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A LABOEATOEY GUIDE
FOE THE STUDY OF
THE PHYSIOLOGICAL ACTION OF DEUGS
CHARLES WILSON gREENE, PH. D.
PROFESSOR OF PHYSIOLOGY AND PHARMACOLOGY, UNIVERSITY OF MISSOURI.
THIRD EDITION, REVISED
WITH 37 NEW ILLUSTRATIONS
P. BLAKISTON'S SON & CO.
1012 WALNUT STREET
Copyright, 1909, by P. Blakiston's Son & Co.
The Maple Press
Instruction in Piiarmacology should be based on a rigid course of
required laboratory experiments. The student in the subject must be given
every opportunity to observe for himself the changes produced by a drug in
the activities of a tissue, of an organ, and of the entire organism. It is only
on such intimate personal experience with the facts that one can reach a
rational understanding of the principles of Pharmacology.
The directions presented here have been formulated during the growth
of the course as presented in the University of Missouri. Under each drug
presented there is given a list of experiments chosen with due consideration
to the facility with which they may be performed by students. This list
is followed by detailed yet brief directions for the execution of the experi-
ments. An occasional type illustration is given to guide the student in his
efforts. It is assumed that the individual student will have time for only a
selected number of experiments on each drug, and the plan of the Guide is
so arranged as to support the instructor in the assignment and execution of
a number of diversified experiments by the average laboratory class.
I am indebted to the Department Teaching Staff, especially to Professor
W. Koch, now of the University of Chicago, to Dr. W. H. Schultz, now of
the U. S. Public Health and Marine Hospital Service, and to Professor
R. B. Gibson for numerous suggestions and much assistance.
I am under special obligation to Mr. G. T. Kline for the drawings of
apparatus, to Professor Gibson and to my students of the past five years
for the majority of the new illustrations presented in this edition of the
Guide. C. W. G.
University of Missouki, April, 1909.
THE ACTION OF DRUGS.
Chloroform . .
Chloral hydrate .
The opium series . .
Caffeine . ...
Strychnine . .
Cocaine . . .
Quinine . . .
Curare . . .
Aconite . .
Veratrine . .
Ergot . . .
Suprarenal gland . .
Nitroglycerine and the
Carbolic acid .
Barium salts . . .
OPERATIONS, APPARATUS AND SPECIAL METHODS.
Physiological solutions . . .64
Anesthesia for dogs, cats, rabbits, and guinea-pigs . 65
The preparation of the ventricular muscle .... . 66
To test the action of drugs on the frog's heart 68
Irrigating and perfusing flasks 69
To test the action of drugs on the blood-pressure, respiration, etc., of
a mammal . . ... .... 70
Method of testing the action of drugs on the reflexes of a frog 71
Method of giving and testing the action of a drug on the frog's gas-
trocnemius muscle . . 72
Transfusion buret for mammals . 72
Apparatus for the study of the isolated mammalian heart .... 73
List of stock solutions 75
Report form for experiments on frogs 76
LIST OF ILLUSTRATIONS.
1. Alcohol perfused through the frog's heart 2
2. Ether on the isolated muscle of the terrapin's ventricle ... . 7
3. Strong ether solutions on the isolated ventricular muscle. 8
4. Ether on tone waves of auricular and sinus muscle. . 9
5. Ether on the irritability of the sciatic nerve 10
6. Chloroform on the rhythm of heart muscle 12
7. Chloroform perfusion of the frog's heart . 13
8. Chloroform on the irritability of nerve . 13
g. Morphine on the rhythm of terrapin's ventricle . 16
10. Morphine on the isolated heart of the cat . . . 17
11. Morphine on the isolated heart of the cat . 17
12. Caffeine on the ventricular muscle . . 21
13. Caffeine on the amount of muscular work 22
14. Strychnine on the ventricular muscle . . . 25
15. Strychnine on the muscular work . 26
16. Strychnine on the isolated mammalian heart . . . 27
17. Cocaine effects on the frog's heart . . 29
18. Cocaine on the muscular work . . 30
19. Quinine on the frog's heart 32
20. Atropine on the heart muscle . . 33
21. Atropine on the isolated cat's heart . 34
22. Nicotine on the ventricular muscle . . 38
23. Nicotine on the isolated cat's heart ... 39
24. Pilocarpine on the blood-pressure and on the respiration of the dog 42
25. Physostigmine on the isolated cat's heart . . . 45
26. Physostigmine followed by atropine on the mammalian blood-
pressure and respiration . 46
27. Aconite on the isolated cat's heart. . . . . . 49
28. Veratrine on the isolated cat's heart . 51
29. Digitalis on the heart muscle ...... 53
30. Digitalis on the isolated cat's heart ... 54
X LIST OF ILLUSTRATIONS.
31. Adrenalin hydrochloride on the isolated cat's heart . . 57
32. Amyl nitrite on the human pulse .... . . 60
33. Calcium chloride on the terrapin's heart muscle ... 62
34. The terrapin's heart, how to cut an apex strip . . 67
35. Apparatus for the study of heart muscular strips . . 67
36. Apparatus for the study of drugs on the frog's heart . . 68
37. Apparatus for the study of the action of drugs on the mamma-
lian heart 74
THE ACTION OF DRUGS.
List of Experiments Showing the Effects of Alcohol. page.
1. On the frog .... i
2. On ventricular muscle . . i
3. On the frog's heart . . 2
4. On the isolated mammalian heart. .... 3
5. On the work of the frog's gastronemius muscle. 3
6. On voluntary work of human muscle. Demonstration . 4
7. On the circulatory and respiratory systems of the mammal . 4
8. On the reaction time of the reflex frog . ... 5
1. Alcohol on the frog. Inject into the dorsal lymph sacs of two
frogs doses of 0.3 c.c. (5 minims) and 0.6 c.c, respectively, of 95 percent
alcohol.' Strong alcohol is quickly absorbed from the lymph sac. The
larger dose is sufficient to produce temporary complete loss of the reflexes,
together with the loss of all respiratory movements. A dose of i c.c.
is toxic for a 40-gram frog. Since the smaller dose is equivalent to 525 c.c.
for a 7c-kilo man, it is evident that the frog is the more tolerant of alcohol.^
2. Alcohol on ventricular muscle. Mount a strip of the ventricle
of a terrapin in 0.7 percent sodium chloride (see page 66 for the method),
and when it is contracting with an even and regular rhythm change to a
solution of 2 percent alcohol in" physiological saline. Return the strip to
'All doses for frogs given in this book are calculated for an animal weighing 40 grams.
Each animal used for experiment should be weighed on a "Harvard" platform balance and
the dose given calculated in proportion to the weight of the animal used.
"A report blank form for the tabulation of observations on the effect of drugs on frogs is
given on page 76.
2 EXPERIMENTAL PHARMACOLOGY.
pure saline solution after two to five minutes. Record the contractions on a
drum moving i mm. a second. Repeat the experiment', using successive
strengths of alcohol of 5 and 10 percent. The alcoholic effect will be dem-
onstrated rather better on a ventricular strip that is contracting regularly
in the weaker Ringer's solution, page 64, but the alcohol must be dissolved
in Ringer's solution of the same composition.
3. Alcohol on the frog's heart. Destroy the brain and spinal cord
of a frog, expose the heart by cutting away the ventral wall from directly
over the ventricle, using care not to lose blood. Do not cut the bridge formed
Fig. I. — Action of 2 percent alcohol on the frog's heart when perfused through
the ascending vena cava. The alcohol was dissolved in Ringer's solution. The
record was taken from the tip of the ventricle by the suspension method. Time in
seconds and minutes. Perfusion began at the first arrow above the second's record and
ended at the second arrow.
by the sternum, but use it as a fixed point to which the heart may be anchored
by a ligature around one of the aortic arches. Take a direct record of the
movements of the ventricle, using a light straw lever of the fulcrum-power-
weight order. Give 1.5 c.c. of 95 percent alcohol in the abdominal
cavity. Take a continuous record during the time of absorption. This
method demonstrates the effects on the volume, and on the type of systole
and diastole. The rate is only slightly changed.
More satisfactory results are obtained by perfusing the heart through a
canula in the ascending vena cava. Perfuse the alcohol from four-ounce
supply flasks provided with constant level tubes. Use a pressure of from
4 to 6 cm. The perfusion strength to use by this method is 2 to 5 percent
alcohol made up in Ringer's solution. Perfuse the heart for from two to
four minutes at a time. Record the contractions of the ventricle. by a thread
from its tip to the vertical arm of a balanced lever, page 68. In this experi-
ment, as in all frog's heart perfusion, it is better to use the weaker Ringer
for the normal solution. The Ringer's solution insures a uniform heart
rate and strength for long intervals, while the sodium chloride solution
will sustain the whole heart in regular and strong rhythm for only a few
4. Alcohol on the isolated mammalian heart. Use a rabbit or a
cat for this experiment. Anesthetize quickly with ether (do not use chloro-
form) , insert a canula in the carotid, bleed completely, defibrinate the blood
and dilute it with nine volumes of Locke's solution. Use this diluted blood
as a normal solution for perfusing the heart. Reserve enough of the solution
for making the drug mixtures, pour the remainder into the perfusion appara-
tus described on page 73, fill the tubes, adjust the apparatus and bring to a
constant temperature of 36 to 37° C. (a higher temperature is unfavorable).
Quickly remove the heart, taking care only to preserve enough of the aorta
for the insertion of the canula without danger of interfering with the semi-
lunar valves. Mount the heart without catching air in the canula, attach the
recording lever of the Guthrie cardiograph and start the perfusion. The
perfusion pressure should be from 80 to 100 cm. of water.
The heart contracts and a uniform rhythm will be quickly established
and may be maintained for several hours. Perfuse the heart with 0.2
percent alcohol in the Locke-blood solution for from 30 to 100 seconds
at a time, allowing full time for a return to the normal after each drug
perfusion. Raise the dose successively to 0.4 percent, i percent, and 2
percent of alcohol. The stronger solutions reduce the amplitude and
ultimately the rate of the heart; the weaker doses, according to Dixon, in-
crease the amplitude.
5. Alcohol on muscular work. Ligate one leg of a frog near the
thigh to exclude its circulation (a quarter-inch rubber tube makes a fine
ligature for this purpose). Inject 0.3 c.c. (5 minims) of 95 percent
alcohol into the dorsal lymph sac. In exactly twenty minutes pith the
frog, pin it out on the frog-board with the ventral side down, and ligate the
alcoholized leg. Quickly prepare the tendon of the normal muscle, keeping
the muscle covered with skin, attach to the muscle lever, and determine the
work it can do by stimulating the muscle directly with a single induction
shock once every two seconds until the muscle is completely exhausted.
Load the muscle with a 30- to 50- gram weight. Record the contractions on
the lower half of a drum with a speed of i mm. a second.
4 EXPERIMENTAL PHARMACOLOGY.
Prepare the second or alcoholized muscle just twenty minutes after it
has been ligated, mount, load, and stimulate in the same manner. Record
the second experiment on the upper half of the same smoked papef and
parallel with the record of the first. Repeat this experiment using a dose
of 1 CO. of 95 percent alcohol, to demonstrate the injurious effects of
alcohol on the work of the muscle. (Lee and Salant, Am. Jour. Physiology,
Volume VIII, p. 6i, 1902.)
A more difficult but more accurate experiment is obtained as follows:
Destroying the brain only of a frog, pin it to the frog-board belly down,
dissect out the right tendon Achilles, and attach to a muscle lever. Isolate
the lumbar plexus on the right of the urostyle, using care not to interfere
with the circulation of the gastrocnemius. Stimulate the right muscle as
above. Now inject alcohol into the abdominal cavity, and after 20 to 30
minutes of absorption measure the work of the left gastrocnemius. This
method has the advantage of maintaining the circulation intact for both the
normal and the alcoholized muscle.
The effect of alcohol on the speed of the contraction can be determined
by one of two methods. Que can measure the simple muscle contraction
before and after alcoholization. Perhaps a better method is to use Lee's
automatic stimulating device (Am. Jour. Physiol., VIII, p. 61) which auto-
matically stimulates a muscle at the moment of complete relaxation. Re-
cord the contractions on a slowly moving drum, 2 mm. per second.
6. Alcohol on voluntary work of hiunan muscle. Demonstration.
Measure the voluntary power of the flexors of the middle finger with a load
of three kilos or more, using Mosso's ergograph. Take two or three normal
records of voluntary contractions at intervals of 20 minutes. Now take a
dose of 20 to 40 c.c. of 20 percent alcohol, according to the susceptibility
of the individual. Remeasure the muscular power after 60, 90, and 120
minutes, respectively. Compute the work done in kilogrammeters. (Lom-
bard, Jour. Physiol., Vol. 13, p. 49; Hallsten, Skand. Arch. f. Physiol. Bd.
16. S. 139.)
7. Alcohol on the circulatory and respiratory systems of the
mammal. Anesthetize a dog with morphine and chloroform, p. 70.
Take the blood-pressure from the carotid artery, and the respiration from a
side branch of a tracheal canula. Expose the saphenous vein and insert
a canula for intravenous injections, and attach it to a 50 c.c. buret.
Students who have attained the requisite skill should take an onkomet-
ric record with the blood-pressure. To prepare for this record open the
abdominal cavity of the dog, remove the outer sheath from the left kidney
and enclose that organ in a renal onkometer. Record the kidney volume
changes by means of a Brodie's bellows, or Roy's piston recorder, page 71.
The anesthetic must be given with perfect regularity, 2 to 6 drops of
chloroform every 30 seconds, the exact amount that will maintain constant
anesthesia to be quickly ascertained for each animal.
Take a record on the continuous kymograph and, when all is in good
working condition and a normal record has been secured, slowly inject 20
percent wanned alcohol from the buret into the vein imtil some decided
effect on the blood-pressure is noted, i.e., after a dose of 20 c.c. or more.
Extreme caution must be observed lest the heart by rapid perfusion be sub-
jected to an overconcentrated solution. The experiment should be re-
peated with different doses.
Since the anesthetics used all depress the irritability of the circulatory
apparatus, this experiment ought to be demonstrated on a decerebrate ani-
mal. In such an animal the medulla being intact will maintain natural
respirations. Any alcoholic stimulation of the medullary centers can easily
be observed. The recommended intravenous dose of alcohol will produce
slowing of the heart, a phenomenon which disappears on section of the vagi,
thus indicating a direct effect on the vagal centers.
8. Alcohol on the reaction time of the reflex frog. Destroy the
brain of a frog, including the medulla, and when it has recovered from the
shock test the normal reaction time to electrical stimuli applied to the toe.
