256
ON THE CONTROL OF THE LEVEL OF ACTIVITY
OF THE ANIMAL BODY
I. THE ENDOCRINE CONTROL OF SEASONAL VARIATIONS
OF ACTIVITY IN THE FROG
BY G. S. CARTER.
(From the Sub-department of Experimental Zoology of the
University of Cambridge.)
{Received 20th December, 1932.)
(With Eight Text-figures.)
HIBERNATION and the other seasonal variations of activity in the vertebrate have been
. the subject of a very large amount of investigation and of an even larger literature.
This has been recently summarised by Pocock (1926) from the standpoint of general
biology, and by Gorer (1931) from that of comparative physiology. Here, it is only
necessary to discuss previous work on those aspects of the subject with which this
paper is immediately concerned.
Seasonal variations of activity are of many different types. An uninterrupted
series may be found between the complete lethargy of hibernating mammals and
reptiles and the relative inactivity which many animals, such as the frog, the subject
of this paper, show in cold conditions. The series may, perhaps, be extended from
these conditions of relative inactivity to the seasonal physiological changes, often
unaccompanied by any obvious inactivity, which occur in many non-hibernating
mammals. Nor is cold the only condition in which animals become inactive;
aestivation is as well-known a phenomenon as hibernation.
It is by no means clear, without further evidence, that conditions of all these
types are due to similar physiological changes within the body. However, in most
of them the body passes into a more or less completely inactive state, and many
show other, more definite, physiological resemblances. Whether it is or is not true
that all such conditions are physiologically similar, it will be advisable, in discussing
the previous work on the aspects of their physiology with which we are concerned,
to consider evidence derived from all these conditions, since it is possible that any of
this evidence may have some bearing on the changes which occur in the frog. This
will be done, but it must not be forgotten that the experimental results to be given in
the paper are concerned only with the seasonal changes of the frog. Generalisation
from these results cannot be justified until other types of seasonal change have been
investigated. It is hoped to extend the observations in later papers.
It will be found that the experimental evidence is in favour of the conclusion
that endocrine changes play a large part in the control of the seasonal changes of the
Endocrine Control of Seasonal Variations of Activity
257
frog. It is not intended to draw from this evidence the further conclusion that other
organs play no part in this control. It is, indeed, probable that the nervous system
is also concerned, but the part played by it and other organs is outside the subject
of the paper and will not be further discussed.
The belief that the control of seasonal variations is endocrine is by no means new.
Even if we restrict ourselves to the more recent literature, we shall find that at one
time or another almost every endocrine organ has been put forward as being actively
concerned in the control of these changes.
Evidence of at least three distinct types has been used to support these conclusions.
Firstly, extracts of the endocrine organs have been injected and their effects on
the activity of the animal observed. In experiments of this type Adler (1920 b) and
Schenk (1922) found that extracts of the thymus, thyroid, adrenal and anterior
pituitary woke the hedgehog from its winter sleep, but their results were criticised
by Zondek (1924) and have not been generally accepted as conclusive. Also,
Dworkin and Finney (1927) found that large injections of insulin induced winter
sleep in the marmot.
Secondly, seasonal changes have been observed in the condition of the endocrine
organs. Adler (1920 b) found regressive changes during the winter in the thyroid
of hedgehogs and, more markedly, of bats. In non-hibernating mammals and birds
the thyroid is larger in the winter (Riddle and Fisher, 1925, who give evidence for
the pigeon and summarise the literature), but, on the other hand, its iodine content
is higher in the summer and to an extent which more than compensates for its larger
size in the winter (Seidell and Fenger, 1913; Kendall and Simonsen, 1928). These
last results show clearly the inadequacy of histological evidence alone, when the
question at issue is, as it is here, the functional activity of a gland. Neither the size
nor the histological appearance of a gland is necessarily correlated with the amount
of its secretion which it is pouring into the circulation.
Mann (1916) was unable to find constant seasonal changes in any of the
endocrine organs of the woodchuck. Inconstant changes in the pituitary and adrenal
were observed.
