A laboratory manual of experimental physiology

Preface

Experimental physiology is now recognized as a fundamental subject in the curriculum of the medical student and as one having a most important place in the training of the student of biology. In the medical course, the physiological laboratory serves as the portal to the clinic; as the testing ground where the student may try for himself in how far the known laws of physics and chemistry can be successfully employed to explain the normal working of the human machine. No more can theoretical study, or demonstra¬ tion, by itself supply a correct understanding of the functions of the living body than could similar methods in training an engineer to understand an engine. Attempts to rectify, by operations or by drugs, functional derangement in the diseased animal without a practical knowledge of the normal working of the various organs, both isolated and as a whole must be as unjustifiable as attempting to repair a complicated piece of machinery would be by any other than a practical engineer.

In the training of the biologist, experimental physiology finds its value because it teaches how to interpret the relationship be¬ tween structure and function. For the advancement of physio¬ logical knowledge it is essential that the functions of the lower animals should be more intensively investigated by those who have been trained in the methods of the experimental physiologist.

In the crowded curriculum, either of medicine or of biology, it is impossible to find an amount of time that is sufficient for the performance of more than a few of the fundamental experiments in each of the main subdivisions of the subject of physiology. It is the duty of the teacher, therefore, to select these experiments with the greatest care so that the experience which the student gains in their performance may guide him aright in building up his knowledge of this subject. The experience gained in the labora¬ tory is to serve as the framework upon which the detailed con¬ struction is to be completed by fitting on to it in their proper re¬ lationship to one another the other facts of physiological know¬ ledge, acquired by theoretical study and demonstration.

Experimental Physiology.

While admitting these principles some have averred that the technical difficulties of experimental physiology are such as to make it impossible for the average medical student to secure a sufficient number of results to justify the expenditure of time and energy required in the laboratory. This is a fallacious criticism for it assumes that every experiment to be of value should be crowned by results that are technically flawless, and it fails to recognize that the performance of the work, if carefully and in¬ telligently done, affords that personal experience without which, in a highly practical science like medicine, theoretical knowledge by itself is valueless and without meaning.

There is, however, no subject of the medical curriculum in which the organization and arrangement of laboratory courses is more difficult for classes of average size than in physiology. In physics and chemistry, as well as the various morphological sub¬ jects, the material for the laboratory course is constantly available, and can be stored away for future use, whereas in physiology living material must be provided afresh for each experiment and there must be constant supervision of the practical work to see that the material is properly used. This requires that the student be adequately directed as to how he should proceed with the experiment without at the same time stifling originality on his part by re¬ quiring him explicitly to follow detailed directions in every step. The experiment is of no value unless it is performed with an inquir¬ ing attitude of mind, and laboratory directions and instruction should be no fuller than is necessary to guide the student in its general performance.

It is with these requirements in view that the present volume has been compiled, and while the authors realize full well its many shortcomings they hope that the practical instruction, not only in their own laboratories, but also in those of other institutions may be assisted by its publication.

The work is arranged in sections, each of which deals with some special part of the subject, this plan being adopted because it has been found the most practicable for courses designed for large elementary classes, as well as for smaller groups of more advanced students. The first section deals with the fundamental experiments in the physiology of isolated muscle and nerve, placed

Preface.

in this position not because this is the simplest part of the subject of physiology to understand, but because it is the easiest to provide material for and therefore the best in which to permit of sufficient practice, so that the student may become familiar with the methods of physiological technique. The two following sections deal with essential experiments illustrating the principles of the heart beat and the circulation of the blood. In selecting experiments in the last mentioned group the endeavour has been made to apply them, as far as possible, to man. A certain number of experiments in which other mammalian material is used is, however, essential in order that the student may be in a position to appreciate the significance of results which will later be demonstrated to him. These demonstrations are described in the last sections of the manual in sufficient detail, so that the various steps may be followed by every student of the general course and the experiments may be performed by small groups of more advanced students.

In the experiments on the central nervous system the decere¬ brate or spinal animal is extensively made use of, as recommended by C. S. Sherrington whose work in this direction must be con¬ sidered as the most important contribution that has recently been made to the advancement of the teaching of practical physiology. In the section on the special senses a relatively larger proportion of theoretical matter is given, along with the directions for the experi¬ ments* partly because the latter, being practically all subjective in nature, cannot be performed unless the student understands fully what he is looking for, and partly because most of them can, and should be performed in the study rather .than the laboratory.