Measure the time of the reaction with a watch, or record it with a writing-
point attached to the foot or leg of the suspended frog. Give a dose of 0.3
c.c. (5 minims) of 95 percent alcohol in the dorsal lymph sac. Retest
the reaction time at exactly 20 and 40 minutes after the injection. Com-
pare the results with experiments i and 5 above.
List of Experiments Illustrating the Effects of Ether. page.
1. On the frog 6
2. On the ventricular muscle 6
3. On the frog's heart 7
4. On the mammalian heart . 7
5. On the irritability of voluntary muscle. 7
6. On the irritability of nerve tissue . . . 9
7. On the blood-pressure and respiration of a mammal 9
8. On the germination of seeds 10
9. On the growth of yeast . . 11
1. Ether on the frog. Inject 0.2 c.c. (3 1/2 minims) of ether' into
the dorsal lymph sac or the abdominal cavity of a frog. Give 0.3 c.c. to a
second frog. The dose can be given more accurately from the hypodermic if
a 50 percent solution of ether in olive oil is used. The first dose will produce
anesthesia in about 10 minutes. The stages most readily observed are : ist,
great excitement shown by rapid respirations, active movements, and
increased reflex irritability; 2d, slower respirations, very sluggish • response
to external stimulation; 3d, loss of voluntary muscular control and sometimes
of respiratory motions. Slight power of reflex response is retained, including
eye reflexes. The voluntary motions will be regained in from 60 to 90
minutes if the animal is kept moist (winter frogs), and complete recovery
in two hours. The frog will recover from the larger dose in from 20 to 24
hours, or it may even fail of recovery.
2. Ether on the ventricular muscle. Mount a strip of terrapin's
ventricle and establish rhythmic contractions in a bath of 0.7 percent saline.
Record on a drum moving i to 2 mm. per second. Immerse the strip in a
bath of I percent ether in saline for two to three minutes, then return
to the physiological saline bath. The sharp decrease in both amplitude
and rate of contractions is recovered quickly in the saline bath.
Repeat the experiment using 2, 4, and 6 percent ether solutions.
The weaker solutions occasionally produce slight but temporary increase
in the rate, the initial excitation stage. Also use strips of auricle and sinus.
'The dose is figured for a 40-gram frog. Proportionate doses should be given for other
weights. In all experiments on frogs that depress their functions, the animal should be retained
in a moist bell jar for as much as 24 hours, if necessary, in order to test the animal's power of
3. Ether on the frog's heart. Pith a frog, expose its heart, insert
a canula in the inferior vena cava and perfuse the heart in place by the
method described for alcohol, experiment 3. This brings the solution into
intimate contact with the entire heart and it responds almost instantly to
any change in the composition of the irrigating fluid. Perfuse the heart
first with Ringer's solution and follow with i percent ether in Ringer's
4. Ether on the mammalian heart. Use the Roy-Adami method
(given by Cushny, Jour. Exp. Medicine, Volume II, page 233) or the
Fig. 2. — The action of ether on isolated heart muscle. In this experiment a strip
of terrapin's ventricle was mounted in physiological saline until a regular rhythmic
beat was established. The saline was then drawn off and the strip left suspended in
moist air. At the point indicated ether vapor was driven through the moist chamber
until the contractions ceased. The ether vapor was then removed with moist air
at the second arrow and the recovery of the rhythm took place as shown.
method described on page 73 and used in experiment 4 of the alcohol
series. Etherize a cat or rabbit, draw the blood, defibrinate it, and dilute
it with nine volumes of Locke's solution and use as a standard Locke-Blood
perfusion fluid. Remove the heart and adjust it in the apparatus, page 74.
Perfuse with the Locke-Blood solution and when the rhythm is established
change to i percent ether in Locke-Blood. Use 2 percent ether in a
5. Ether on the irritability of voluntary muscle. Mount a gas-
trocnemius of the frog in the moist chamber, arrange to stimulate the
muscle directly with a current of medium intensity but which produces a
maximal contraction. Adjust a vapor apparatus containing saturated
ether water ready for quick connection with the gas tube of the moist
chamber. Stimulate the muscle with single induction currents once every
30 seconds throughout the entire experiment, whether contractions are
secured each time or not. Record on a drum having a rate of 2 mm. per
Take records, three or four normal contractions, then turn on the ether
vapor for five minutes. Quickly remove the vapor with a current of fresh
air. The muscle's irritability will decrease to a point at which the stimulus
is submaximal or even subminimal, but when the ether vapor is removed
the contractions quickly reappear and attain their former amplitude. Use
small muscles for this experiment.
Fig. 4. — The action of ether on isolated strips from the auricle (upper trace) and
from the sinus (lower) of the terrapin. The strips were giving tone contraction waves,
but no fundamental rhythm when bathed with normal sodium chloride solution. At the
mark "on" the sodium chloride was changed to one percent ether in normal saline
for seven minutes, then again to the normal. During the ether bath the tone waves
disappear, a condition which lasts for three minutes after the ether is removed. Fol-
lowing the ether there is a renewal of the tone waves which are even more rapid than
in the normal. Time in minutes.
One may with this preparation also demonstrate that the muscle has a
diminished power to do work when etherized, method page 72.
6. Ether on the irritability of nerve tissue. Prepare a muscle
nerve of the frog, isolating the entire sciatic with a piece of cord, and with the
skin covering the muscle. Mount the preparation with the nerve in the
moist chamber and on the electrodes, but with the muscle hanging through
the hole in the floor of the moist chamber and on the outside so that it will
not be etherized. Close the hole with a sheet of moist filter-paper. Proceed
exactly as in experiment 5 above testing the irritability of the nerve through
its effect on the muscle.
The influence of ether on nervej irritability may also be demonstrated
directly from the nerve by the action' current method. For a description of
the method, see Am. Jour. Physiol., Volume I, p. 104.
7. Ether on the blood-pressure and on the respiration of the
mammal. Anesthetize a dog with morphine and ether. Introduce an
lO EXPERIMENTAL PHARMACOLOGY.
arterial canida in the carotid for taking the blood-pressure record, see page
70 for details of method. Insert a tracheal canula and take a record of intra-
tracheal pressure from a T-tube attached to the canula. Give ether from
an ether bottle connected to the end of the tracheal tube. Take a continuous
record and ultimately give excess of ether, then allow partial and guarded
recovery. Give ether to the point where respirations cease, a point attained
with difficulty except when the animal has a large dose of morphine. The
blood-pressure is an index of safety, for it has been shown that respiratory
impulses are quickly re-established when the blood-pressure remains high.
Saturated ether in saline as an intravenous injection in doses of 20 c.c.
and more, given along with a uniform administration of ether by the trachea.
Fig. 5. — The influence of ether vapor upon the irritability of the sciatic nerve
in the muscle-nerve preparation of a frog. The muscle-nerve preparation was sus-
pended in a moist chamber with the muscle hanging through the opening in the cham-
ber. The first two contractions are normal. The next eight are successive contrac-
tions at intervals of ten seconds during the passing of ether vapor. At the X the
ether vapor was removed by a stream of moist air, the nerve being stimulated at inter-
vals of ten seconds until recovery of the irritability as shown by the contractions at the
last part of the experiment. A gap of two minutes occurs at X, during which the nerve
was not irritable. Time in seconds.
will often demonstrate the characteristic circulatory and respiratory effects
of the drug.
The rectal temperature should be recorded at intervals to demonstrate
the fall of temperature under anesthetics. Note also the state of dilatation
of the pupil.
8. Ether on the germination of seeds. Arrange two eight-ounce
wide-mouth bottles with stoppers each fitted with two glass tubes, letting
one tube extend to near the bottom of the bottle. Suspend in each, by
means of cotton, a dozen seeds — corn, wheat, clover, beans, etc. — and in-
troduce just enough water to maintain a saturated vapor. Set both bottles in
a window. Twice a day for a week, pass through one saturated ether
vapor, through the other air. The seeds in both will swell from the ab-
sorption of the water, but only the seeds in the bottle with pure air intro-
duced will grow. Reverse the two. The sprouting grain will have its
growth checked and the etherized seeds will begin to grow.
9. Ether on the yeast. Take two fermentation tubes of active yeast
culture, add 2 c.c. pure ether to one. Note the relative rate of gas liberation.
List of Experiments Illustrating the Effect of Chloroform. page.
1. On the frog. 11
2. On the heart strip. 11
3. On the frog's heart. 11
4. On the mammalian heart. 12
5. On muscular irritability. . 12
6. On nerve irritability. 12
7. On the blood-pressure and respiration of a mammal. 12
8. On the kidney secretion. 14
9. On germinating seeds. 14
1. Chloroform on the frog. Inject 0.06 c.c. of pure chloroform
or 0.3 c.c. of 20 percent chloroform in oHve oil into the dorsal lymph
sac or into the abdopiinal cavity of a frog. The anesthesia is more pro-
found and the reco^'ery less rapid than in the case of ether. Determine the
relative intensity of action of chloroform and ether by your own experiments.
2. Chloroform on the heart strip. Proceed as with ether in ex-
periment 2, page 6, using a 0.05 percent solution of chloroform in 0.7 percent
saline for one to three minutes. The contractions cease almost at once.
Recovery in saline takes place very slowly. In comparison with ether the
period of anesthesia is long. The amplitude of the first contractions to
reappear is very slight and the recovery rate slow and irregular. The
original character of activity is not restored within 20 to 40 minutes. Re-
peat using a solution of o.i percent chloroform.
An instructive picture is given by parallel records of experiments on
strips from the same heart showing the effects of 4 percent ether and of
o.i percent chloroform for two minutes, both in saline.
3. Chloroform on the frog's heart. Proceed as in the siinilar ex-
periment with ether, page 7, using 0.5 percent chloroform in physiolog-
ical saline to irrigate the outer surface of the heart. Or perfuse the heart
bo o <N
■2 S <„
(U <4-( XI
g g «
with 0.05 percent chloroform in saline
through the vena cava. Care must be
used not to prolong the action of the drug.
The amplitude is reduced to one-half and
the rate markedly slovi^ed or entirely sup-
pressed. Both rate and amplitude are
recovered, but not so quickly as with ether.
4. Chloroform on the mammalian
heart. Determine the action of chloroform
on the isolated cat's heart, using the method
described for the alcohol experiment 4,
page 3. Perfuse the heart with chloro-
form 0.02 to 0.05 percent in the Locke-
blood. The chloroform perfusion must be
for short periods and be guarded closely.
5. Chloroform on the muscular
irritability. See ether experiment 5, page
3. Use o.i percent chloroform water
in the vapor apparatus. Care must be
taken to remove the saturated chloroform
vapor from the vapor apparatus just before
using, otherwise the muscle, or the nerve
in the next experiment, will be over anes-
thetized and will not recover its irritability.
There is no danger with ether from this
6. Chloroform on the irritability of
the nerve. Repeat ether experiment 6.
Use o.i percent chloroform water in the
7. Chloroform on the blood-
pressure and on the respiration of a
mammal. Proceed as with ether experi-
ment 7, page 4, using chloroform to anes-
thetize the dog or cat (rabbits are too
sensitive to chloroform for use in this ex-
periment except in practiced hands). Re-
member that chloroform is said to be
about forty times as strong as ether in its
general effects on the animal body. If the vagi are intact and the animal
is anesthetized without tracheotomy there will be marked slowing of the
heart rate together with a sharp fall of blood-pressure. This effect is
Fig. 7. — Chloroform perfusion of the frog's heart. Ringer's solution used for the
normal, o.i percent chloroform between "on" and "off." Perfusion pressure
4 cm. Record from the suspended apex. Time in seconds.
eliminated by section of the vagi. With extreme care chloroform anesthesia
may be pushed to the point where respirations cease, and the animal be
recovered without artificial respiration. Often, however, in 5 to 10 seconds
Fig. 8. — The effect of chloroform on the irritability of the nerve in the muscle-
nerve preparation. The first two contractions represent the normal amplitude upon
stimulating the nerve of a preparation mounted in a moist chamber. The muscle was
allowed to hang through the opening in the floor of the moist chamber, so as to protect
it from chloroform vapor. At the point indicated by the word "on" chloroform vapor
was driven through the moist chamber. The nerve was stimulated at regular inter-
vals until no further contractions occurred. The chloroform was next removed
with moist air and the stimulations continued. After a short interval contractions
gradually resumed until they reached their normal maximum.
after respirations cease, the blood-pressure will suddenly sink to a low
level, and the heart will become weak and slow (see experiment 8 below).
14 EXPERIMENTAL PHARMACOLOGY.
a state from which recovery can be secured only by rapid and vigorous
Give chloroform intravenously in doses of lo to 20 c.c. of 0.5 percent
solution in saline, allowing plenty of time for recovery in each test. Com-
pare with alcohol and ether.
8. Chloroform on the secretion of the kidney. Anesthetize a dog
with morphine, i c.c. of i percent, and chloroform, avoiding deep anesthesia
during the preliminary preparations. Insert a ureter canula and connect
it with a horizontal glass tube mounted on a graduated scale, or see methods,
page 70. Take a continuous record of the arterial pressure.
Now determine the normal rate of secretion of urine per 10 minutes
for at least 40 minutes, keeping the dog under light but constant anesthesia.
Inject intravenously 10 c.c. of 0.5 percent chloroform solution in saline.
Double the dose if necessary until profound anesthesia with low blood-
pressure and weak heart is obtained. Or produce deep anesthesia by means
of the respiratory inhalations. Recover and maintain light anesthesia for
an hour or more. The circulation is quickly re-established in good condi-
tion, hilt the secretion of urine which is suppressed during the stage of
deep anesthesia is more slowly brought up to the normal with the re-establish-
ment of good circulation.
9. Chloroform on germinating seeds. Repeat the experiment
described for ether, page 11, passing air from saturated chloroform water into
one bottle of seed, and pure air into the other. After the seeds in air have
sprouted, reverse the bottles. Both seeds and young growing plants are
anesthetized by chloroform. The seeds may not grow later, as the drug kills
plant protoplasm when given beyond a rather narrow limit of both time
Experiments Showing the Effects of Chloral Hydrate. page.
1. On the frog. . 14
2. On the rabbit or cat. . . 15
3. On the heart of the frog. . . 15
4. On the heart muscle. . 15
I. Chloral hydrate on the frog. Give a hypodermic injection of
0.5 c.c. of 2 percent chloral hydrate dissolved in 0.7 percent saline.
Keep the animal in a moist battery jar until complete recovery. Give
particular attention to the effects on the circulatory and the nervous systems.
THE OPIUM SERIES. 1 5
2. Chloral on the rabbit or cat. Give a hypodermic injection of
2 c.c. of 2 percent chloral hydrate per kilo of body weight in saline.
Repeat in sixty minutes if necessary to produce the chloral narcosis. Make
close comparison with the effect of morphine and strychnine.