Thirdly, the seasonal changes in the physiology of the animal may be compared
with the known effects of over- and under-activity of the various glands. Thus
Britton (1928, 1930, 1931) has compared the phenomena of hibernation in the
mammal with the results of lowered activity of the adrenal. On the whole, however,
in spite of his arguments, it seems to be true that the physiological condition of the
winter state agrees more closely with the condition of hypothyroidism than with any
other abnormal condition of the endocrine system. The respiratory quotient, basal
metabolism, pulse rate and body temperature all alter in the same direction in
hibernation and in hypothyroidism. Certainly, the condition of hibernation is not
that which is to be expected from any possible rise in the insulin content of the
circulation. Neither the fall of the respiratory quotient nor the characteristic
reduction of the fat content of the body are to be expected from this change, so long
as it remains within the range which is likely to occur. The known physiological
258
G. S. CARTER
effects of the thymus and most of those of the pituitary (v. Bugbee, Simond and
Grimes, 1931) show no definite correlation with the changes associated with hibernation. An exception with regard to the anterior pituitary will be discussed in the
succeeding paper of this series.
At first sight one other line of evidence seems to bear on the subject under
discussion. There is evidence that the condition of the endocrine organs varies in
races living under different conditions. Thus, Adler (1920 a) found that frogs
(R. temporaria) living continuously in cold conditions have abnormally large and
active thyroids. But such races must remain active in the cold conditions in order
to maintain their life, and their needs are very different from those of animals which
can become inactive when the temperature falls, passing into hibernation. Adler's
evidence cannot therefore be considered to indicate activity of the thyroid in
hibernation. Rather, it indicates the opposite conclusion—that the thyroid is active
when the animal is forced to be abnormally active to maintain its life in cold
conditions.
From this survey of the literature it may be concluded that there is considerable
evidence that the endocrine organs are concerned in the control of seasonal changes,
but that the evidence is not yet sufficient to decide which of these organs is responsible for their control. On the whole the most reliable evidence suggests that
the thyroid and the adrenal are concerned, but it cannot be said that this has been
established. Very probably more than one organ plays a part in this control, but
it is hardly likely that all those for which evidence has been put forward are equally
concerned. There can be little doubt that the reason why positive results have been
obtained with so many of these organs is that most of the experimental work has
been carried out within the body, where the interactions of the endocrine system are
extraordinarily complex. When the endocrine balance in the body is disturbed in any
way, it is extremely hard to decide which of the observed results are directly due to
the primary alteration and which to secondary alterations induced in other organs.
If evidence could be obtained from experiments carried out outside the body, it
would seem to be of more value in the discussion of these problems. Recently an
opportunity of gaining evidence of this type has been provided by the results of
Barcroft and Izquierdo (1931). They found that the temperature-pulse rate curve of
the excised heart of the frog is of different form according as the experiment is
carried out in the summer or the winter. The curve given by the winter heart is
exponential between 5 and 20° C , whereas that of a summer heart is almost straight
over the same range of temperature and can be shown, when plotted logarithmically,
to be composed of two exponential curves crossing at or slightly above io° C. They
found the intact heart of the summer frog to give a curve of the same form as that
given by the excised heart in the summer. In the winter they failed to get concordant
results from the intact heart.
Here we have a very definite seasonal variation in the behaviour of an organ, and
one which can be observed outside the body. It is therefore a phenomenon on which
the effects of various substances can be tested without danger of secondary effects
due to other, induced, changes in the system. The experiments to be discussed in
Endocrine Control of Seasonal Variations of Activity
259
this paper consisted in determining the effects of various endocrine and related
substances on the form of this curve. The results of such experiments should give,
at least, some indication of the changes within the body which cause the difference
in the condition of the heart.
MATERIAL AND METHODS.
Both the common frog (Rana temporaria) and the edible frog (R. esculenta) were
used as material for these experiments. There is no difference in the behaviour of the
hearts of these two species in the phenomena which were being investigated, except
that the heart of R. esculenta is able to support a slightly higher temperature without
Fig. 1. Apparatus for circulating fluids through the frog's heart. The different
parts of this figure are not drawn to the same scale.
damage. In most of the experiments a temperature range of 5-200 C. was used for
the heart of R. temporaria and 5-240 C. for that of R. esculenta. Exposure to the
highest of these temperatures for the necessary period did not alter the rate of beat
at lower temperatures. It was therefore assumed that no damage had been done to
the hearts by the high temperature.
The heart was excised (the two anterior venae cavae being bound) and mounted
in the apparatus illustrated diagrammatically in Fig. 1. Cannulae C and B were
inserted in the truncus arteriosus and the inferior vena cava, so that a current could
be passed through the heart from the inner tube E and out through the tube F.
