon
dioxide on, 13; See also Blaze
current; Electrical sign of life.

Electrical current, 37.

Electrical resistance, changes in,
after stimulation, rot.

‘Electrical sign of life. See Blaze
current; Electrical resistance.

Electricity: measure of, 3; ve-
locity of, 8.

Electrodes, 12, 13, 40.

Electromotive force, in nerve, 35.

Electropositive, 58.

Energy, source of, in living matter,
Q, II.

Ether, 25, 61; for effect of, on
carbon dioxide production, see
Carbon dioxide production.

Ethyl urethane, 25, 63. See also
Carbon dioxide production.

Excitability: degree of, 59; de-
pression of, see Anesthetics;
its relation to conductivity, 57,
chap. iv; its relation to metab-

olism, 59; three criteria of,
59; transmission of, 41.° Seealso
Irritability.

Faraday, 3

Fat, 10.

139

Fatigue: as metabolic sign, 40;
lack of, in nerve, 8, 46.

' Fermentation, 10, 23, 25, 27.

Film, of barium hydroxide, 15, 16.
Fletcher, 30, 41.
Forgetting, phenomenon of, 35.

Frog. See Carbon dioxide pro-.
duction.

Frodhlich, 45.

Functional changes, 4; in the
nerve impulse, 7; invisible, 4;
See also Electrical changes;
Chemical changes.

Fundulus hectroclitus, 110.

Galen, 11.

Galvani, 5, 13.

Galvanometer, 3, 5, 13, 23.

Ganglion, 31; comparison with
metabolism of nerve fiber, 31;
heart ganglion, 32. See also
Carbon dioxide production.

Glass plate, 20, 37; diagram, 38.
Gotch and Burch, 47.

Gradient: metabolic, 79; in
afferent fiber, 76; in efferent
fiber, 72; in sensory dendrite,
77; Yelation of, to direction of
impulse, 78, 80; structural, 75.

Green pigment, 2.
Growth, 10, 35.

Guinea-pig. See Carbon dioxide
production.

Haberlandt, 54.
Heart, 31.
Heat coefficient, 52.

Heat formation: in brain, 36; in
nerve 7, 49; its relation to
metabolism, 5.

Helmholtz, 50.
Herrick, 71.
Hill, 30; on heat formation, 50.

Homarus vulgaris. See Carbon
dioxide production.

Hopkins, 41.

beth nerve. See Nerve.

Impulse: in plant, 2; nerve, see
Nerve impulse.

Inhibition: by heat, 58; non-
transmissibility of, 58.

Injury, 41, 91.

Insect, wings of, 47.

Invertebrate, 18.

Irritable response, 103.

Irritability: definition of, 4;
metabolic condition for, 85;
nature of, 104; origin of physi-
cal theory of, 9; relation of, to
conductivity, 57; relation of, to
metabolism, 104; two phases in
protoplasmic, 57.

Katabolism, 48.

King crab. See Limulus poly-
phemus.

Lateral line nerve. See Nave.

Lavoisier, 11.

Learning, phenomenon of, 35.

Lens, 15, 16.

Libinia canaliculata. See Carbon
dioxide production. _

Life: chemical sign of, chap. v;
definition of, 95; quantity of,
107.

Light, 35.

Limax, 22.

Limulus. See Limulus polyphemus.

Limulus polyphemus. See Carbon
dioxide production.
Living process, 103; material

changes in, 3; physical state of,
105.

Living things: property of, 3; two
signs of, 4. _

Lobster. See Homarus vulgaris.

‘

Medullated nerve, 8, 21. 5

_ Memory, 35.

also Carbon dioxide p

oduction.

Metabolic gradient. See Gna
dient. .

Metabolism: function of, 85, ‘ot :

increased, on stimulation, 34; ys y

in different tissues and organ-
isms, see Carbon dioxide pro-
duction; indirect evidence for,
in nerve, 12; its meaning 5°,
104; method of study of, in
nerve, see Biometer; relation to
behavior, 81; resting, 33, 80.

Methylene blue, 37.

Motor nerve. See Nerve.

Mouse, 22.

Muscle: acid production in, 91; __
contraction of, as sign of nerve
impulse, 23; increased metab-
olism of me st 353
smooth, 51; voluntary, 18.

Narcosis. See Anesthesia.

Narcotics. See Anesthetics.

Negative phase, 13.

Negative response, influence of
carbon dioxide, 14.

Nerve: afferent, 58; claw, 18;
efferent, 58; ‘hypoglossal, 793
lateral line, 22; motor, 21;
oculomotor, 21; olfactory, 21;
optic, 22; sciatic, az, 22, 67%
sensory, 22, 583 nerves of
different animals. See also
Carbon dioxide production.