3. Chloral hydrate on the frog's heart. Pith a frog and take trac-
ings of the ventricle when irrigated over the surface with i percent chloral
hydrate. Or perfuse the heart with 0.2 percent chloral hydrate in
Ringer's solution and take tracings of the ventricle by the method described
on page 69. Note that the recovery period is unusually long.
4. Chloral hydrate on heart muscle. Prepare a ventricular strip
of the terrapin and establish contraction in a bath of 0.7 percent sodium
chloride as usual. Change to a bath of o.i percent chloral hydrate
in sodium chloride. Stronger solutions may suppress the rhythm entirely.
THE OPIUM SERIES.
Experiments Illustrating the Effects of Morphine, Codeine and The-
1. On the frog. 15
2. On the mammal. 15
3. On ventricular muscle. 16
4. On the frog's heart. 18
5. On the mammalian heart. 18
6. On the reflex reaction time. . 18
7. On the volume of air breathed. ... 18
8. The morphine group on the circulation and respiration in
the mammal. 18
1. Morphine on the frog. Give a dose of i c.c. of 2 percent mor-
phine acetate in physiological saline in the dorsal lymph sac. Keep the
animal under observation for two or more hours to secure the later efiects
in the frog. An instructive comparison is had by giving a dose of 0.5 c.c.
(8 minims) of o.i percent strychnine nitrate to a second frog at the same
time. Keep the frog under observation until complete recovery.
Give a second frog an injection of i c.c. of i percent codeine in phy-
siological saline. Compare with morphine.
The dose of thebaine for the frog is i c.c. of i percent.
2. Morphine on the mammal. Give a hypodermic injection of 1.5
c.c. of 2 percent morphine under the skin of the shoulder, see anes-
thesia, p. 65. Keep the animal
under observation for at least
two hours and note at intervals
the temperature, irritability,
respiratory, ocular, and other
changes under the influences of
the drug. If vomiting is pro-
duced a concentrated watery ex-
tract of the vomit will show the
presence of morphine, thus
demonstrating that excretion of
morphine takes place into the
3. Morphine on the ven-
tricular muscle. Prepare a
strip of the ventricle of a terrapin
and get it into regular contrac-
tions by a bath of 0.7 percent
sodium chloride. Change the
strip to a bath' of i percent
morphine in sodium chloride
for five minutes, or until a
marked change in the rate and
amplitude of the contractions
occurs, after which the strip
should be returned to saline.
Record the contraction on a
smoked drum moving with a
speed of I to 2 mm. a second.
This experiment shows that the
contractions of cardiac muscle
are weakened and slowed under
morphine. Repeat, varying the
conditions according to the re-
sults of the previous experiment.
A stronger solution of morphine
will inhibit all heart muscle
activity for many minutes or
even hours. The proper
strength of codeine for this
THE OPIUM SERIES.
lo EXPERIMENTAL PHARMACOLOGY.
experiment is, 0.5 percent, in physiological saline, of thebaine about i
percent soliition. V ' ^ i'"
4. Morphine on the f fog's heart. PitH at fi-og, expose tKe heart
and take a record of' its contrafetions, either by the perfusion or by, the sus-
pension method. Test the irritability of the vagus' t^unk. If the circulation
is still effective give, a lymph saC injection of 0,5 c.c. of 10 percent mor-
phine acetate of apply drops ol this solution directjy to the heart from the
dropping bottle. Retest the effectiveness of thei Vagus stiniulation. - If the
heart is perfused, the effective solution to use is 0.5 percent of morphine
acetate in Ringer's solution. ; '
5. Morphine on the mammalian heart.— Etherize a cat, collect its
blood and isolate its heart as described in alcohol experiment 4. Fill the
tubes of the mammalian heart perfusion apparatus, bring the apparatus to a
temperature of 36° C, adjust the heart in it and begin the normal perfusion.
When the heart is contracting with a regular and relatively even rhythm
perfuse it for two minutes with 0.5 percent morphine in Locke-blood solu-
tion. Repeat the experiment using i percent solution. If the perfusion
is too prolonged the heart depression will be removed only with the greatest
6. Morphine on the reflex reaction time. Test the reaction time
in a reflex frog in the usual way, see methods page 71, first on the normal
animal, then 20 and 40 minutes after a dose of i c.c. of 4 percent morphine
7. Morphine on the voltmie of air breathed. Anesthetize a rabbit
(or cat) with two grams urethane and ether, administering the latter at
perfectly regular intervals and with a constant number of drops. Keep a
continuous record of the respiration rate per minute either by counting or by
recording on a smoked drum. Measure the respiration volume as follows:
Insert a tracheal canula and connect it with an apparatus for measuring the
volume of expired air. Ether can be given through the open' tube of the
apparatus except when actually 'measuring the air volume. Fill the grad-
uated cylinder with water. Then measure the voliime of eight or ten expi-
rations, according to the volume of the apparatus used, and repeat several
times to secure reliable averages, of the expiratory volume of the etheriz;ed
animal. Compute the expiratory volume per minute. Now give i c.c. of
2 percent morphine hypodermic. Remeasure the respiratory rate and
expiration volume at intervals of 10 minutes.
8. The morphine group on the circulation and on respiration in
the mammal. Anesthetize a dog with chloroform (no morphine), take the
THE OPIUM SERIES. I9
blood-pressure from the carotid, insert a tracheal canula and take the
respiration by the intratracheal method. Insert a canula into the saphen-
ous vein and connect with a buret for intravenous injections. (One may
readily insert a canula into the ureter and follow the secretion of urine
under the morphine. Consult the instructor.)
Give an intravenous injection of 2 c.c. of 2 percent morphine in saline.
The injected solution should be about the temperature of the body. Repeat
after ten minutes, using 4 c.c. When equilibrium is reestablished give
2 c.c. of I percent codeine. Give 2 c.c. of i percent thebaine. Give
thebaine first if there is an opportunity to make the test on a second animal.
Now cut the vagus nerves and repeat the dose of 2 c.c. of 2 percent
The anesthetic must be gradually diminished according to the amount
of morphine, etc., that has been injected. Use artificial respiration if
20 EXPERIMENTAL PHARMACOLOGY.
Experiments Illustrating the Effects of Caffeine. page.
1. On the frog . . .... 20
2. On the ventricular muscle . . 20
3. On the frog's heart . . .20
4. On muscular irritabiUty and on muscular work. . 21
5. On voluntary work of human muscle . . .21
6. On the reaction time in man . 22
7. On the reflex reaction time in the frog. . . 22
8. On the mammalian heart . . . . . . 23
9. On the circulation and respiration in the mammal 23
10. As a diuretic . . 24
1. Caffeine on the frog. The dose is i,c.c. of 0.5 percent caffeine
in the dorsal lymph sac. There is usually a great increase in the irritability
together with muscular cramps in the later stages, and finally paralysis.
There may be considerable opisthotonus from the direct muscular effects at
the area of injection. Note the recovery stages when the frog is kept in a
moist battery jar.
2. Caffeine on the ventricular muscle. Record the contractions of
a strip of terrapin's ventricle beating in physiological saline on a slow-speed
drum. Change the strip from saline to o.i percent caffeine in saline for
five minutes or less, then back to saline until the contractions become uni-
form and typical of the saline curve. Repeat, varying the tim'ig of the
immersion in the drug. Increase the strength of the solution to 0.5 per-
cent. Ref. — Lingle; Am. Jour. Physiol., VIII, 75.
3. Caffeine on the frog's heart. Expose the heart of a pithed frog
and adjust a balanced lever on the ventricle. Irrigate the surface of the
heart with physiological saline from a dropping bottle for a few minutes, then
change the irrigating fluid to i percent caffeine in saline for five minutes,
after which return to saline irrigation. Repeat once or twice, then apply
caffeine continuously until the maximum effect is obtained. Compare
especially the auricle with the ventricle in the later stages of the caffeine
When the heart is perfused through one of the veins then the solution
of caffeine should not be stronger than o.ixto 0.2 percent, and the record
should be taken by the apex suspension method. This method yields the
more accurate results.
4. Caffeine on muscular irritability and muscular work. Lay
a tight ligature around the thigh of a frog to close off the circulation in one
leg. Give a dorsal lymph sac or abdominal injection of i c.c. of 0.5 percent
caffeine. Allow twenty minutes for absorption, then pith the frog and
ligate off the caffeinized gastrocnemius. Determine the irritability by
the minimal stimulus method and, second, determine the amount of
work the muscle will do when stimulated directly once in two seconds
Fig. 12. — Action of caffeine on the ventricular muscle of the terrapin. Between
the marks X-X the strip was immersed in 0.5 percent caffeine in saline. Before and
after the caffeine the strip contracted in normal saline.
until completely fatigued. Use a constant load of 50 grams in this experi-
ment. Prepare first the normal gastrocnemius as quickly as possible and test
its irritability and the amount of work it will do with uniform load and
method of stimulation. Repeat the irritability test and the determination
of work using the caffeinized muscle. If the records are taken on the
same recording paper, one above the other as in experiment 5, page 3,
the comparison is very sharp.
5. Caffeine on the voluntary work of human muscle. Measure
the amount of work of the flexors of the middle finger by means of a Mosso's
ergograph while lifting a 3 to 4 kilo weight, one that exhausts the muscle in
about 50 contractions. Repeat in thirty minutes. Consider these as
normals. Take two cups of strong coffee or 0.3 gram of caffeine in
sweetened warm water. Remeasure the work of the flexors as directed
above 60 and 90 minutes after taking the caffeine. The amount of the
muscular work is usually markedly increased by caffeine. See the next
6. Caffeine on the reaction time in man. Arrange a tuning fork
vibrating one hundred times per second and a set of signal keys for measur-
ing the reaction time to touch. (For details see directions in StirHng's
Practical Physiology, page 325.) Determine the normal reaction, then the
reaction time at 30, 60, 90 and 120 minutes after a dose of 0.3 gram
Fig. 13. — Caffeine effect on the amount of work of the gastrocnemius of the frog.
The upper tracing is the normal, the lower the opposite muscle after the absorption of
caffeine in water. This experiment may be performed together with the
7. Caffeine on the reflex time in a frog. Prepare a reflex frog
by destroying the brain and the medulla. After the shock has passed away
determine the reflex reaction time to electrical stimuli applied to the toe of
the suspended frog. This time is easily recorded by a paper writing point
attached to some part of the foot itself. Give a dose of i c.c. of 0.5 percent
caffeine in the dorsal lymph sac. After 30 minutes redetermine the reflex
time. Draw conclusions from averages here, as the error of procedure is
8. Caffeine on the mammalian heart. Etherize a cat, bleed it,
and dilute the blood with Locke's solution as in the alcohol experiment 4.
Prepare the apparatus and raise its temperature to 36° C. Isolate the heart
and quickly insert it in the cardiograph and start the circulation of the
Locke-blood solution. When the rhythm is regular, perfuse with o . o 2 to 0.2
percent caffeine in Locke-blood solution. It is better to begin with a
weaker perfusion solution of caffeine and increase the strength through two
or three grades, say from o.i to 0.5 percent.
Fig. 13 (Continued).
The stimulus was repeated at regular intervals once in two seconds until exhaustion.
0.5 c.c. I percent caffeine.
9. Caffeine on the circulation and respiration of the mammal.
Give a dog a hypodermic of i c.c. of 2 percent morphine and anesthetize
with chloroform. Tracheotomize. Take the arterial blood-pressure from
the carotid and the respiratory pressure from the trachea. Insert a canula
in the saphenous vein for intravenous injections. Take records on the
continuous paper kymograph. After securing a normal record of both the
blood-pressure and the respiration give an intra veous injection of 5 to 10 c.c.
of 0.5 percent caffeine in physiological saline. Give slowly from a warmed
buret. This experiment should begin with the small doses and the doses be
gradually increased. See the next experiment on diuretic action.
24 EXPERIMENTAL PHARMACOLOGY.
10. Caffeine as a diuretic. Prepare a dog as in the preceding experi-
ment. Take the blood-pressure and the respiratory rate. Insert a caiiula
for venous injections. Open the abdomen in the median line, seek out one
ureter near its connection with the bladder, ligate and insert a urethral
canula, taking care to make an unobstructed connection. After the flow
of secretion has been established, a 2 to 3 mm. rubber tube is connected with
the canula and the abdominal opening sewed up. Connect the rubber tube
with a I c.c. pipet graduated in o.i c.c. Mount the buret horizontally.
Read the rate of secretion by injecting a small bubble of air each five min-
utes into the rubber tube at its connection with the buret.
Determine the rate of secretion under constant anesthesia both before
and after 5 to 10 c.c. of 0.5 percent caffeine. Take readings every five
minutes for a period of about two hours.
List of Experiments Showing the Effects of Strychnine. page.
1. On the frog ... . .24
2. On a mammal, demonstration .... . . 24
3. On the ventricular strip ... -25
4. On the frog's heart and cardiac nerves 25
5. On the irritability and work of voluntary muscle .... 26
6. On reflex irritability and reaction time. . . . . 26
7. Local action on the spinal cord . 26
8. Spasms depend upon cutaneous stimulation 27
9. Absorbed slowly from the stomach or bladder and readily
from the intestine or peritoneum . . 27
10. Stored in the spinal cord 27
11. On the mammalian heart 28
12. On the blood-pressure and respiration rate of mammals. 28
1. Strychnine on the frog. Give a frog a toxic dose of strychnine
nitrate, 0.3 c.c. (5 minims) of o.i percent. Give in the dorsal lymph sac.
Note the time of the appearance and the successive stages of increased
irritability, convulsions, and paralysis. Take fresh frogs and determine the
limits of the therapeutic or non-toxic dose, i.e., determine the dose per gram
of body weight that will just fail to produce convulsions.
2. Strychnine on the mammal. Demonstration. Give a rabbit a
hypodermic injection of 0.4 c.c. of 0.1 percent strychnine nitrate per
kilo of body weight. Consult Cushny's Pharmacology for symptoms.
Meltzer gives the toxic dose for rabbits as 0.5 mgr. per kilo. Reference,
Am. Jour. Physiol., Volume IX, page i.
3 . Strychnine on the ventricular strip. Suspend a strip "of terrapin's
ventricle in physiological saline and, when it is contracting regularly, subject
it to a bath of o.i percent strychnine nitrate in saline for five minutes.
Return the strip to physiological saline until the contractions are unques-
tionably of the saline type. The rhythm and amplitude are both reduced.
Fig. 14. — Action of strychnine solution on the ventricular muscle of the terrapin
In trace a the strip was contracting in physiological saline. At the vertical arrow it
was transferred to 0.5 percent strychnine. The contractions are gradually slowed and
weakened. Tracings b to g are successive records around a six-inch drum during the
recovery which is very prolonged in this experiment.