Thus, the heart received a current of fluid which did not mix with the fluid in the
outer vessel (D), in which it was suspended. Both E and D received fluid from the
upper vessel K through the tubes G and H, and the levels of the fluid in E and D
260
G. S. CARTER
were maintained constant by the siphons L and M. The heights of these siphons
were adjusted so that the heart was under a venous pressure of about 3 in. The liquid
in the vessel D could be stirred by a current of air through the tube P. The fluid
which had passed through the apparatus was replaced from the dish O into K by
a suction device. In the course of its passage between these two dishes it was well
aerated in the tubes of this device. In most of the experiments a litre of fluid was
used and this passed round the apparatus in 1-2 hours. The temperature of the
heart was altered by immersing the vessel D in a much larger vessel of warmer or
colder water. It was kept constant at any value by filling this larger vessel with
water of the required temperature.
Barcroft and Izquierdo (loc. cit.) found difficulty in obtaining the same rate of
beat at any given temperature when the temperature was rising as when it was falling.
They found it necessary to allow the heart to beat outside the body for some hours
before they could get this result. It was found that this difficulty was almost entirely
overcome by keeping the temperature of the fluid constant for at least five minutes
before the observations of rate of beat were made. All the experiments were carried
out in this way. It was found necessary, however, to allow the heart to beat for at
least one hour after the apparatus had been set up, and for at least half an hour after
it had been disturbed in any way, as, for instance, in adding substances to the fluid.
The procedure of keeping the temperature constant for five minutes before each
observation and the fact that several curves were needed in many of the experiments
(and therefore not more than 1-2 hours could be given to each) made it necessary
to observe fewer points on the curve than were observed by Barcroft and Izquierdo
in some of their experiments, but the smaller errors in each observation at least
compensated for this disadvantage. The experimental error is clearly proportional
to the rate of beat. In several determinations it was found to be less than 0-5 beat
per minute at the lowest temperatures and 2 beats per minute at the highest. An
error of about this amount may be assumed throughout. On the logarithmic plot
this is equivalent to an error of log"1 0-02 throughout the range of temperature.
The fluid circulating through the apparatus was a Ringer solution of the following
constitution:
/
/U
NaCl
/o
0-65
NaH 2 PO 4
o-ooi
KC1
CaCl2
0-014
0-012
Glucose
Lecithin
O-OOO2
O-2
The lecithin was added on the evidence of Clark (1913) that it prolongs the life of the
heart. The/>H of this solution was brought to that required by adding the necessary
amount of a strong solution of NajHPO4. The amount necessary to give a />H of 76,
which was used in almost all the experiments, was equivalent to about 0-012 per
cent. NajHPOj.
It was found that the heart would beat for at least 12 hours in this solution,
and often for 24 hours, with little reduction in the rate of beat (see Figs. 3, 5). The
phosphates were used to buffer the solution, in place of the more usual bicarbonates,
Endocrine Control of Seasonal Variations of Activity
261
in order that the control of />H might be more effective. In spite of this, there was
always a slight movement of the />H in the acid direction during the experiments
(to />H 7-3-4 after 2-3 hours). The />H was readjusted to pH 7-6, whenever the
medium was altered, i.e. after each curve had been obtained. The effects of variations
in pH were observed in experiments recorded below.
When the action of synthetic substances was to be observed, these substances
were dissolved in N/10 NaOH and added to the medium in the correct proportion.
Adrenaline alone was dissolved in N/10 HC1. ThepH of the medium was readjusted
after these additions. Extracts of various organs were made by grinding the tissue
in Ringer solution and precipitating the proteins by means of tri-chlor-acetic acid.
The only commercial extract used was that of pituitrin. The extracts were added
directly to the medium.
Each result was confirmed in a series of at least three experiments. Wherever the
results were doubtful or contradictory, a longer series of experiments was carried
out, and this was also done where the experiments seemed to be of especial importance, for example, the experiments on thyroxine and adrenaline, and on the effect of
long-continued beat in unmodified Ringer solution.
EXPERIMENTAL RESULTS.
(1) The summer and winter form of the pulse rate-temperature curve.
Typical examples of the two forms of the curve are given in Fig. 2. In this and
all the succeeding figures the observations of pulse rate are directly plotted against
the temperature in the lower part of the figure, and smoothed curves are drawn
through these points. In the upper part of the figure these smoothed curves are
redrawn on a plot of which the abscissa is the reciprocal of the absolute temperature
and the ordinate the logarithm of the pulse rate. Although the lines of the upper
part of the figure are drawn from the smoothed curves of the lower part, the observed
points have been replotted in order that the goodness of the fit may be estimated.