A great difficulty has always been felt by instructors in physio’ogy in attempting to supply simple experiments bearing on the chemistry of respiration and yet there is no part of physiology in which practical experience is more important, if the student is to understand the principles of this difficult subject. A section of the manual is devoted to experiments in which simplified and inex¬ pensive apparatus for gas analysis is employed.

Throughout the book, besides the directions for the experi¬ ments, a brief statement is given of the theoretical matter which bears on them. This is done to enable the student to appreciate the object of the experiment and to guide him in the interpretation

Experimental Physiology.

of the result; but the latter is not described in detail, this being left for the student to determine for himself.

While the general plan and arrangement of the book is the work of the authors jointly, each author is responsible for certain chapters as indicated by the initials given in the table of contents.

The authors wish to express their thanks to Miss Marion E. Armour for the care and patience which she bestowed in the pre¬ paration of the drawings for the illustrations, many of which are original while others are copied from other texts, acknowledgment for which is given in the legends.

They wish also to thank Miss Jean Halliday for her assistance in arranging the index and correcting the proof. They are indebted to Dr. J. P. Eisenberger for a final reading of the proof.

Lois McPhedran Fraser.

F. A. Hartman.

J. J. R. Macleod.

The Universities of Buffalo and Toronto.

Section I. MUSCLE AND NERVE

Chap. Page.

Preface . 3

I. Preliminary Technique. By F. A. Hartman . 9

II. Voluntary Muscle. By F. A. Hartman . 13

III. Smooth Muscle and Ciliated Cells. By F. A. Hartman. .. . 41

IV. Physiology of Nerve. By F. A. Hartman . 45

Section IIA.

CIRCULATION.

V. Cardiac Muscle. By F. A. Hartman . 56

VI. Blood Pressure. Effect of Chemical Substances on the

Blood Vessels and Heart. By F. A. Hartman . 65

Section IIB.

CIRCULATION.

VII. Haemodynamics. By J. J. R. Macleod . - . 71

VIII. Vaso Motor Nerves. Depressor Nerve. Circulation Time.

By J. J. R. Macleod . 80

IX. P ol ysph y gmogra ph Tracings. By J. J. R. Macleod . 84

Section III. REFLEX ACTION

X. Reflex Action in the Frog. By J. J. R. Macleod . 89

XI. Reflex Action in Man. Reaction Time in Man. By J. J. R.

Section IV. RESPIRATION

XII. Analysis of Air and Collection of Alveolar Air. By J. J. R.

XIII. Respiratory Exchange in Man. By J. J. R. Macleod . 102

XIV. Determination of the Gases of Blood by the Pump Method.

By J. J. R. Macleod . 106

XV. Determination of Blood Gases by the Chemical Method.

By J. J. R. Macleod . 112

XVI. The Dissociation curve for Oxygen and the C02 Combining

Power of Blood. By J. J. R. Macleod . 116

Section V. SPECIAL SENSES

XVII. Vision. By Lois McPhedran Fraser . 123

XVIII. Errors in Refraction. By Lois McPhedran Fraser . 133

XIX. Examination of the Refraction of the Eye and of the

Interior of the Eyeball. By Lois McPhedran Fraser.. . 142

XX. Retina. By Lois McPhedran Fraser . 147

XXL Colour Vision. By Lois McPhedran Fraser . 154

XXII. Simultaneous Contrast. Visual Judgments. By Lois Mc¬ Phedran Fraser . 162

XXIII. Hearing. By Lois McPhedran Fraser . 166

XXIV. Skin Sensations. By Lois McPhedran Fraser . 175

Contents.

Section VI. DEMONSTRATIONS

CIRCULATION AND RESPIRATION.

Chap. Page .

XXV. The Circulation of the Blood and Lymph and the Re¬ spiration. By J. J. R. Macleod . 182

XXVI. The Direct Demonstration of Vasoconstrictor by the

Plethysmographic Method. By J. J. R. Macleod. . . . 185 XXVII. Perfusion of the Excised Mammalian Heart. By J. J. R.

XXVIII Measurement of the Blood Flow Through the Hands and Feet by the Calorimetric Method. ByJ. J. R.

XXIX. Lymph Formation. By J. J. R. Macleod . 197

XXX. Experiments to Demonstrate the Pumping Action of the Heart and the Action of the Valves. By J. J. R.

Macleod . 201

Section VII. DEMONSTRATIONS

DIGESTIVE SYSTEM.