4. Strychnine on the heart and cardiac nerves. Pith a frog, expose
the heart, take a direct tracing of the movements of the ventricle. Test
the vagus activity by stimulating the vago-sympathetic trunk with a current
that produces complete inhibition. Next irrigate the surface of the heart
from a dropping botde with o.i percent strychnine nitrate in physiological
saline. While continuing the irrigation stimulate the vagus trunk at inter-
vals of 5 to 10 minutes. Look for a progressive effect on the beat, the rate,
and on the cardiac nervous mechanism as demonstrated by th€ results of the
5. Strychnine on the irritability and work of voluntary muscle.
Ligate the thigh of one leg of a frog to occlude its circulation, give a dose
of 0.2 c.c. of o.i percent strychnine nitrate. In twenty minutes ligate
off the other leg, pith the frog (pith immediately if tetanic contractions
appear earlier), and determine the irritability of the normal and of the
drugged gastrocnemii by the minimal stimulus method.
Follow the above test by measurement of the work the muscles will
do, testing first the normal, and second, the strychnine muscle. Use the
method described for experiment 5 under alcohol. The irritability and
the work of voluntary muscle are both greatly increased in the therapeutic
stage, in sharp contrast to the effect of strychnine on heart muscle.
I O ,3 C - 0,X.J- H Q
■ Son -t-Cof/r/r! -J/Zi/oJi
Fig. 15 — Showing the action of strychnine on the muscular work and on amplitude
of the single contractions in the frog's gastrocnemius. The lower series is of the normal
muscle, the upper of the strychninized muscle. The dose was 0.25 c.c. of o.i percent
strychnine hydrochloride injected into the dorsal lymph sac, after ligation of one leg at
the thigh. Absorption was allowed until the first spasms when the frog was pithed.
Weight of frog 40 grams. Load 200 grams. One stimulus in three seconds.
6. Strychnine on reflex irritability and reaction time. Prepare a
reflex frog, i. e., destroy only the brain including the medulla. After the
shock has passed away, one hour or more, determine the reflex reaction time
to electrical stimulation of the toe by the method given on page 71. Now
give 0.5 c.c. (8 minims) of 0.02 percent strychnine nitrate. After each
ten minutes take the reaction time to electrical stimulation until spasms
appear, which ought to be under 60 minutes.
7. Local action of strychnine on the spinal cord. Cut the cord of a
frog at the base of the medulla and destroy the brain. Free the cut end of
the cord from the surrounding tissue, and carefully paint it with i percent
strychnine nitrate solution. Muscular spasms will be produced or will
follow stimulation of the toes of the fore leg. Or stimulate the toe of the
hind leg — reflexes of an orderly nature occur, where no general tetanic con-
vulsions have been induced by the dose. Pith the cord, all spasms cease.
8. Strychnine spasms depend also upon cutaneous stimulation.
Strychninize a frog and when the spasms are strong and continuous paint
the skin with a 2 percent cocaine solution. The cocaine paralyzes the
cutaneous sensory apparatus whereupon the convulsions cease. Dip the
frog in water to remove the excess of cocaine, its local effect will disappear
in about 10 minutes and the strychnine convulsions will reappear.
9. Strychnine is absorbed very slowly from the stomach or blad-
der, but very readily from the intestine and body cavity. Anesthetize
a half-grown fasting cat. Ligate both the cardiac and the pyloric orifices
ton ■OlJ.Sir-ycknins in Locke-Blocl
I [ I I 1 I I 1 I
Fig. 16. — Strichnine on the isolated mammalian heart. The heart was perfused
with Locke's solution containing 10 percent of the animal's own blood. Between the
points marked on and of, a total of 50 seconds, the heart was profused with o.oi percent
strychnine hydrochloride. The stock solution of the drug had been carefully neutralized
to eliminate any trace of acid effects. Temperature 34° C. Time in seconds.
of the stomach. Inject 10 c.c. of o.i percent (10 mgrs.) of strychnine
nitrate into the stomach. If no spasms occur in 30 minutes then cut the
pyloric ligature apd run the stomach content into the intestine. Spasms
may be looked for in two minutes or less. If enough strychnine is absorbed
from the stomach to produce muscular spasms repeat the second half of the
experiment on a second animal, injecting the drug directly into a portion of the
Compare absorption from the urinary bladder and from the abdominal
cavity in the same manner.
10. Strychnine is stored in the spinal cord (Lovett, Jour. Physiol.,
Volume I, p. 99). Inject 10 mgs. strychnine nitrate into the dorsal
lymph sac of a large bull frog. Allow 30 minutes for absorption. Then
remove the skin and wash away all traces of strychnine that may re-
28 EXPERIMENTAL PHARMACOLOGY.
main unabsorbed. Take the cord, also an equal amount of other tissue,
macerate each in 0.7 percent saline. Inject equal portions of the extracts in
the dorsal lymph sacs of two frogs. Allow hours, if necessary, for the symp-
toms to develop. This method will detect traces of strychnine too small for
1 1 . Strychnine on the mammalian heart. Arrange the apparatus
for the isolated mammalian heart, bring it to a temperature of 34° to 36° C.
Anesthetize a cat or rabbit with ether, bleed and defibrinate the blood and
dilute it to ten volumes with Locke's solution. Quickly cut out the heart,
insert the canula into the aorta and ligature it, attach the cardiograph, and
start the perfusion of Locke-blood solution. Perfuse with 0.005 percent
strychnine hydrochloride for 30 secods. The dose may be increased to
o.oi or even 0.02 percent but recovery from the latter is very slow and
12. Strychnine on the blood-pressure and on the respiration rate
of mammals. Anesthetize a lo-kilo dog. Take a continuous record of the
blood-pressure from the carotid and of the respiration rate from the trachea.
Give an injection of i c.c. of o.i percent solution of strychnine nitrate
from a hypodermic into the saphenous vein. One should give close atten-
tion to the symptoms of this mild dose which will produce little more than
the therapeutic effects. Repeat this injection until the cumulative dose pro-
duces convulsions of a mild character. Note that the convulsions may be
suppressed here by giving more chloroform. Give especial attention to the
blood-pressure conditions during the tetanic spasm, so as to eliminate the
strictly passive mechanical factors.
List of Experiments Showing the Action of Cocaine.
1. On the frog. ...
2. On local sensory surfaces.
On the heart muscle.
On the frog's heart. .
On muscle work. . .
6. On the circulatory and respiratory systems of the mammal
1. Cocaine on the frog. Give a frog a dorsal lymph sac injection
of 0.4 CO. of 0.5 percent solution of cocaine hydrochlorate in physio-
logical saline. Observe the symptoms in the usual way. Also examine
the vifhite corpuscles of the blood for motility ais compared with the unpoisoned
2. Cocaine on local sensory surfaces. Paint one-half the surface
of your own tongue with a brush wet in 2 percent solution of cocaine
Fig. 17. — Cocaine effects on the frog's heart when perfused in o.ooi percent
Ringer's solution. Time in seconds.
hydrochloride. Use care not to swallow any of the solution. In 8 to 10
minutes compare the sensitiveness of the two halves of the tongue to electri-
cal currents by the minimal stimulus method. Test for taste sensations of
sweet, of salt. Note also the personal sensations of any character result-
ing from the experiment.
Give one drop of i percent cocaine in the right eye; repeat in
three minutes. There is a loss of sensitiveness and the eyeball may be
30 EXPERIMENTAL PHARMACOLOGY.
touched without pain. Compare the pupils as to size, as to reaction to
light. Determine the acuteness of vision of each eye separately at the read-
ing distance. Cocaine is an analgesic, but not a perfect mydriatic.
3. Cocaine on the heart muscle. Prepare a strip of terrapin's
ventricle and vs^hen it is beating in physiological saline in good rhythm
submit it for from three to five minutes to a bath of o.oi percent cocaine in
saline. Record the contractions on a slow drum.
Fig. 18. — Effect of cocaine on the muscular work of the frog's gastrocnemius.
4. Cocaine on the frog's heart and its nervous mechanism. Pith
a frog, expose the heart. Take a continuous record of its contractions by
the usual method. Irrigate its surface with physiological saline. Test the
irritability of the vagus trunk using a medium strength stimulus. Now
irrigate the heart for one minute with 0.2 percent cocaine in saline followed
by saline. When the cocaine contractions have somewhat recovered, retest
the inhibitory power of the vagus. Repeat the test,using two or three
drops of I percent solution, and do not wash it off afterward.
Perfuse the heart with 0.002 percent solution of cocaine in Ringer's
weaker solution. This method gives more constant results than does the
irrigation and it is to be preferred.
5. Cocaine on muscle work. Ligate one leg of a frog at the thigh, or
use the method described under alcohol. Give a dose of 0.4 c.c. (7 minims)
of 0.5 percent cocaine in the dorsal lymph sac. Allow 20 minutes for
absorption then ligate the cocainized leg. Load with a 50-grain weight.
Take records of the contractions of the normal muscle on a drum with a
speed of i mm. per second. Stimulate with single break induction shocks
once in two seconds until exhausted. Prepare the cocainized gastrocnemius,
mount and stimulate with the same rate and load. If the two records are
parallel on the same paper, see figure 18, it will demonstrate the comparative
difference in work done. Calculate the amount of work per gram of muscle
in each of the two preparations.
Fig. 18 (Continued).
The endurance of the cocainized muscle is greatly increased.
6. Cocaine on the circulatory and respiratory systems of the
mammal. Give morphine and chloroform to a dog. Insert a tracheal
canula. Take the blood-pressure and respiratory records on the continuous
paper kymograph. Insert a canula and connect a buret with the saphenous
vein. Inject 2 c.c. of i percent cocaine very slowly while watching
the blood-pressure as an indicator.
Experiments on the Effect of Quinine.
1. On the frog. . . . .
2. On the frog's heart.
3. On the striated muscle . . .
1. Quinine on the frog. Inject i c.c. of o.i percent solution of
hydrochlorate of quinine into the dorsal lymph sac. In addition to the
usual observations, examine the blood of this frog as regards the motility
of the white corpuscles. Compare with the blood of a normal frog.
2. Quinine on the frog's heart. Pith a frog, expose the heart, and
take a record of its contractions with physiological saline while perfusing
it from a perfusion bottle. Change the Ringer's fluid to 0.05 percent
quinine hydrochlorate in Ringer for about one minute. Repeat after
Fig. 19. — Action of quinine on the frog's heart. The perfusion method was used.
Normal rate with Ringer's solution. Strength of quinine 0.05 per cent. Timei in
recovery, using' i percent quinine. Continue this perfusion until no further
contractions are secured. Examine the condition of the ventricle at the close
of the experiment.
Vary this experiment by pithing the frog, taking care to lose little blood
and making a record from the ventricle.- Now give a lymph sac injection
of I c.c. of I percent quinine and take a continuous record through 20 to
3. Quinine on the striated muscle. Ligate one leg of a frog at
the thigh, inject i c.c. of o.i percent quinine into the dorsal lymph sac.
In just 20 minutes ligate off the other leg and pith the frog. Determine
the irritability, first of the normal then of the drugged muscle. Determine
the work each gastrocnemius will do.
Experiments Showing the Action of Atropine. page.
1. On the frog . 33
2. On the heart muscle 33
3. On the frog's heart and on the cardiac nervous apparatus . 34
4. On the secretory nerves of a mammal 34
5. On the isolated heart of the cat . . -35
6. On the circulatory and respiratory systems of the mammal . 35
7. On the eye 36
8. On man in the therapeutic dose 36
9. As secreted by the kidney 36
10. Scopolamine on the frog . 36
I. Atropine on the frog. Give a frog an injection of i c.c. of i
percent of atropine sulphate. Keep it under observation in a moist
battery jar until complete recovery. The toxic dose is i c.c. of 3 percent.
Atropine -001^ i^^
Fig. 20. — Action of atropine on the heart muscle of the terrapin's ventricle.
The normal solution is sodium chloride, the strength of atropine o.ooi percent. The
decrease in amplitude is usually not so great as in this tracing, or is even absent entirely
Time in seconds.
2. Atropine on the heart muscle. Mount a ventricular strip from
the terrapin in 0.7 percent sodium chloride, and when it is contracting with a
uniform amplitude and regular rhythm change to o.ooi percent atropine
in physiological saline for five minutes, see figure 17. Recover the charac-
teristic rhythm in saline and repeat using 0.002 percent atropine in saline.
3. Atropine on the frog's heart and cardiac nervous apparatus.
Pith a frog, expose the heart and take a tracing. Determine an effective
strength of stimulus for the vagus. Now irrigate the heart for one minute
from a dropping bottle containing o.i percent atropine in saline. Stimu-
late the vagus immediately and once every five minutes or less. Atropine
eliminates the vagus control of the heart by poisoning the peripheral endings.
The atropine effect is antagonized by physostigmine, page 44, experiment 3,
and by muscarine.
4. Atropine on the secretory nerves of a manunaL Anesthetize
a 10 jkilo dog with morphine and chloroform. Expose and tie a canula in
I Fig. 2j:. — Action of atropine on the isolated heart of the cat. Locke-blood solution
the arrows was .001 percent. Temperature 39° C. Pressure 85 cm. of water. The
the subinaxillary' duct. Expose and stimulate the chorda tympani nerve
in the hilus of the gland, noting the rate of secretion by the drops of saliva
per minute from the canula. Give a hypodermic injection of 1.5 c.c. of
I percent atropine. Stimulate the chorda tympani nerve again. No
secretion is obtained even though the nerve is stimulated down close to the
hilys of the gland.
This demonstration may be made in part as follows: Produce a rapid
flow of saliva in the dog by a hypodermic of i c.c. of 0.2 percent pilocarpine.
Observe the .flow by turning out the dog's upper lip. Follow with a hypo-
dermic dose of I c.c. of I percent atropine. The secretion stops.
5. Atropine on the isolated heart of the cat. Anesthetize a cat
with ether. Bleed it and prepare the Locke-blood perfusion fluid. Isolate
and suspend the heart in the perfusion apparatus. Obtain a normal record
then perfuse with .001 percent atropine in Locke-blood solution.
6. Atropine on the circulatory and respiratory systems in the
mammal. Anesthetize a lo-kilo dog as in experiment 4 preceding. Place
an arterial canula in the carotid and insert a tracheal canula. Take a con-
tinuous record of the blood-pressure and of the respiration. Stimulate the
peripheral end of the sectioned vagus with a stimulus that produces complete
inhibition of the heart. Stimulate also the central end of the vagus. Now
was used for the normal perfusion fluid,
time in seconds.
Fig. 21 (Continued).