The procedure of plotting the figures logarithmically has been carried out because
it seemed that the alteration of the shape of the curve could be most easily recognised
in the logarithmic plot. It is not intended to discuss whether or not the form of
the logarithmic curve has any biological meaning.
The curves of Fig. 2 are redrawn from results published by Barcroft and
Izquierdo (loc. cit.). Both curves refer to isolated hearts.
The curve of the winter heart differs from that of the summer heart not only in
its exponential form but also in that the rate of increase of the beat begins to fall off
towards the upper limit of the range of temperature at a lower point. As a result of
this the winter curve is often S-shaped within the temperature range, and the
summer curve is not. This is shown in Fig. 2, and is also evident in Figs. 3, 5, 7, 8.
This being so, the points on the uppermost part of the winter curve have been
neglected in drawing the logarithmic plot, since it is clear that in this region other
conditions are controlling the rate of beat than those which control it in the central
region of the range.
G. S. CARTER
262
Results will be given below (Figs. 3,5,6) which show that no general increase or
decrease in the rate of beat can be observed as the heart passes from one seasonal
condition to the other. When plotted on the same ordinates and abscissae, the summer and winter curves lie over each other. It is for this reason that in all the figures
except Fig. 2 the ordinates have been stepped down for each curve, so that the
curves may not become confused with each other.
0-0036
I
10
25°C
Fig. 2. Pulse-rate curves of the summer and winter forms, R. temporaria. Redrawn
from Barcroft and Izquierdo, 1931, pp. 149, 152. S, summer; W, winter.
15
20
On the other hand, comparison of the figures will show that there is considerable
variation in the rate of beat at the same temperature from one heart to another.
(2) Preliminary experiments.
(a) Long-continued beat in Ringer solution.
It was necessary, before examining the effects of adding substances to the heart,
to observe whether the form of the temperature-pulse rate curve underwent any
alteration, when the heart was allowed to beat in unmodified Ringer solution for the
period for which the experiments would last (5-12 hours). No change in the form
of the curve given by the winter heart during this time could be observed, but the
curve of a summer heart showed a very slow change towards the winter form. This
Endocrine Control of Seasonal Variations of Activity
263
was hardly recognisable after 5-6 hours, but became evident after 12 hours
(Fig. 3)These results are explicable if some substance is present in the summer heart in
greater concentration than in the winter heart, and is gradually reduced by being
washed out of the heart or used up. They are very difficult to explain if the difference
consists in greater concentration of a substance in the winter heart. Thus, if the
endocrine theory is true, they are strongly in favour of greater activity of some
organ in the summer.
0-0036
B ~"
1-8
0-0035
0-0034 1/r
10
1-81-6
1-6-
•3
1-41-2
1-26060-
5050-
g 40
I 30
40
30-
20
20-
10
15
20
25°C
Fig. 3. R. esculenta. Pulse-rate curves of the same heart in Ringer solution. Curve B,
I2i hours after curve A. A, 5.4.32, 10-11 p.m.; B, 6.4.32, 11.30 a.m.-i2.3O p.m.
(b) Effects of variation of the pH of the medium.
It has been stated that small variations of the />H of the Ringer solution during
the experiments could not be avoided. These variations did not exceed pH 0-2-0-3 in
any experiment, and were nearly the same in all experiments, but their existence
made it necessary to determine the effects of changes of pH on the form of the curve.
In Fig. 4 curves given by the same heart ntpH 7-6,7-2 and 8-o are shown. The general
rate of beat was slightly less at/>H 7-2, but the figure shows that pH changes even of
this extent, i.e. greater than any which occurred during the experiments, had
practically no effect on the form of the curve. All three logarithmic curves are almost
264
G. S. CARTER
equally bent, and the differences in form between them are probably within the
experimental error. It may, therefore, be assumed that the variations of />H which
occurred during the experiments had no appreciable effect on the form of the
curves.
(c) The action of tri-chlor-acetic acid.