XXXI. The Innervation of the Salivary Glands. By J. J. R.

XXXII. The Control of the Pancreatic Secretion. The Secre¬ tion of Bile. By J. J. R. Macleod . 206

XXXIII. Experiment on the Normal Secretion of Saliva and

Gastric Juice. By J. J. R. Macleod . . . 209

XXXIV. The Movements of the Oesophagus and Intestine. By J. ].

R. Macleod . 214

Section VIII. DEMONSTRATIONS

THE CENTRAL NERVOUS SYSTEM.

XXXV. Reflex Action in the Mammalia. By J. J. R. Macleod . 217

XXXVI. Cerebral Localization. Decerebrate Rigidity. Reci¬ procal Innervation. Functions of Spinal Roots in

the Mammal (Dog). By J. J. R. Macleod.. . . 221

XXXVII. The Decerebrate Cat I. By J. J. R. Macleod . 225

XXXVIII. The Decerebrate Cat II. The Decerebrate Pigeon.

By J. J. R. Macleod . 229

XXXIX. The Spinal Cat. By J. J. R. Macleod . 233

Appendix . 236

Index . 239

Chapter I. Preliminary Technique

Electrical Stimulation. — In experimental physiology it is necessary in many instances to employ an artificial stimulus in order to cause action of the tissue studied. Many kinds of stimuli are capable of this, but most of them have their objections when used as stimuli of nerve or muscle. Mechanical, chemical and thermal stimuli are all more or less injurious. Moderate electrical stimulation on the other hand brings these tissues into action without appreciable injury. As this is the means by which the tissues are usually stimulated, we will discuss briefly the principles involved in the more important pieces of electrical apparatus employed.

For a source of current either a dry cell or a dynamo may be used. A dry cell is essentially a large zinc cup serving as container in which is suspended a carbon cylinder. A small amount of water containing ammonium chloride held by gelatin or some inert substance fills the rest of the container. A dry cell is essentially a wet cell, but with the water protected from spilling by the gelatin “sponge”. Whenever the carbon and zinc are con¬ nected by a conductor, electrical energy is set free by the solution and is transmitted along the conductor. It has been found that if a live muscle is placed in the electrical circuit that the tissue will be stimulated at the time the circuit is made and again when it is broken provided the current is great enough.

Two important factors must be kept in mind in the use of an electric current, the resistance of the tissue and the “pressure” of the current. The unit of “pressure” is the volt. Therefore the amount of pressure is called the voltage. By increasing the voltage the penetrating power is increased and thus the physiological effect augmented. If the resistance of the tissue is high as in dry skin, a

Experimental Physiology.

greater voltage is necessary than if the skin had been moistened, because moist skin is a better conductor. Dry cells are made to give in the neighbourhood of one or two volts. Greater voltage in a circuit can be obtained by connecting two or more cells in series, i.e., zinc to carbon.

In most of your work the current generated by dynamos in the power plant will be used. At each working place two wires from the main line are connected with a metal ribbon of high resistance. Terminals are arranged so that different proportions of this ribbon can be used in the circuit, thus varying the amount of voltage. The amount of voltage obtained can be estimated by adding the total voltages between the two terminals employed. The following diagram gives the voltages between terminals. T represents the terminals as arranged on the table.

T8T8T4T4T4T2T2T abcdef gh

To illustrate the method of finding voltage, connecting a to f gives 28 volts; f to h, 4 volts; d to e, 4 volts.

Besides the direct current one can use the induced current for stimulation. It is well known that if one wire is placed near another wire which carries a current, a brief current is induced in the first wire at the time of breaking the circuit in the second. Likewise a current is induced at the closing or making of the circuit. The wire carrying the direct current is called the primary circuit, while that in which the current is induced is called the secondary circuit. Greater effects can be produced by having a coil of well insulated wire surrounding a soft iron core as the primary circuit element and another coil of insulated wire as the secondary element. These two coils are arranged on a stand for convenience, the whole apparatus being termed an inductorium (Fig. 1). The chief advantages of the induced over the direct current for purposes of stimulation are; first, the voltage is very much greater so that high resistances can be overcome and, secondly, it can readily be altered in strength.