The strength of atropine perfused between
give an intravenous injection of i c.c. of o.i percent atropine. Note
the exact time of the injection on the record by a signal pen. When the
equilibrium is again established, re-stimulate the ends of the sectioned vagus
with the same strengths of stimulus used before atropine was given. Note
that the heart rate is no longer slowed on stimulation of the peripheral
vagus, but that the pupil still actively dilates after this dose when the
central end of the vagus trunk is stimulated. Atropine also destroys the
vagus control over the smooth muscle of the alimentary tract thus decreasing
Physostigmine is an antagonist to atropine. Try i c.c. of o.i percent
36 EXPERIMENTAL PHARMACOLOGY.
intravenous. Use artificial respiration if necessary. Give a second injec-
tion of atropine later. See figure 23 for a reverse antagonism.
7. Atropine on the eye. Drop i or 2 drops of i percent atropine
in the right eye of a dog or cat. The pupil will be widely dilated in a few
minutes. Keep the animal under observation until the effect entirely dis-
appears, often only after several days. Atropine destroys the power of
accommodation and it is used for this clinical purpose in eye practice.
Students should not use atropine on their own eyes, but a mild dose of hom-
atropine, 2 or 3 drops of i percent, the effect of which passes off in 24
to 36 hours, may be tested in one's own eye. In such experiments test the
accommodation, light reflex, and size of the pupil.
8. Atropine on man in therapeutic dose. Test on yourself the
action of a dose of 1/120 to 1/60 grain of atropine by way of the mouth.
Note the effects on the heart rate, pulse character, respiration, size of pupil,
light reflex and sensations.
9. Atropine is secreted by the kidney. — ^This may be demonstrated
on the rabbit which is very tolerant of the drug. Give a rabbit urethane.
Collect the urine from a bladder canula. Give a large hypodermic injection,
2 c.c. of 2 percent atropine, and test the rabbit's urine on the eye of a cat or
dog. The atropine may be extracted (Binz). Concentrate a large amount
of urine, add ammonia, shake up with chloroform, evaporate, dissolve the
residue and test on the eye of a cat or a dog.
10. Scopolamine on the frog. Give a dose of i c.c. of i percent
scopolamine in the dorsal lymph sac of a frog. Compare with the effects
of an equal dose of atropine in experiment i above.
Experiments Illustrating the Action of the Nicotine. page.
1. On the frog. . . -37
2. On the ventricular muscle. . 37
3. On the frog's heart and its nervous apparatus. . . 37
4. On the nerve fiber and on nerve ganglia. . . -37
5. On the mammalian heart. 38
6. On the circulatory system and on the respiratory nervous
mechanism . .... 38
7. On muscle irritability . . . 39
1. Nicotine on the frog. Give an injection of a 0.5 c.c. of 0.2
percent nicotine into the dorsal lymph sac of a frog.
2. Nicotine on the ventricular muscle. Prepare a terrapin's heart
strip and when it is contracting rhythmically in 0.7 percent physiological
saline, immerse the strip in a 0.05 percent solution of nicotine in saline
for two minutes, then return to the saline bath. Repeat. If the solution is
too strong the strip will exhibit a strong tonus with incomplete relaxations.
The amplitude and the rate are markedly increased.
3. Nicotine on the frog's heart and its nervous mechanism.
Pith a frog, expose the heart and take tracings on a drum with a speed of
2 mm. per second. Stimulate the vago-sympathetic with an interrupted
current that just causes complete inhibitions. Now irrigate the heart from
a dropping bottle with o.i percent nicotine in 0.7 percent saline, and
stimulate the vagus at intervals of two minutes. If the nerve stimulation
ceases to be effective, then apply the electrodes directly to the sinus.
To demonstrate the stronger effects on heart muscle prepare a second
frog. Take a tracing of the heart. Apply a few drops of i percent solu-
tion of nicotine.
4. Nicotine on the nerve ganglia and on the nerve fiber. Anes-
thetize a rabbit (or cat or dog), dissect out the cervical sympathetic and
the superior cervical ganglion. Stimulations of the nerve or of the ganglion
lead to vasoconstriction in the ear and dilation of the pupil. Paint the
nerve below the ganglion with i percent nicotine. Stimulation at a point
still lower down shows that the nerve impulses still pass undisturbed.
Now paint the ganglion itself. Stimulate the nerve below the gangUon, also
38 EXPERIMENTAL PHARMACOLOGY.
the ganglion directly. What Conclusions ? See also the next experiment.
(Ref.: Langley and Dickinson, Journal of Physiol., Volume II, page 265.)
5. Nicotine on the mammalian heart. Prepare the mammalian
heart perfusion and recording apparatus and bring it to a temperature of
36° C. Etherize a cat, bleed, defibrinate the blood, and dilute to one-in-ten
of Locke's solution. Quickly take out the heart, suspend it in the apparatus
and start the perfusion. When the heart is beating well, perfuse it with
o.ooi percent of nicotine in Locke-blood solution. There is a constant
Fig. 22. — Action of nicotine on the ventricular muscle from the terrapin. The
strip was contracting in physiological saline when transferred to 0.2 percent nicotine in
saline at the point marked. The solution was too strong and was removed after about
forty seconds. Time in seconds and half-minutes.
sharp increase in amphtude with a more slowly developed increase in
rate. The after amplitude may remain greater than the preceding normal.
6. Nicotine on the circulatory and respiratory nervous mechan-
ism. Anesthetize a dog or cat (the animal used in experiment 4 above
may be used for this experiment also). Take a blood-pressure from the
carotid and a respiration tracing from the trachea, (i) Determine an effect-
ive stimulus for the heart and respiration. (2) Now inject 5 c.c. of o.l
percent solution of nicotine into the saphenous or jugular vein. Repeat
the dose if necessary, until distinct effects are produced on the heart rate and
blood-pressure. (3) Stimulate the vagus at first with the strength of stimu-
lus used before the injection, then with successively stronger stimuli. An
instructive picture is obtained by dissecting down to and stimulating the
cardiac branches from the annulus of Vieussens, which may be done in
the dog on the left side without opening the thorax.
Fig. 23. — Action of nicotine on the isolated cat's heart. The heart was contract-
ing in Locke-blood solution when it was perfused with o.ooi percent nicotine between
the points marked by the arrows. The increase in amplitude is more marked when
the heart is beating weaker at the time of perfusion. Time in seconds.
7. Nicotine on muscle irritability. Lay a ligature around the
thigh of one leg of a frog and then give i c.c. of o.i percent nicotine
in the dorsal lymph sac. After 20 minutes test the irritability of the normal
and of the nicotinized gastrocnemius muscles by the minimal and maximal
40 EXPERIMENTAL PHARMACOLOGY.
Experiments on the Effect of Curare. page.
1. On the frog. .... 40
2. On the motor nerve endings. ... 40
' 3. On the heart muscle and on the cardiac nervous mechan-
ism. . 40
4. Poisons the motor endings before the other portions of the
reflex arc. . . . . 41
5. On the mammal. . 41
1. Curare on the frog. Give a frog a hypodermic of 0.3 c.c. (5
minims) of 0.2 percent curare. The motor apparatus is paralyzed, but
the circulation continues and the frog will recover in from one to three days,
respiration being maintained through the moist skin if the animal is kept in
a covered jar.
2. Curare on the motor nerve endings, Bernard's experiment.
The specific action of curare was demonstrated by Claude Bernard to be on
the motor nerve end plates. Ligate one leg of a frog to shut off the circula-
tion, give a hypodermic of 0.3 c.c. of 0.2 percent curare. When general
paralysis is secured, perform the following tests, interpreting the results
through the effect on the gastrocnemius muscles:
a. Stimulate the sciatic nerve on the unligated leg.
b. Stimulate the gastrocnemius of the unligated side.
c. Stimulate the sciatic nerve on the ligated side above the ligature.
d. Below the ligature.
e. The corresponding muscle. Conclusions.
3. Curare on the heart muscle and on the cardiac nervous appa-
ratus. While minimal doses of curare suffice to poison the motor end plates,
it takes relatively large doses to paralyze the cardiac nervous apparatus.
The paralysis apparently affects the ganglionic nerve endings first and then
the cardiac motor endings and muscle. Pith a frog, expose the heart, pre-
pare the vagus trunk for stimulation and adjust a heart lever for record.
Allow physiological saline from an irrigating bottle to run over the heart.
Take a normal record and then stimulate the vagus nerve. Now irrigate
slowly with 0.2 percent curare in saline and stimulate the nerve 10 seconds
at a time at intervals of 10 minutes for several tests.
4. Curare poisons the motor endings before the other portions
of the reflex arc. Tie a ligature on a frog's leg at the thigh, inject 0.3
c.c. of 0.2 percent curare. Just as voluntary activity ceases stimulate the
skin of the poisoned leg. The unpoisoned gastrocnemius will contract.
Rapidly expose and stimulate the poisoned sciatic. The poisoned gastroc-
nemius will not contract, while the unpoisoned one will, owing to reflex
stimulation through the cord.
5. Curare on the mammal. Morphinize and chloroform a dog.
Take blood-pressure. Introduce a tracheal canula and take the respiratory
record by the intra-tracheal method. Arrange the apparatus for artificial
respiration when needed. Inject into a vein 5 c.c. of i percent curare.
All movements of voluntary muscles will quickly cease including respiratory
movements. The heart rate and the blood-pressure remain good, and if
artificial respiration is applied the circulation can be maintained for several
hours, or until the drug is eliminated and recovery occurs.
Experiments Showing the Action of Pilocarpine.
1. On the frog
2. On the mammal
On the ventricular muscle . ....
On the frog's heart 43
On the circulatory and respiratory systems of the mammal . 43
I. Pilocarpine on the frog. Give a frog a hypodermic injection
of 0.6 c.c. of 10 percent solution of pilocarpine nitrate. Keep the
frog in a moist battery jar until normal again. The toxic dose is I c.c. of
10 percent solution of pilocarpine.
Fig. 24. — Blood-pressure and respiratory effects of an intravenous injection of i
c.c. of I percent pilocarpine in the dog. Injection between the points "on" and
"off." Time in seconds. Pressure in mercury. Reduced to four-fifths the original size.
2. Pilocarpine on the mammal. Give a dog a hypodermic injection
of 0.3 c.c. (5 minims) i percent pilocarpine. This dose produces a
marked secretion by glandular structures. Examine the flow of saliva by
turning back the upper lip, drying it and noting the accumulation of drops
at the mouth of the salivary duct. An injection of i c.c. of o.i percent
atropine in a vein antagonizes pilocarpine and stops the secretion.
Give additional drops of i percent pilocarpine in the eye. Drops
of I percent atropine on the eye will overcome the action.
3. Pilocarpine on the ventricular muscle. Mount a strip of the
terrapin's ventricle in physiological saline. When the contractions are
regular transfer to a o.i percent pilocarpine solution in saline. Allow it
to act only i minute then renew the physiological saline bath. As a final
test give the strip a continuous bath of 0.1 percent pilocarpine and note
the successive effects.
4. Pilocarpine on the frog's heart. Pith a frog, expose the heart
and take tracings of the ventricle. Test the activity of the vagus with a
strong interrupted current. Now irrigate the surface of the heart with
drops of I percent pilocarpine for two minutes. Stimulate the vagus trunk
one minute after pilocarpine and at successive intervals of five minutes. If
the drug is strongly active the heart will beat slower. In the early stages
the stimulation will result in acceleration, but in no inhibition as in the
normal. Applying the electrodes directly to the sinus gives no inhibition
showing that the pilocarpine has acted on the vagus endings and not on the
ganglionic connections. Drops of i percent atropine sulphate will restore
the heart beat after pilocarpine, these two drugs being antagonists.
5. Pilocarpine on the circulatory and respiratory system of the
mammal. Anesthetize a dog with morphine and chloroform. Introduce a
tracheal canula and be prepared to use artificial respiration if necessary.
Take blood-pressure and respiration records on a continuous paper kymo-
graph. Test the activity of the vagus. Give an intravenous injection of
I c.c. of I percent pilocarpine nitrate. Retest the activity of the vagus
after the pilocarpine. Atropine antagonizes the pilocarpine effect. Ex-
amine the pupils from time to time. Also note the increased rate of salivary
44 EXPERIMENTAL PHARMACOLOGY.
Experiments on the Action of Physostigmine. page.
1. On the frog ... . . 44
2. On cardiac muscle . . 44
3. On the frog's heart and its nervous apparatus . . . . 44
4. On the heart of the cat ... 44
5. On the respiratory medullary center and on the circulatory
system of the mammal . . .... 44
6. On the eye . . . . 47
1. Physostigmine on the frog. Give a frog a dorsal lymph sac
injection of i c.c. (17 minims) i percent physostigmine. The effects
produced are diminished irritability, loss of muscular tone, paralysis of the
respiratory center, loss of reflexes, and death, or, at best, a very slow and
2. Physostigmine on cardiac muscle. After a ventricular strip
from the terrapin has begun beating regularly in physiological saline, transfer
it to o.i percent physostigmine in saline for two to three minutes. Physi-
ological saline recovers the normal contractions after several minutes.
Compare the results with those from pilocarpine and muscarine. A bath of
0.002 percent atropine antagonies the physostigmine effects.
3. Physostigmine on the frog's heart and its nervous apparatus.
Pith a frog and take a record of the heart beat. Determine the minimal
effective stimulus of the vagus trunk for inhibition of the heart rate. Now
irrigate the surface of the heart with drops of o.i percent physostigmine
from an irrigating bottle for two minutes. Redetermine the minimal stimulus
for the vagus trunk beginning with a very weak induction current. If
the contractions are at a slow rate or have ceased, irrigate the surface of the
heart with i percent atropine. Atropine antagonizes physostigmine.
Compare with pilocarpine. If the perfusion method is used then 0.0 1
percent physostigmine is the proper solution strength for the frog's heart.
4. Physostigmine on the isolated heart of the cat. Prepare an
isolated cat's heart by the method used in the nicotine experiment 5. When
it gives regular contractions, perfuse it with o.oi percent physostigmine
in Locke-blood solution.
5. Physostigmine on the respiratory medullary center and on
the circulatory system of the mammal. Anesthetize a dog, insert a trach-
eal canula and be prepared for artificial respiration. Take a continuous
record of the blood-pressure and of the respiratory movements. Insert a
canula in the saphenous vein and connect it with a buret containing the drug.