It has been mentioned that tri-chlor-acetic acid was used to precipitate proteins
from the extracts of the endocrine organs used in these experiments. Since a
0-0036
A B C
-
1-8
0-0034 W
0-0035
15
10
Log. pulse rate
1-8
1-8-
1-6 -
1-8
1-6-
1-4
1-4
1-4-
1-2 -
1-2
1-2-
50 -
50
50-
40 40
30 -
per
|
40-
30
1
20 -
a
30-
20
10 -
20-
10
10
10
15
20
25 °C
Fig. 4. R. esculenta. Pulse-rate curves of the same heart in Ringer solution of different pH.
Curve A, pH 7-6; B, pH 7-2, z hours after curve A; C, pH 8-o, 1J hours after curve B.
trace of this substance may remain in the extract, it was thought advisable to determine whether it had any effect upon the form of the curve. The medium in which the
experiments were carried out was always atpH 7-6, and, for this reason, the experiments on this substance were also carried out at />H 7-6. It was therefore not the
effect of the acid but that of the organic radicle which was determined. The concentration used was 1 in 10,000, a higher concentration than could have been present
in any of the rest of the experiments. Even in this high concentration, no effect upon
the form of the curve was found. Later addition of thyroxine to the same heart
gave the typical effect of this substance (p. 265 below). It is therefore clear that the
Endocrine Control of Seasonal Variations of Activity
265
failure of tri-chlor-acetic acid to produce any effect was not due to some damage of
the heart caused by its high concentration.
(d) The action of tissue extracts of organs other than the endocrine organs.
It will be found that not all the extracts used in the following experiments had
any effect upon the form of the curve given by the heart. Thus the results are probably sufficient by themselves to exclude the possibility that tissue extracts of any
organ might contain an effective substance. In order that this point might be more
definitely established, a few experiments were carried out with extracts of rabbit
muscle and salivary glands. No alteration of the form of the curve was observed,
although the ability of the heart to react to thyroxine was not damaged by these
extracts.
(3) The action of endocrine substances,
(a) Thyroxine.
In Figs. 5 and 6 the effect of adding thyroxine to the winter hearts of R. esculenta
and R. temporaria are shown. The same effect is shown in curve C of Fig. 8. These
experiments are examples of a long series, all of which gave similar results.
A B C0-0036
1-8'1-
0-0035
0-0034
</T
1-8
e
1-6-
1-81-6
3
a 1-4}.
1-6-
,3
V2
1-2
60
1-2-
60
50-
40
40
40-
30
30
1
3
30-
20
20
20-
10
10
10'
10
20
15
25°C
Fig. 5. R. etculenta. A, pulse-rate curve of a heart in the winter condition; B, curve given by the
same heart after beating for 1 J hours in Ringer solution containing thyroxine, 1 in 5 x io'; C, 13 hours
after return to Ringer solution.
IBB-xiii
18
266
G. S. CARTER
It will be seen that the substance produces a definite alteration of the curve
towards the summer form. The effect is produced rapidly and can always be obtained
when the heart has been beating in the presence of thyroxine for one hour. No
change in the form of the curve could be found when a heart in full summer
condition was treated with thyroxine. When the heart was allowed to beat for some
hours in unmodified Ringer solution after treatment with thyroxine there was some
return towards the winter condition (Fig. 5), presumably due to loss of the thyroxine.
A
1-8
B,0-0036
1-8-
0-0035
0-0034
25 °C
1-6
1-6-
3
1-4
1-41-2
60 1-260-
50
5040
c
40-
•§
t 30
a
S
3020
« 20
10
10
15
20
25 °C
10
Fig. 6. R. ttmporaria. A, pulse-rate curve of a heart in almost complete winter condition in Ringer
solution; B, curve given by the same heart after beating for i \ hours in the same solution with the
addition of thyroxine, i in 2 x 10*.
The lower limit of the effective concentration of thyroxine was approximately
i in io7. At this concentration the substance produced a complete effect in two
experiments, but in others it was ineffective. All concentrations between i in io 6
and i in io 7 were effective. When the concentration was above i in io6, the beat was
slightly slowed and in some experiments the effect seemed to be less or absent.
The range of the effective concentration is approximately equivalent to the
change in the concentration of the thyroid secretion which may be expected to
occur in the blood, i in io 7 of thyroxine is equivalent to about 6y of iodine per
ioo c.c. Values of 5-157 per 100 c.c. have been observed in normal human blood,
and much larger variations occur in the female during the sexual cycle. It may
Endocrine Control of Seasonal Variations of Activity
267
therefore be expected, if the thyroid is concerned in the control of seasonal variations,
that changes of at least 6y in the iodine content of the blood will occur. Thus, the
evidence indicates that the protein secretion of the thyroid is no more effective than
thyroxine in this effect upon the heart.