Nerve fibers: Changes of weight
under anesthesia, 62, 65; chem-
ical change in, chaps. ii and iii;
chemical sign of, 55; different

ited, 24; met

g ient in, 72; metabol-

1 on stimulation, 55; oxygen

’ ion by, 53; physical

. in, 7; production of

» acid cid in, 25; property of, 6;

| ' ae resting metabolism in, 22;

| structural changes, 7; — struc-

' ~——s tural gradient in, 75; use of

, @ De ioatel, 23.

_ Nerve impulse, 6; direction of,

| 72; direction of, and metab-

olic gradient, 77; effect of

salt on, 82; effect of tempera-

ture on, 83; nature of, 85;

4 nature of material basis of, 107;

' ~—-_s velocity of, 8; velocity of, and
| resting metabolism, 80.

__ Nitrogen, electrical response in,
-@ 45.

i Non-medullated nerve, 18.
Nucleus, cell, on oxidation, 16.

Oculomotor nerve. See Nerve.
Olfactory nerve. See Nerve.
B: Optic nerve. See Nerve.
4a Organic compounds, 22.

) Osterhout, 92.

Oxidation: measure of, see Metab-
olism; rdle of nucleus on, 16.

Oxygen: as essential to life, 11;
atmospheric, 15; consumption
by brain, 36; consumption by
linseed oil, 35; consumption by
nerve fibers, 53; consumption
by stimulated nerve, 50, 53;

_ consumption under anesthesia,
60; deficiency of, on nerve me-
tabolism, 27; discovery of, 11;
réle of, on excitability, 54, 60;
réle of, in salt stimulation, 26."

Painting, chemical process in, 34.
Peas, 31.

response in, 2. See eon Seed. .
Pneumatic spirit, 11. .
Positive phase, 13.

Positive response, influence of
carbon dioxide on, 14.

Potassium chloride, 42.

Potassium cyanide, 75.

Priestly, 11.

Propagation, of excitability.
Nerve impulse.

Proteins, ro.

Protoplasm, 48; physical condi-
tion in, 8.

Protoplasmic respiration, 35.

Psychic change, 3.

Psychic life, evolution of, 2; in
child, 2; in seed, 3; physical
basis of, 3.

See

Psychic process, physical process,

accompanying, 4.
Psychism, 3; indirect measure of,
6.

Psychometer, 3.

Rabbit. See Carbon dioxide pro-
duction.

Raia erinecia. See Carbon diox-
ide production.

Raia ocallata. See Carbon dioxide
production.

Ramus lateralis accessorius. See
Nerve.
Ramus lateralis vagis. See Nerve.

Rana esculenta. See Carbon di-
oxide production.

Rana pipiens. See Carbon dioxide
production.
Rana temporaria. See Carbon

dioxide production.

Rat. See Carbon dioxide pro-
duction.

study, 15.

wait metabolism. sige metab-

Riggs, 42.
Ringer’s solution, 28.
Rolleston, 50.

— permeability of, to nerve,
8; effects on nerve impulse, 82:

Ringer’s solution, 28; ‘sodium,
on metabolism, 43.

Sciatic nerve. See Nerve.

Secretion, 8.

Seeds: blaze current in, 5, 88;
chemical signs in, 88; dete-
rioration of, 87; effect of
anesthetics on, 91; electrical
sign in, 5, 88; increased metab-
olism of, on injury, 90;
invisible response in, 4; metab-
olism in killed, 91; old Egyp-
tian, 88; psychic life in, 3;
vitality of, at low temperature,
87.

Sensory dendrite. See Dendrite.

Sheath. See Medullary sheath.

Sisyphus, 84.

Skate. See Raia.

Smooth muscle. See Muscle.

Snyder, 51.

Sodium chloride, 42.

Spider Crab. See Carbon dioxide
production.

Squiteague. See Carbon dioxide
production.

| Seaaiaey metabolism, how to

Temperature: effect on

Tait, 48.
Turtle, 22.

effect on ne

impulse, 82;
metabolism, 83.
Thorner, 45.
Transmission, of excitability. See
Nerve impulse.
Twelfth dynasty, 88. -

Universe, 2
Unstable equilibrium, 105.
Urethane. See Ethyl urethane.

Velocity: its relation to resting
metabolism, 80; of the nerve
impulse, different salt concen
tration on, 82; of various crus-
tacean nerves, 81; temperature
on, 83.

Vertebrates.
production.

Viscera, nerve to, 18.
Voluntary muscle. See Muscle.

Waller, 11; metabolism of nerve,
12; on blaze current, 5; on
changes of electrical resistance,
ror; on longevity of nerve, 24;
on protoveratrin, 48; on stair-

See Carbon dioxide

case, 13.
Water, as end product of metab-
olism, tI: .

Weston cell, 52.

Yohimbin, on refractory period,
48.

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