Give an intravenous injection of o.i percent physostigmine slowly until the
first effects are noticed on blood-pressure. Note the amount and mark
the time of the dose on the record by a signal pen. Usually there is a pro-
Fig. 26. — Action of physostigmine and its antagonism by atropine in the mammal,
showing the effects of an injection of 8 mgr. of physostigmine just before the part of
of respiration and the circulation effects upon the injection of 2 mgr. of atropme be-
At a, b and c are shown portions of the trace at 30-second intervals. Time in seconds.
gressive paralysis of the respiratory center accompeinied by a great slowing
of the heart and a fall of blood-pressure by one-half or more. The heart
continues to beat long after respiration ceases. Use artificial respiration
until the blood-pressure improves' and the anesthesia becomes Hght. This
will not restore automatic respiratory movements as it does after heavy
anesthesia. If at this time a venous injection of 0.5 c.c. of i percent
atropine be given from a hypodermic syringe the respiratory movements
will be quickly established and the slow heart and low pressure will give
way to the rapid heart and strong pressure following primary injection of
atropine, experiment 6, page 35. The vagus inhibitory apparatus is effective
under physostigmine, but not after atropine. Repeat the experiment. It
takes a larger dose of physostigmine to overcome the atropine and pro-
I ' I j^ i
> 1 1 1 1 1 1 1
Fig. 26 (Continued).
Parts I, iX and iT give the respiration trace, blood-pressure and time, respectively,
the trace shown. The parts of the trace numbered 2, 2X and 2T show the recovery
tween the arrows. A few second's gap intervenes between the two parts of the figure.
duce the characteristic effects. Examine the pupil before and after the
6. Physostigmine on the eye. Give 2 drops of i percent phy-
sostigmine in one eye of a dog or a rabbit, at intervals of five minutes. It
is better to use one of the experimeter's own eyes. Strong contraction of
the pupil follows. A decrease in intraocular pressure has also been proven,
48 EXPERIMENTAL PHARMACOLOGY.
and to produce this effect is the chief therapeutic use of the drug. A strik-
ing comparison is obtained by dropping i percent atropine in the unused
eye of the dog after the physostigmine effect has come on in the other eye.
Physostigmine will overcome the atropine dilation of the pupil. The experi-
menter may show the antagonism between homatropine and physostigmine
on his own eyes, but it is recommended that one eye always be reserved.
Experiments on the Action of Aconite. page.
1. On the frog 48
2. On the circulatory system of a mammal. 48
3. On the frog's heart 48
4. On the mammalian heart. . . . 48
1. Aconite on the frog. The dose is 0.5 c.c. of o.i percent aconite.
Compare with digitalis.
2. Aconite on the circulatory system of the mammal. Take a
continuous tracing of the blood-pressure of a dog. Give i c.c. of o.i
percent aconitine crystals. Note particularly the progressive effects on
the nervous and muscular elements of the circulatory apparatus.
3. Aconite on the frog's heart. Destroy the cerebrum and optic
lobes only of a frog, -expose the ventricle and take a tracing. Give an in-
jection of 0.5 c.c. of O.I percent aconitine in the lymph sac. One may
expect a progressive stimulation of the accelerator and vagus nervous
apparatus followed by paralysis of nerves and muscle.
4. Aconite on the mammalian heart. Prepare an isolated heart
as described for the nicotine experiment 5. When the heart is contracting
regularly with the Locke-blood perfusion then perfuse for 10 seconds with a
0.0002 percent aconite solution. A prolonged perfusion or perfusion
with a stronger concentration of aconite will quickly set up incoordinate
contractions and fibrillation.
50 EXPERIMENTAL PHARMACOLOGY.
Experiments on the Action of Veratrine. page.
1. On the frog. . .' . . -5°
2. On the mammal. . .... 50
3. Veratrine on heart strip. . . . 50
4. On the frog's heart. . . . 50
5. On the isolated mammalian heart. ... 50
6. On the form of the simple muscle contraction 50
7. On the circulatory and respiratory systems of the mammal. . 50
1. Veratrine on the frog. The dose for a frog is about 0.5 c.c.
of a I percent solution of the fluid extract veratrum viride or 0.3 c.c. of
o . 1 percent veratrine . Compare with the effects of aconite and of barium.
See experiment 4.
2. Veratrine on the mammal. Give a cat or rabbit i c.c. of o.i
percent veratrine hypodermically, or i c.c. of i percent for a dog. Keep
under observation for a considerable time.
3. Veratrine on the heart strip. Subject the contracting strip of
ventricle to 0.005 to 0-05 percent vera tine in sahne.
4. Veratrine on the frog's heart. Pith a frog, expose the heart and
take a tracing when perfused with 0.005 percent veratrine in Ringer's
solution (o.oi percent destroys coordination).
5. Aconite on the isolated mammalian heart. Prepare the appara-
tus for the isolated heart experiment, isolate a cat's heart and perfuse with
0.0002 percent veratrine in Locke-blood solution. See Fig. 25.
6. Veratrine on the simple muscle contraction of the frog. Ligate
one leg of a frog and give a hypodermic of 0.5 c.c. of o.i percent vera-
trine. After 15 minutes prepare the veratrinized muscle and take simple
muscle contractions to show the form of the contraction wave, using a
tuning fork to record the drum speed. Compare this curve with that of
the undrugged muscle.
The frog of experiment i may be used to show the veratrine effect on
muscle work. Stimulate once in three seconds in this experiment, since the
relaxation may not be complete in an interval of two seconds.
7. Veratrine on the circulation and respiration of a mammal.
Take a record of the blood-pressure from the carotid of an anesthetized dog.
Tracheotomize and take respiratory tracings. Give i c.c. of i percent
veratrine in the abdominal cavity. When marked cardiac slowing appears
cut the vagi and note the effects on the heart.
52 EXPERIMENTAL PHARMACOLOGY.
Experiments Showing the Action of Digitalis. page.
1. On the frog. ... 52
2. On the ventricular muscle. ... . . . 52
3. On the frog's heart. . . 52
4. On the atropinized frog's heart. . 52
5. On the mammalian heart . . . 52
6. On the circulatory and respiratory systems of the mammal. 53
7. Digitalis as a diuretic 53
1. Digitalis on the frog. Give a dose of 0.5 c.c. of 0.2 percent
of soluble digitalis. The digitalis effects develop slowly. Note the heart
rate, and particularly the circulation in the web. Keep in a moist battery
jar for at least two hours. Examine the heart if death occurs.
2. Digitalis on the ventricular muscle. Treat a strip of terrapin's
ventricle contracting in saline to a bath of 0.002 percent digitalis in saline.
Follow with pure saline. Repeat with a 0.005 percent digitalis. Still
stronger solutions may be used, but a marked tone will result as shown in
Digitalis solutions may be used made up in the weaker Ringer, but
as the rate is slower and the amplitude much greater than that in sodium
chloride solutions the picture will be quite different, though the same in
kind. Delirium cordis of the strip is produced by the stronger solutions act-
ing for several minutes.
3. Digitalis on the frog's heart. Pith a frog and take a record of
the contractions of the ventricle when irrigated with physiological saline.
Irrigate slowly with drops of 0.2 percent digitalis for two minutes, then
wash off with saline.
A more effective method is to perfuse the heart from a canula in the
vena cava. Use a much weaker solution for perfusion, i.e., 0.0005 to
o.ooi percent digitalis in Ringer. These effects should be compared with
the effects on cardiac muscle above.
4. Digitalis on the atropinized frog's heart. Atropinize the frog's
heart to eliminate the car'diaic nervous control, then repeat experiment 3
5. Digitalis on the mammal heart. Use the method described
on page 70. Isolate and perfuse the cat's heart with the normal solution
of Locke-blood, then with o.oooi percent digitalis (soluble digitalis)
in Locke-blood. Increase the strength to 0.0005 percent. The stronger
solution will usually produce a great increase in amplitude followed by
fibrillation. The weaker solution, see figure 30, produces a typical mild
therapeutic effect on the heart. Compare with the results of experiment 6.
6. Digitalis on the circulatory and respiratory systems of the
mammal. Anesthetize a dog and take continuous kymographic records
of the blood-pressure and of the respiration. Slowly inject into the saphe-
nous vein 2 c.c. doses of 0.5 percent digitalis at five-minute intervals
Fig. 29. — Experiment showing the action of digitalis on the rhythm and tone of a
strip of terrapin's ventricle. The strip was contracting in physiological saline. Between
the words "on" and "off" it was subjected to 0.06 percent of digitalis in saline.
until the three stages of digitalis effect on the heart and blood-pressure are
obtained. The anesthetic must be perfectly constant. One may give the
maximal dose of 4 c.c. of i percent digitalis at once. In this instance the
three stages are passed through rapidly and the animal will usually die in
10 to 20 minutes. Read Cushny's Pharmacology, pp. 430-435.
7. Digitalis as a diuretic. Morphinize and chloroform a dog.
Take the blood-pressure. Isolate the ureters near the bladder and insert
canulas, using care not to occlude the ureters by twisting or otherwise. Con-
nect the ureters by means of a T-tube with a horizontal 2 c.c. pipet graduated
to 1/50 c.c. Close the abdomen with sutures. Insert a venous canula and
connect with a transfusion buret. Establish the normal secretion per 10
minutes, cutting off the column of secreted urine by injecting a bubble of air
into the mouth of the buret by inserting a hypodermic needle through the
rubber connecting tube. Now inject 5 c.c. of o.i percent digitalis or
strophanthin into a vein and take the secretion in successive 10 minute
periods until the flow is constant. Repeat the dose once or twice at long in-
tervals. Mark the secretion intervals on the blood-pressure record.
Compare these results with those observed on other diuretic drugs —
caffeine, urea, inorganic salts, etc.
Experiments on the Action of Ergot. page.
1. On the frog 55
2. On the heart muscle. 55
3. On the arterioles of the frog. 55
4. On the blood-pressure and heart rate of a mammal. 55
1. Ergot on the frog. Give 0.5 c.c. of the fluid extract.
2. Ergot on the heart muscle. Change a contracting heart strip
from saline to a i o percent solution of Squibbs' fluid extract of ergot in saline
solution. Allow it to act for five minutes. Take a continuous record.
3. Ergot on the arterioles of the frog's web. Wrap a frog in
a wet cloth and fasten to a frog-board for examining the web. Give a
lymph sac injection of 0.5 c.c. fluid extract of ergot. Select a good field
of small arterioles and measure their diameter at once. The relative
change in diameter of small vessels can be determined by selecting a field
in which pigment spots mark the borders of the vessels. Sketch such a
vessel and spots for the normal. Re-sketch after the drug. Re-measure at
intervals of five minutes as the ergot is absorbed.
4. Ergot on the blood-pressure of a mammal. Give an intravenous
dose of 0.5 c.c. fluid extract of ergot to a mammal while taking a record
of the blood-pressure.
56 EXPERIMENTAL PHARMACOLOGY.
The commercial preparation of the active principle of suprarenal gland,
adrenalin hydrochloride, presents the same physiological action as the gland
extract and has the special advantage of preparation in definite and known
strengths. It has come into general use for therapeutic purposes and is,
therefore, used in these experiments.
Experiments Showing the Action of Adrenalin Hydrochloride, page.
1. On the frog. 56
2. On the ventricular strip 56
3. On the frog's heart 56
4. On the isolated mammalian heart .... -56
5. On the simple muscle contraction . 58
6. On muscle work . . • 58
7. On the local mucous surfaces . . 58
8. On the size of the blood-vessels in the frog's web 58
9. On general blood-pressure and peripheral vaso-constriction 58
1. Adrenalin on the frog. Give 0.5 c.c. o.i percent in the dorsal
2. Adrenalin on the ventricular muscle. Transfer a terrapin's
ventricular strip contracting in physiological saline to o.oi percent adren-
alin in saline. Change after two to five minutes. The drum speed should
be I cm. per minute. Suprarenal extract has also been shown to increase
the amplitude and the rate of the ganglion free ventricular muscle of the
dog. Cleghorn, Amer. Jour. Physiol., Volume III, p. 273.
3. Adrenalin on the frog's heart. Use the perfusion method, page
68, with the heart in place and the inflow canula in the ascending vena cava.
Follow physiological Ringer perfusion with o.ooi percent adrenalin
hydrochloride in Ringer. The drum speed should be 2 mm. per second.
Direct application to the surface of the heart requires a strength of at least
0.05 percent adrenalin hydrochloride.
4. Adrenalin on the isolated heart. Perfuse a cat's heart in the
usual way with Locke-blood solution for a normal, then change to a o.oooi
percent adrenalin hydrochloride in Locke-blood. If the heart be beating
feebly it often happens that the contractions will increase in amplitude by
200 percent and more.
58 EXPERIMENTAL PHARMACOLOGY.
5. Adrenalin on the simple muscle contraction. Ligate one leg
of a frog and give 0.5 c.c. of 0.05 percent adrenalin. Allow ten minutes
for absorption. Compare the simple muscle contractions of the two gastroc-
nemii as repards a, amplitude, and b, the time of the simple contraction.
The muscular power of patients with Addison's disease has also been shown
to be greatly improved by giving the extract of suprarenal gland.
6. Adrenalin on muscle work in the frog. Prepare a frog as in
experiment 5 above and test the work performed by the two muscles. For
details of procedure see alcohol experiment 5.
7. Adrenalin on mucous surfaces. Paint one-half the tongue with
0.1 percent adrenalin hydrochloride. At intervals of five minutes drop
0.0 1 percent in saline (sterilize by boiling) in one eye. Compare the two
halves of the tongue and the two eyes as to vascular condition. Examine
the size of the pupils. Test for possible differences as to the sensitiveness
of the conjunctiva. Try the effect in the eye of a cat or dog.
8. Adrenalin on the size of the blood-vessels of the frog's web.
Use a dose of 0.5 c.c. of o.i percent as a hypodermic. See ergot experi-
ment 3, page 55; nitroglycerine experiment 3, page 59. Or apply drops of
o.i percent directly to the web under the microscope.
9. Adrenalin on general blood-pressure and on vaso-constriction
in a manunal. Prepare a dog for blood-pressure. Adjust an onkometer
to the left kidney and record the change in volume with a Brodie's bellows
or Roy's piston recorder. Give 2 to 4 c.c. of o.oi percent adrenalin
hydrochloride slowly in a vein. Give 2 c.c. of 0.1 percent atropine to
eliminate the vagus action on the heart and repeat the adrenalin. Compare
with digitalis, page 53; ergot, page 55; veratrine, page 50. Drugs of
antagonistic action are nitrites and potash salts.
NITROGLYCERINE AND THE NITRITES. 59
NITROGLYCERINE AND THE NITRITES,
Experiments on the Action of Nitroglycerine and the
1. On the frog. 59
2. On the heart muscle. . 59
3. On the arterioles of the frog 59
4. On the circulation volume 59
5. Amyl nitrite on the pulse . 59
6. Nitrites on mammalian blood-pressure . 60
Nitroglycerine and the nitrites affect primarily the peripheral circu-
lation, causing vaso-dilation with fall of blood-pressure. The specific action
is on the muscular tissue.
1. Nitroglycerine on the frog. Give a frog a dose of 0.5 c.c. of
o.i percent nitroglycerine in the dorsal lymph sac.