The figures also show that there is no general increase of the rate of beat in the
presence of thyroxine. Barcroft and Izquierdo (1931) obtained similar results in
untreated hearts of summer and winter frogs (e.g. Fig. 2). In this respect, as in
the change of the form of the curve, the effect of thyroxine resembles the natural
change. A general increase of the rate of beat is the effect which has most often
been looked for in experiments on the action of thyroxine on the heart. These
results are therefore in agreement with those of the large proportion of the
previous experiments which have given negative results, when an increase of the
rate of beat was expected.
(b) Adrenaline.
The addition of adrenaline to the heart results in a general increase in the rate
of beat without any alteration of the form of the curve. This effect is shown in Fig. 7.
The differences in the form of the logarithmic curves in this figure are within the
experimental error. The increase amounted at a maximum to about 20 per cent, of
0-0035
Q.QO36
c B
1-8 •
1-8
pulse rat
V
<f
O-op34
10
1-8-
1-6
1-6
1-4 -
1-6-
1-4
1-2-
1-4-
60 1-2
an
ov
60-
50
50
per
•UIUl
!
&
40 -
50-
40
30 -
40-
30
20
3020
20
25 °C
15"
20
Fig. 7. R. etculenta. Effect of adrenaline. Pulse-rate curves of the same heart in Ringer solution (A);
the same solution containing adrenaline, 1 in 10* (B); and containing adrenaline, 1 in 5 x 10* (C).
B, 2 hours after A; C, i\ hours after B.
18-2
10
268
G. S. CARTER
the original rate of beat. A practically complete effect was obtained when the
concentration of adrenaline was as high as i in io7, and a smaller effect could be
recognised with a concentration of i in io 8 . All these effects were immediate.
These results are in apparent contradiction to those of Gellhorn (1924), who
found a rise of the Qlo of the pulse rate of muscle strips of frog's heart on the
addition of adrenaline. It is impossible to give a complete explanation of this
discrepancy, but it may be remarked that the concentration of adrenaline used in his
experiments was very large (1 in 1-500,000), and very far outside the concentration
in the blood (Schlossmann, 1927, 1 in 2 x io 8 at the absolute maximum).
(c) Insulin.
A definite modification of the form of the curve towards the winter condition
was observed in the presence of this substance. The effect was, therefore, opposite
to that of thyroxine. The effective concentration was higher than that of thyroxine,
and the effect was smaller. A concentration of 1 in io 8 of the dry substance gave
a slight effect in some, but not all, of the experiments: a concentration of 1 in 3 x io 5
gave a somewhat larger, but still small, effect. The concentration of insulin in blood
is 5-6 units per 100 c.c. (mammal, Brugsch and Horsters, 1930), equivalent to a
concentration of 0-2-0-3 mg. of the dry substance per 100 c.c. or 1 in 3-500,000. The
necessary change for the production of the effect is therefore large, but not impossibly so. It is more important that the effect produced was always small, and
necessitated an increase of the insulin content of the blood in the winter (cf. p. 263
above). These facts together with the lack of agreement, noted above (p. 257),
between the physiological changes of hibernation and those to be expected from
an increase of insulin in the circulation, and make it very unlikely that insulin is the
natural effective agent in these changes.
(d) A general pituitary extract.
A Ringer extract of a whole pituitary of a frog was made and its effect determined
in 250 c.c. of the medium. It produced a definite change in the form of the curve
towards the winter condition, and therefore an opposite effect to that of thyroxine.
This is shown in Fig. 8. It will be seen that the effect could be reversed by means
of thyroxine. The effect was larger and more rapid than could be accounted for by
the change in the form of the curve which results from continued beat in Ringer
solution alone (p. 262 above), and was considerably greater than the experimental
error. It must therefore be believed to be a real effect, but the dilution was great and
the substance present must be very active.
(e) Pituitrin.
No attempt was made to locate the effective substance in the pituitary by means
of Ringer extracts of its various parts, but some experiments were carried out with
a commercial extract of the posterior pituitary, which contained both the oxytocic
and the pressor principles. This extract was found to produce a definite expansion
of the pigment cells of the frog's skin at a concentration in the circulation of about
0-0008 unit per c.c, and a slight expansion at 0-00008 unit. A concentration of
Endocrine Control of Seasonal Variations of Activity
269
0-02 unit per c.c. in the Ringer solution circulating through a heart produced no
alteration of the curve towards the winter form. The heart responded to potassium
bromide, which will be shown in the succeeding paper of this series to transform
the curve from the summer into the winter form, after treatment with this pituitary
extract.
(/) Extract of the thymus gland.
It would seem improbable that this organ, which normally decreases in size in the
vertebrate after puberty, is the effective organ in controlling changes which continue
<u
1-6
1-8-
3
1-6
a
M
25°C
10
1-8
V
cd
i*
0-0034
0-0035
0-0036
B
i4
1-8
1-6-
1-4
1-4
V4-
1-2
60 1-2
60 1-2-
60-
50
50
50-
per
40 40
30 -
4030
=
30-
20 20
20
T
10
15
20
25°C
Fig. 8. R. esculenta. Effect of pituitary extract. Curves given by a heart in Ringer solution (A); the
same solution containing pituitary extract (B); the same solution with the addition of thyroxine,
i in z x io* (C). B, 2 hours after A; C, three hours after B.
throughout the life of the animal. It may also be remarked that the thymus is also
very small in the frog.
Because of the small size of the frog's thymus, extracts were made of rabbit
thymus, and their effect upon the frog's heart was determined. Extracts from about
o-5 c.c. of the gland were added to 500 or 1000 c.c. of the medium. They had no
effect upon the form of the curve, and, after treatment with these extracts, the heart
was found to be capable of responding to thyroxine. It may be concluded that the
thymus of the vertebrate does not contain a secretion which is capable of controlling
the behaviour of the heart in this respect.
270
G. S. CARTER
(4) Substances chemically related to thyroxine.
A series of experiments with various substances related more or less closely to
thyroxine gave the following results:
(a) Des-iodo-thyroxine. This substance gave a definite effect of the same type as
the thyroxine effect at a concentration of 1 in io8, doubtful effects at 1 in 2 x io 6 and
no effect at 1 in 5 x io8. At 1 in io 8 its effect was as large as the effect normally
produced by thyroxine. It was therefore effective, but at a concentration about five
times as high as the effective concentration of thyroxine.
(b) Inorganic iodine. Iodine dissolved in five times its concentration of potassium
iodide produced definite effects of the same kind as those of thyroxine in concentrations of iodine of 1 in io 8 and 1 in 2 x io8, but none, or a doubtful effect, at 1 in
5 x io 8 . Inorganic iodine was therefore, like des-iodo-thyroxine, effective but at a
higher concentration than thyroxine.
(c) Di-iodo-tyrosine. This substance was used in concentrations between 1 in
2-5 x io 6 and 1 in 2 x i o \ Its effects were never more than doubtful.
(d) Tyrosine and tyramine had no effect at a concentration of 1 in 2 x io5,
phenyl-ethyl-amine none at concentrations of 1 in 2 x io 6 and 1 in io5.
DISCUSSION.
1. The experiments on the effect upon the heart of long-continued beat in
unmodified Ringer solution (p. 262 above) show clearly that, if the difference between the summer and winter conditions is due to a change in the endocrine system,
the organ responsible must be more active in the summer.
2. Of the endocrine substances and extracts used in the experiments the only
one which produced a condition of the heart similar to the natural summer condition
by an increase of its concentration in the medium was thyroxine. Thus, the thyroid
is the only endocrine organ of those investigated which is capable of controlling the
difference between the summer and winter condition in agreement with the observed
facts of this difference. Further, the exactness of the similarity between the effect
of increased concentration of thyroxine and the observed difference between the
summer and winter conditions makes it very probable that the thyroid is not only
capable of controlling this change, but does actually do so. This conclusion is further
strengthened by the fact that the concentration of thyroxine which was found to be
effective in these experiments is approximately equivalent to the change in the
concentration of the thyroid secretion which is likely to occur in the circulation.
We must conclude that the control of this seasonal change is endocrine and that
the thyroid is the endocrine organ which controls it. This last conclusion is, as we
have seen, in agreement with the balance of the evidence provided by previous work.
3. Both insulin and some secretion of the pituitary were found to be capable of
producing the opposite effect—a change towards the winter condition by increase of
their concentration in the circulation.