2. Sodium nitrite on the heart muscle. Test the action of 0.02
percent sodium nitrite on the contracting ventricular strip.
3. Nitroglycerine on the arterioles of a frog. Bind a frog for the
microscopic examination of the web circulation. Then give i c.c. of o.i
percent nitroglycerine in the lymph sac. Immediately measure the
smaller arterioles in a favorable field and re-examine every two minutes as
absorption progresses. Try direct application of drops of 0.1 percent to the
4. Sodium nitrite on the circulation volume. Pith a frog or small
terrapin. Insert a canula in the aorta or one of its branches, snip the
veins with the scissors to allow free perfusion, set the frog-board at an angle
to facilitate drainage of liquid. Perfuse the blood-vessels with a weaker
Ringer's solution for a normal. Follow with o.oi percent soditun nitrite
in the weaker Ringer, keeping a uniform pressure of the perfusion liquids
of from 6 to 10 cm. Measure the perfusion rate in drops per minute, or
collect the outflow in a 25 c.c. graduate.
Test the amount of outflow when irrigated with 0.0005 percent of
soluble digitalis, then follow with o.oooi percent sodium nitrite, both in the
5. Amyl nitrite on the pulse. Take normal pulse records with one of
the standard sphygmographs. Break an amyl nitrite pearl on a handkerchief
and breathe deeply the fumes. Pulse tracings taken 5 and 10 minutes later
will show the usual signs of dilated blood-vessels with accompanying low
pressure. Slight headaches sometimes follow the use of amyl nitrite.
6. Nitrites on mammalian blood-pressure. Anesthetize a lo kilo
dog and take the blood-pressure. Give intravenous doses of nitrites in the
following order, repeating with larger doses if necessary and always allowing
full time for recovery: i c.c. of o.i percent nitroglycerine, 3 c.c. of o.i
percent; 2 c.c. of 0.1 percent amyl nitrite; 6 c.c. of 0.1 percent
Fig. 32. — Action of amyl nitrite on the human pulse. One amyl nitrite pearl was
crushed on a handkerchief and the fumes inhaled deeply. Trace i is the normal pulse.
Trace 2 immediately after amyl nitrite fumes. Traces 3 and 4 are stages of recovery.
Time in seconds.
sodium nitrite. The blood-pressure remains low for a long time after
sodium nitrite. A dose of 2 c.c. to 5 c.c. of 0.2 percent digitalis or 2 c.c.
of o.oi percent adrenalin hydrochloride will antagonize this effect.
Give 2 c.c. of O.I percent atropine to eliminate the action of the car-
diac nervous apparatus and repeat the above doses of nitroglycerine and
CARBOLIC ACID. 6l
Experiments on the Action of Carbolic Acid. page.
1. On the frog. . . 6i
2. On the growth of yeast and bacteria . . . . . 6i
3. On the circulatory and respiratory systems of a mammal 61
1. Carbolic acid on the frog. Give a dose of i c.c. of i percent.
2. Carbolic acid on the growth of yeast and of bacteria. Prepare
six fermentation tubes of active yeast culture and as many test-tubes of
inoculated bouillon. Keep one tube of each for a normal and to the others
add enough 10 percent carbolic acid to make a series of o.i, 0.5, i, 2, and
4 percent solutions. Keep at laboratory temperature and observe through a
period of several days.
3. Carbolic acid on the circulatory and respiratory systems of the
mammal. While taking records of blood-pressure and respiration by the
usual method give an intravenous injection of 10 c.c. of 0.5 percent carbolic
acid. When the collapse stage is far advanced inject i percent soditmi
sulphate slowly. Judge the amount required by the action in overcoming
the carbolic acid depression of the respiratory apparatus.
Experiments Showing the Action of Potassixmi Salts. page.
1. On the heart muscle ,61
2. On the reaction time in the reflex frog. . . 61
3. On muscle irritability and muscle work in the frog . . 61
1. Potassium chloride on the heart muscle, A ventricular strip
contracting in physiological saline solution is transferred to 0.06 percent
potassium chloride in saline for two to five minutes. Contractions return
in saline even after stronger doses of potash.
2. Potassium bromide on the reaction time in the reflex frog.
Compare the reaction time of a reflex frog before and 20 to 40 minutes after
0.3 c.c. of 5 percent potassium bromide in the dorsal lymph sac.
3. Potassitxm chloride on muscle irritability and muscle work
in the frog. Compare the two gastrocnemii as to irritability and as to
amount of muscular work done. Dose 0.3 c.c. of 5 percent solution given
hypodermic after one leg is ligatured.
Experiments Illustrating the Action of Calcium Salts. p'Age.
1. On heart muscle 62
2. On the frog's heart . . 62
3. On the blood-pressure and the respiration in the mammal. 63
I. Calcium chloride on heart muscle. Transfer a ventricular strip
from physiological saline to 0.03 percent calcium chloride in saline for
three to five minutes. Record on a drum speed of 2 cm. per minute.
Repeat, using 0.06 percent. The rate is increased and the amplitude often
Fig. 33. — Terrapin's heart muscle as influenced by a solution of 0.06 percent cal-
cium chloride in physiological saline. A weaker solution produces much less tone.
Time in seconds.
doubled. The stronger solution produces great increase in tone which
sometimes passes into delirium cordis. Potash salts antagonize. Read
Ringer, Jour. Physiology, 1883.
2. Calcium chloride on the frog's heart. Perfuse the frog's heart
through the vena cava with 0.7 percent sodium chloride and follow with
0.03 percent calcium chloride in 0.7 percent sodium chloride. Recover
BARIUM SALTS. 63
the sodium chloride type of contractions, then perfuse with o.oi percent
barium in sodium chloride.
3. Calcium chloride on the blood-pressure and the respiration
in the mammal. Give an intravenous dose of lo c.c. of i percent for a
dog. Cut the vagi and repeat the dose. Alternate the dose with potassium,
2 to 4 c.c. of I percent.
Experiments on the Action of Barium Salts. page.
1. On the frog . . 63
2. On the heart muscle 63
3. On the circulation and on the respiration movements in
mammals . . ..... 63
1. Barium on the frog. Dose, i c.c. of i percent barium chloride.
2. Bariimi chloride on the heart muscle. Transfer a contracting
ventricular strip from 0.7 percent sodium chloride to o.oi percent bariiun
chloride in saline. Short immersions increase the rate, but long baths show
that this salt does not sustain contractions as do calcium salts. A o.i percent
solution in saline delays contractions with prevention of relaxation. Con-
tractions still take place in i percent barium chloride. Compare with
3. Baritmi chloride on the circulation and on respiratory move-
ments in mammals. The effect on the heart and blood-pressure and on
respiration in a mammal is demonstrated by an intravenous dose of 5 c.c.
of 0.2 percent given slowly. This dose should be repeated several times
both before and after section of the vagi. Barium salts act as strong poisons
to the nerve centers, especially those in the medulla.
OPERATIONS, APPARATUS AND SPECIAL
The lymph and blood plasma in which the tissues develop are the true
Artificial solutions imitate lymph in its isotonicity — ^its physical character,
and in its composition — its chemical character. Sodium chloride in 0.6 per-
cent solution, used first by Nasse in 1869 on frog's muscle, and by Bow-
ditch in 1871 on the frog's heart, was supposed to prevent injurious changes
in the tissue by virtue of its isotonicity. Ringer in 1883 and Locke in 1885
introduced the solutions which bear their names. They showed that the
chemical factors play a fundamental part in the effects of these solutions
on the tissues. At the present time we recognize that exact isotonicity is not
nearly so fundamental as at first supposed, and that these solutions are
chemically active in relation to the living protoplasm.
At the present time much attention 'is being given to the effects of
asphyxiation on the physiological activity of living tissues that are isolated
from the normal circulation. The artificial solutions can be made more
efficient by shaking with air before using, or by shaking with pure oxygen.
Isolated mammalian hearts give much more constant characters in their
response to artificial solutions which contain defibrinated blood, prefer-
ably from the animal supplying the heart. Even with five to ten percent of
blood such solutions are strikingly more efficient, supposedly because they
are much better oxygen carriers. For cats' hearts Locke-blood solutions
aerated by a stream of oxygen are very efficient indeed.
1. Physiological salt solution or normal saline. Sodium chloride
in distilled water 0.7 percent. More exact isotonicity is secured by 0.6
percent for frogs, 0.7 percent for terrapin and 0.9 percent for mammals.
2. Ringer's solution. The Ringer's solution that imitates the blood
serum in its effects on heart tissue is made up in this laboratory in the
Sodium chloride, 0.7 percent.
Potassium chloride, 0.03 percent.
\ Calcium chloride (cryst. computed water free), 0.026 percent.
For heart work where a more rapid rate is desired the amount of potas-
sium must be reduced to that in Ringer's original formula.
Sodium chloride, 0.7 percent.
Potassium chloride, o.oi percent.
Calcium chloride (cryst. computed water free), 0.026 percent.
3. Locke's solution. Locke's solution is a mixture of the salts in
Ringer's solution with dextrose added to make o.i percent.
Sodium chloride, 0.7 percent.
Potassium chloride, 0.03 (or o.oi) percent.
Calcium chloride (cryst), 0.026 percent.
Dextrose, o.i percent.
4. Locke-blood solution. . Add 5 to 10 percent of defibrinated whole
blood to the above Locke's solution.
The mammals usually available for laboratory experimental purposes
are dogs, cats, rabbits and guinea-pigs, each of which can best be anesthe-
tized by a special treatment of its own.
Dogs. Give a lo-kilo dog i c.c. (17 minims) of 2 percent mor-
phine under the skin of the shoulder, holding its head firmly between the
operator's legs while the hypodermic injection is being given. Allow 15
minutes or more for the morphine to take effect. The morphine should be
followed by chloroform or chloroform and ether in equal parts. Give it
by means of a small nose hood made by sewing a cheese-cloth, that has been
folded in the form of a blunt cone, to a wire ring, or use a Senn's inhaler
mask. When the voluntary movements have about ceased, tie the dog to a
holder and take it to the experimental table. The tests of good anesthesia
are: i, loss of voluntary movements; 2, no cutaneous reflexes; 3, slight
corneal reflexes or none in deep anesthesia ; 4, even and fairly deep respira-
tion; 5, medium blood-pressure and pulse. This condition of anesthesia
is maintained by giving chloroform from a dropping-bottle at abso-
lutely regular intervals of 30 seconds by the watch. The number of
drops necessary for each animal will quickly be found by trial. In the
experience of this laboratory it is from 3 to 6 drops per 30 seconds. The
success of most pharmacological experiments on dogs depends upon main-
taining an absolutely even anesthesia.
Cats. A mixture of equal parts of chloroform and ether is the most
66 EXPERIMENTAL PHARMACOLOGY.
practical anesthetics for cats. These animals are anesthetized most conven-
iently by putting them in a box of about two cubic feet in dimension and
provided with a close cover. A very convenient box is the tin display
cracker box with glass window obtained of the grocer. Drop in the box with
the cat a small strip of cheese-cloth saturated with chloroform-ether mix-
ture, ID c.c. in broken doses will anesthetize a cat in lo minutes. As
soon as the animal falls down under the influence of the anesthetic it
should be taken from the box, fixed in the holder, and the anesthetic given
from a cloth in the manner and with care prescribed above for the dog.
Cats do not survive pure chloroform in the hands of the ordinary student
Rabbits. Give rabbits 2 grains of urethane by the mouth. Follow
with light and careful use of ether. Or pure ether may be given without the
urethane. Give the ether in the manner and with the regularity recom-
mended above for giving chloroform to dogs. Do not use chloroform or
even chloroform mixtures with rabbits.
Guinea-pigs. These little animals when they must be used for
pharmacological purposes are anesthetized best with pure ether or ether
followed with a little morphine.
THE PREPARATION OF THE VENTRICULAR MUSCLE.
Destroy the brain of a terrapin, remove the plastron and open the peri-
cardium. Grasp the left angle of the base of the exposed ventricle with a
forceps and cut with a scissors from this point around the apex to the oppo-
site side, thus removing a piece about one inch long and the size of the half
of a small lead pencil. Split this strip into two or three smaller ones for
To mount the heart strip tie silk threads to each end, one with a loop
one-half inch long and the other with a loop about four inches long. Place
the short loop on the hook of the glass-rod support provided for the purpose,
and the long loop over the recording lever. Use a straw lever of the power-
fulcrum-weight order mounted in a muscle lever holder. A total tension of
one gram is best for developing the contractions of the ventricular strip.
The holder mentioned above is made of a glass rod 4 to 5 mm. diameter
and 15 cm. long. Bend it at a right angle in the middle and then draw out
and turn a hook on one end, the hook being turned back on the rod. The
apparatus set up complete consists of a single iron stand with three clamps,
the top one to support the lever holder, the middle the glass rod, and the bot-
THE PREPARATION OF THE VENTRICULAR MUSCLE.
Fig. 34. — The terrapin's heart, ventral view, showing how to cut an apex strip
for experimental purposes and how to split this apex into smaller pieces.
Fig. 35. — Apparatus as set up to demonstrate the contractions of the apex muscle
of terrapin's ventricle. The glass L-shaped holder should be set on the stand high
enough to allow of easy change of solution tubes. The figure shows the tube of physio-
logical saline and other details for the better illustration of the mounting of the heart strip
torn one a platform on which rests the footed test-tube (1x3 inch specimen
tube) to contain the solution surrounding the strip. The ventricular strip
mounted in this apparatus with a tension of one gram and bathed in a solu-
tion of 0.7 percent sodium chloride will begin rhythmic contractions in from
10 to 40 minutes. These contractions will continue about two hours,
growing constantly smaller for the entire time. The strip may then be
revived by a bath of Ringer's solution or by serum, and may again be used
in the sodium chloride bath.
TO TEST THE ACTION OF DRUGS ON THE FROG'S OR
Two methods are used in this laboratory for the study of the action of
drugs on the frog's heart, both permitting of permanent records. The
Fig. 36. — Showing the method of recording the action of the frog's heart in place
n the body cavity. If the perfusion method is used the canula can be inserted with
greater ease if the frog is reversed on the support.
most convenient method is to pith the frog, open the thorax and expose
the heart, adjust the foot of a delicately poised heart lever on the venticle
and, while the record is being taken, irrigate the surface of the heart with the
IRRIGATING AND PERFUSING FLASKS. 69
drug. Dissolve the drug in physiological saline and always take a previous
normal record under saline irrigation. This method requires the use of
relati\'ely strong solutions. The most convenient irrigating bottles are four-
or eight-ounce aspirator bottles with tubed foot for rubber connector. These
are each provided with a small-mouthed canula attached by a short rubber
connection, and the flow is regulated by a screw compress. Fit these
flasks with Marriotte stoppers and support them on a stand by a universal
buret clamp about the neck.