For the reasons which have been already given (p. 268 above), it is very improbable that insulin is responsible for the natural control of these changes. Why it
Endocrine Control of Seasonal Variations of Activity
271
should be able to produce changes of the same nature as those produced by thyroxine,
although opposite in direction, is a subject into which we need not enter here.
Nor can the pituitary secretion control these changes. The experiments, however,
provide evidence for the presence of a substance in the pituitary, distinct from
pituitrin, which is somehow related to the thyroid secretion in its action. It will be
shown in the succeeding paper of this series that there is evidence for interaction
between the thyroid and the pituitary, especially the anterior pituitary. It need not
therefore cause surprise that secretions of these glands produce effects which are
similar, although opposite.
4. So far we have only considered the temperature-pulse rate curve of the heart and
its seasonal changes, and this is by no means the only seasonal change in the physiology of the animal. Many of the other seasonal changes are those which might be
expected to result from a reduction in the amount of the thyroid secretion in the circulation, but it is possible that some of them are controlled in other ways. The experiments give no evidence for or against seasonal variations in the activity of any organ
of which the secretion has no effect on the form of the temperature-pulse rate curve.
However, our consideration of the previous evidence led to the conclusion that
there is no reliable evidence for seasonal variations in the activity of any endocrine
organ except the thyroid and, perhaps, the adrenal, which Britton believes to play
a part in the causation of hibernation in the mammal. The adrenal is therefore the
only other organ which we need consider.
It has been stated that no general increase in the rate of beat was observed as the
heart passed into the summer condition. If, as Britton suggests for the mammal, the
adrenal is in an inactive condition in the winter animal, such an increase in the rate
of beat should occur, for our experiments with adrenaline showed that the pulse
rate was increased over the whole range of temperature by the addition of this
substance. It is possible, however, that the experimental conditions were not sufficiently exact to allow us to form a definite conclusion on this point. The action of
adrenaline is very rapid, and it is possible that the handling of the frog in killing it,
although this was done as quickly as possible, may have stimulated the adrenal to
activity before death. If so, hearts taken from winter frogs would have contained
more adrenaline than is normal in the winter condition. Thus, it is possible, and may
seem probable in view of the general inactivity of the animal in the winter, that the
winter condition is characterised by lowered activity of the adrenal, but the experiments of this paper give no proof that this is so.
5. The final conclusion of this argument must be that the winter condition of
the frog is one in which the amount of the thyroid secretion and, perhaps, also
of adrenaline in the circulation is reduced. We have no reason to believe in the
occurrence of any other endocrine changes. We do not know how the endocrine
changes which do occur are brought about. It is probable that the endocrine organs
are themselves controlled by the nervous system.
272
G. S. CARTER
SUMMARY.
1. Barcroft and Izquierdo(i93i) have shown that the form of the temperaturepulse rate curve of the excised heart of the frog differs seasonally. The effects
produced by endocrine substances and extracts of endocrine glands on the form of
this curve have been investigated with a view to determining the nature of the
seasonal change within the body.
2. When the heart of a winter frog is allowed to beat for some hours in Ringer
solution, there is no alteration in the form of the curve. If the heart is that of a
summer frog, there is a gradual alteration of the curve towards the winter form. It
is concluded that, if the control of this change is endocrine, the organ responsible
is more active in the summer.
3. Thyroxine, at a concentration equivalent to the possible change in the amount
of the thyroid secretion in the circulation, transforms the curve given by a winter
heart into the summer form, but has no effect on the curve of a summer heart.
4. Adrenaline, pituitrin, thymus extract and extracts of non-endocrine organs,
such as muscle and salivary glands, have no effect on the form of the curve.
5. Insulin, in large doses, and a general extract of the pituitary have an effect
opposite to that of thyroxine, altering the curve from the summer to the winter
form.
6. It is concluded (1) that the control of this phenomenon is endocrine and
(2) that the thyroid is the effective organ, by increase of its activity in the summer.
Although the adrenal does not control the phenomenon investigated, it is not
excluded that it may control other seasonal changes in the frog and other vertebrates.
7. Of several chemical substances related to thyroxine, des-iodo-thyroxine and
inorganic iodine produced effects on the form of the curve similar to those of
thyroxine, but higher concentrations of these substances were necessary.
My thanks are due to the Government Grant Committee of the Royal Society
for a grant by means of which some of the expenses of this research were defrayed.
Endocrine Control of Seasonal Variations of Activity
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