The second method, that of perfusion, is carried out best as described
by Walden in the American Journal of Physiology, Volume III, page 123.
Insert a canula into the inferior vena cava for an inflow and one in the aorta
for an outflow, or merely cut one aortic branch and let the outflow go free.
The canula is connected with two supply bottles, one for physiological saline,
the other for the drug in solution. The Marriotte stoppers should be set
at exactly the same pressure levels. Connect the two flasks with the inflow
canula by a T-tube brought as close as possible to the heart in order that
the solutions may be changed quickly with only a short connecting tube to be
washed out. Very weak solutions of drugs are required by this method of
perfusion. The frog's heart is quickly exhausted in pure saline solutions,
so, for certain prolonged experiments it is better to use the weaker Ringer's
solution for dissolving the drug.
Record the contractions of the ventricle by a thread from its apex to
the ventrical arm on a balanced horizontal lever. A flexible paper or
celluloid writing point will add to the accuracy and beauty of the records.
These writing points should be 2 to 3 cm. long by 0.5 cm. wide and made
of Hght-weight but hard note paper.
IRRIGATING AND PERFUSING FLASKS.
The quarter- and half-pint aspirator bottles manufactured by Whitall,
Tatum & Co., with tubed foot for attaching a rubber tube are particularly
adapted to both irrigation and perfusion of the heart. For use in irrigation
these bottles are clamped to a heavy based stand by a universal buret clamp
on the neck. Insert a tight-fitting rubber stopper with a 2 mm. glass tube,
to give a constant pressure level. A short heavy rubber connector provided
with a small screw compress and a glass dropper serves to regulate the speed
of the outflow. Such a flask attached to an independent stand and set at a
level so that the fluid drops only a few millimeters is an exceptionally satis-
factory method of applying solutions directly to the surface of the heart.
70 EXPERIMENTAL PHARMACOLOGY.
Two perfusion bottles may be connected together by a T-tube for per-
fusion work. In this case the inflow canula is connected by a very short
(6 to lo cm.) tube of small caliber and is supported firmly by a clamp on
the T-tube. Or a Y-canula can be used and the flasks attached directly to
its limbs. In either case it is better to insert a small T-tube with light
spring clamp in order to wash out any drugs in the tubing. This canula,
when provided with an overflow, as shown by Gibson and Schultz in an
article now in manuscript, permits change from one solution to another
without a break in the pressure of the fluids. The connecting tubes for
the flasks should be 25 to 30 cm. long to permit adjusting. Set screw or
spring compresses near the bottle. Fill one flask with the normal solution,
the other with the drug. A very small amount of the fluid can be applied
by means of these perfusion flasks.
TO TEST THE ACTION OF DRUGS ON THE BLOOD-PRESSURE,
RESPIRATION, ETC., OF A MAMMAL.
1. The anesthetic. For anesthesia methods see page 65.
2. The operations. Blood-pressure is taken from one of two arteries,
the right common carotid, or the femoral artery. The femoral is practical
only for the dog. To expose the common carotid make a three-inch cut
over the trachea from the proximity of the larynx to the manubrium. Sepa-
rate the muscles down to the trachea, and then along the side of the trachea
till the common carotid artery and vagus come into view. Use the scalpel
handle and tear rather than cut the facias and muscles involved. Avoid
the veins and the laryngeal arteries. No blood need be lost after the skin is
cut. Separate the fascia binding the artery and vagus, using care not to injure
the latter. Place a bulldog forceps on the artery well toward the thorax.
Ligate the cephalic end. Lay and tie a ligature loosely about the intervening
stretch of artery for the canula. Grasp the artery at the cephalic ligature
and use the tip of the scissors to make a V-shaped cut two-thirds through
the artery wall and directed toward the heart. Insert the canula and ligate
it firmly with the ligature already laid.
The femoral artery is exposed by a 5 cm. cut over the artery where
the pulse can be felt near Poupart's ligament. The artery is prepared and
the canula inserted as described for the carotid.
The saphenous vein or the jugular are used for injecting drugs.
Insert a small washout canula toward the heart choosing the vein exposed
by the previous operation. Keep the vein closed with a bulldog forceps in
ACTION OF DRUGS ON THE REFLEXES OF A FROG. 7 1
order to prevent small clots in the mouth of the canula, except when in-
jections are to be made.
Tracheotomy should generally be performed for all student work on
the mammals used in blood-pressure experiments in pharmacology. Free
the trachea immediately below the thyroid cartilage and insert a metal
canula made especially for the dog, or insert one limb of a glass T-tube of
as large size as the trachea will take. Tie it firmly with small stout twine.
The apparatus consists of a continuous paper kymograph (Ludwig's
weight-driven pattern arranged to run the paper in the right-handed direction
is the most satisfactory instrument) ; mercury manometer for measuring the
blood-pressure; respiration tambour (Marey's form); signal pen to record
stimulations, injections and other events; time signal; stimulating coil and
accessories complete; and a jacketed buret for transfusing warm solutions
into the vein. The recording pens of the manometer, tambour, signals, etc.,
must all be adjusted to the kymograph in an exact vertical line. Fill the
lead tube of the manometer with lo percent magnesium sulphate from a
pressure bottle, take the zero level of the manometer, set the time signal to
write on this level, connect with the canula, and fill to a pressure of 130 mm.
mercury. Connect the respiration tambour directly with the side branch of
the tracheal tube. Start the kymograph, ink all the pens, remove the arterial
bulldog clamp, and the experiment is ready to begin.
A renal onkometric record should be taken with blood-pressure in the
investigations on certain drugs. Open the abdomen along the entire median
line, cut the wall transversely for two to three inches over the left kidney.
Strip the kidney of its fat and looser coverings and enclose it in a renal
onkometer. Adjust the overflow from the onkometer to the exact kidney
level and take a record of the variations with a small sized Brodie's bellows
recorder adjusted in line with the recorders mentioned abo\'e.
METHOD OF TESTING THE ACTION OF DRUGS ON THE .
REFLEXES OF A FROG.
Carefully destroy the brain and the medulla, but not the cord. Pre-
vent the loss of blood. Suspend the frog to a horizontal rod on a stand,
using a card hanger or a loop of string on the upper jaw. Stimulate the tip
of the toe with acid or with platinum electrodes and measure the reaction
time by counting seconds until the foot is withdrawn. The reaction time
may be recorded on a kymograph. Attach a horizontal writing point of
mucilaged paper to the leg above the foot. Take the speed of the drum with
72 EXPERIMENTAL PHARMACOLOGY.
one magnet beating seconds, and record the instant of stimulation with a
second and independent magnet controlled by a contact key.
Take the normal reaction time first, then give the drug as an injection
in the dorsal lymph sac and allow about 20 minutes for absorption. Re-
measure the reaction time and repeat at intervals of 10 minutes to get the
progressive effects of the drug.
METHOD OF GIVING AND TESTING THE ACTION OF A DRUG
ON THE FROG'S GASTROCNEMIUS MUSCLE.
One should always compare the drugged muscle with a normal or un-
drugged muscle from the same frog. This may be done in one of two ways.
I St. With the circulation undisturbed. Pin the frog face down on a frog-
board, isolate and attach the tendon Achilles to a muscle lever, isolate and
stimulate the sciatic at its origin in the lumbar plexus using care not to dis-
turb the circulation, or stimulate the muscle directly. After a normal record
is secured then give the drug in the usual way and take a record of the other
or drugged muscle. 2d. Lay a ligature about one leg near the thigh tight
enough to stop its circulation. Give the drug by injection into the dorsal
lymph sac, or abdominal cavity, and after absorption is complete and the
tissues have been acted on by the drug (20 to 30 minutes) dissect out the
gastrocnemii and test. Always use the undrugged muscle first and the
drugged one immediately following. While the normal muscle work is being
• tested the drugged leg should have its circulation stopped by ligature. This
leaves the two muscles in more nearly the same state of nutrition and
asphyxiation. It is usually best to stimulate the muscle directly. There
are three tests that can be applied: i. Irritability, by the minimal stimu-
lus method; 2, Rapidity of the simple muscle contraction; 3, The amount
of work a muscle will do with simple contractions at constantly repeated
intervals. In this latter test stimulate once in two seconds, record on a
drum with speed of i mm. per second.
TRANSFUSION BURET FOR MAMMALS.
Transfusions of several cubic centimeters of liquid should be warmed
to body temperature. Inclose a 50 c.c. buret in an ordinary Liebig's con-
denser jacket and mount vertically on a heavy base stand. Mount and
connect a 6-inch funnel with the upper side tube of the condenser. Attach
a rubber tube fitted with a spring compress clamp on the lower side tube to
ARRARATUS FOR THE STUDY OF THE ISOLATED MAMMALIAN HEART. 73
regulate the outflow of the warm water introduced by the funnel to keep the
perfusion liquid at the proper temperature. Mount a thermometer inside
the condenser with its bulb near the lower end of the apparatus. The buret
connections with the transfusion canula should be as short as possible and
their tubes should be provided with light screw-compresses. Where only
I or 2 c.c. of liquid is to be introduced it is unnecessary to warm it. In fact
a hypodermic syringe is most convenient where the volume of the injection
does not exceed 1.5 c.c.
APPARATUS FOR THE STUDY OF THE ISOLATED MAMMALIAN
The mammalian heart isolated completely from the body can be
maintained in constant activity for several hours. It gives constant responses
to drugs in solution in the perfusion liquid best adapted to maintain its
life, i.e., Locke's solution with a small quantity of the animal's defibrinated
blood. Cats and rabbits are especially well adapted to this experiment.
The smaller size of the cat's or rabbit's heart makes it preferable to that of
The points to be secured in the isolated heart apparatus are: i. A
uniform temperature of about 37° Centigrade. 2. An adjustable pressure
for the perfusion fluid. 3. A device for quickly shifting from the normal
perfusion to the drugged perfusion fluid without change in temperature,
pressure or any other factor than the presence of the drug. 4. An accurate
The apparatus shown assembled in Fig. 37 accomplishes all of the
above points. The gas water heater connected as shown will maintain a
uniform temperature in the water jacket through which the perfusion tubes
run to the heart canula. The overflow from the water jacket is conducted
into a pan in which the perfusion fluid reservoirs receive preliminary warm-
ing. The heart is attached to a very short canula beneath the warming
jacket and the overflow of perfusion fluid maintains a temperature of the
heart only slightly below that of the warming jacket.
The pressure on the heart, i.e., on the perfusion fluid, is accomplished by
connecting the perfusion bottle with an air or oxygen reservoir, and this in
turn with a water reservoir which can be raised or lowered. The flow of
water from the pressure bottle into the closed system produces the desired
pressure on the perfusion system. At the same time the perfusion fluids
are aerated by the air (or oxygen) as it is forced into the reservoir, a result
accomplished by conducting the perfusion bottle inlet tubes to the bottom
of the containers.
A uniform pressure is secured on both the normal and the drugged per-
fusion fluids by the system of tubes shown. If the clamp is removed from
Fig. 37. — Illustrating the assembly of apparatus for the pharmacological study of
the isolated heart of a mammal. The legends on the apparatus are self-explanatory.
the outflow tube of the drugged perfusion fluid at the exact moment a second
clamp is placed on the tube from the normal fluid reservoir (or vice versa) ,
the shift will be accomphshed without change of pressure on the heart. The
tubes run independently to the canula which is itself so short that the time
LIST or STOCK SOLUTIONS. 75
from the moment of turning a perfusion fluid on or off is reduced to a mini-
mum. The canula is provided with a side washout tube.
The Guthrie cardiograph shown is very adjustable in all essential fea-
tures. It gives satisfactory and accurate records, if care is used in inserting
the lever tips into the walls of the heart. This apparatus permits a direct
record on the ordinary kymograph. It also permits one to surround the
heart with a warm cup or jacket where greater constancy of temperature is
desired, as in research work.
LIST OF STOCK SOLUTIONS.
Make the solution up in 0.7 percent sodium chloride solution and in Ringer's
solution. Special solutions must be prepared for the mammalian heart experiments.
Aconite o.i percent.
Adrenalin hydrochloride o.ooi percent, o.oi percent, 0.05 percent, o.i percent.
Alcohol 95 percent, 2 percent, 5 percent, 10 percent, 20 percent.
Amyl nitrite 0.1 percent, pearls.
Atropine o.ooi percent, 0.002 percent, 0.1 percent, 0.2 percent, i percent, 2 per-
cent, 5 percent, and 1/120 grain tablets.
Barium chloride o.oi percent, 0.1 percent, 0.2 percent, i percent.
Caffeine 0.1 percent, 0.2 percent, 0.5 percent, e percent.
Calcium chloride 0.03 percent, 0.06 percent, i percent.
Carbolic acid 0.5 percent, i percent, 10 percent.
Chloral hydrate 0.1 percent, i percent, 2 percent.
Chloroform 0.05 percent, 0.1 percent, 0.5 percent, 20 percent in oil, pure.
Cocaine hydrochlorate o.oi percent, 0.2 percent, 0.5 percent, i percent, 2 percent.
Codeine 0.5 percent, i percent.
Curare 0.2 percent, i percent.
Digitalis 0.0005 percent, o.ooi percent, 0.002 percent, 0.1 percent, 0.2 percent,
0.5 percent, i percent.
Ether i percent, 2 percent, 4 percent, 6 percent, 8 percent, pure.
Ergot Squibb's fluid extract, 10 percent of fluid extract.
Hyoscyamine i percent.
Morphine acetate i percent, 2 percent, 10 percent.
Nicotine 0.02 percent, 0.1 percent, 0.2 percent, i percent.
Nitroglycerine 0.1 percent.
Physiological saline 0.7 percent.
Physostigmine 0.1 percent, i percent.
Pilocarpine nitrate 0.1 percent, i percent, 10 percent.
Potassium chloride 0.03 percent, i percent, 5 percent.
Potassium bromide 5 percent.
Quinine hydrochlorate 0.1 percent, i percent.
Ringer's solution, weak, strong.
Sodium nitrate o.oi percent, 0.02 percent.
Sodium sulphate i percent.
Strychnine nitrate i percent, 0.1 percent, .02 percent.
Thebaine 0.5 percent, i percent.
Veratrine 0.05 percent, 0.1 percent, i percent, i percent of fluid extract.
76 EXPERIMENTAL PHARMACOLOGY.
REPORT FORM FOR EXPERIMENTS ON FROGS.
I. Normal Reactions.
3. Respiration 4. Reflexes 5. Eye
I. Position, Activity,
2. Circulation, Heart,
Skin, Mucous Mem.
II. Action or the Drug.
Time Dose How given Dose per kilo.
h. m .s. ■ Apfjyijipg 2. Circulation 3. Respiration 4. Reflexes, etc. 5. Eye
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