It has been found that the least induced current is produced when the secondary is at right angles to the primary; from this position the current is increased more and more as the secondary becomes more nearly parallel. In addition to the angular relation-

Preliminary Technique.

ship, the distance between the two coils determines the strength of current induced, the closer they are the greater the effect. Experiment 1. — Try the following. — 1. Connect the electrodes to two terminals on the table so as to obtain 2 volts. Touch the tongue with the electrodes and then make and break the circuit by means of the switch. Repeat with increasing voltage. Describe the effects at make, at break and during the passage of the current.

2. Connect the binding posts 1 and 2 (Fig. 1) of the primary coil to 4 volts on the table, with a simple key in the circuit.

shocks.

Connect the electrodes to the end of the rods upon which the secondary slides. With the secondary coil far out and parallel to the primary coil stimulate the tongue by making and break¬ ing the primary circuit. Move the secondary closer and closer to the primary coil and note the effect on the current produced. Again move the secondary out into such a position that it just clears the primary coil. Now study the effect of tilting the secondary coil.

Experimental Physiology.

The Graphic Record. — By the use of a lever it is possible to magnify muscle contraction. The movement of this lever is re¬ corded on smoked paper. The kymograph is devised for carrying the smoked paper.

Glazed paper is wrapped around the drum of the kymograph so that the glaze is outward. The gummed end is fastened to the opposite end so that a tight fit is made and the paper will not slip. The drum is held in the smokiest part of a gas flame while being rotated rapidly on the brass tube which is maintained horizontally by hand. In this way a uniform coating of carbon can be obtained on the paper. A light chocolate colour is to be preferred to a deep black because there is less danger of burning the paper.

A small triangular piece of photographic film or waxed paper is fastened by a bit of wax to the end of the lever in order to serve as a writing point. The lever is always on the right hand side of the drum so that when the latter is rotated it pulls away from the writing point. The lever must be at a tangent to the curved surface of the cylinder in order to prevent the writing point leaving the smoked surface when it rises. The writing point may be bent inward to keep the rest of the lever free of the drum.

When ready to remove the paper from the drum, it is cut at the overlap by a sharp knife in such a way that the knife strikes the under paper and not the drum. A corner of the paper is grasped against the edge of the drum with the other hand so that it will not fall. At this stage the record may be placed on the table and lettering or other explanatory matter inscribed. It is well to place your name and date on each record. The record, with smoked side up, is passed once through a rosin-alcohol solution (120 gm. rosin to 1,000 c.c. of 95% ethyl alcohol) and then hung over the tray to drain. When dry the parts desired are to be care¬ fully cut out and preserved in the note book.

Instruments for Dissection. — Each student should "supply himself with the following: Two pairs of scissors, one heavy the other fine; two pairs forceps, one heavy the other fine; one dental probe, one strabismus hook, one scalpel, one small serre fine and one haemcstat. The fine scissors and fine forceps are to be used only in delicate dissection. In order to do the best work the cutting instruments must be kept sharp.

Chapter II. Voluntary Muscle

Animal life differs essentially from plant life in the power of movement. Whatever the power which certain plants may have in this respect is extremely limited. Animals on the other hand not only contain organs which are more or less constantly moving, but also possess the extremely useful power of locomotion. This power to move resides in contractile tissue. Muscle is the most important type of this kind of tissue in the higher and many of the lower animals. What can an animal do without muscle? There would be no respiration, no circulation, no movement. Truly muscle is a master tissue.

A portion of the muscle of the body is under the control of the will. Upon this muscle we depend for locomotion and the major movement in the organism. Such muscle is composed of fibres with a characteristic cross striation. These fibres are by far the most active in the body, not only working rapidly, but doing the most work.

We could study muscle by watching it contract or by finding out how much it could do. The knowledge gained by such means would be very limited. We should find that a muscle shortens and thickens when it contracts; we might discover that heat is produced in the process, but many changes would escape observa¬ tion. A simpler method and one by which more accurate informa¬ tion can be obtained is to isolate a single muscle and study its responses to artificial stimuli.

STIMULATION.

Various kinds of stimuli will cause muscle to contract, but before we decide upon the best form to use we must know where to apply the stimulus. Normally the muscle fibres are called into action by impulses passing along the nerve fibres. Artificial stimuli can

Experimental Physiology.

be effectively applied along the same path. Thus mechanical, thermal, chemical and electrical changes can cause a muscle to act through its nerve. Any of these changes to be effective must not only be of sufficient magnitude but must be abrupt. A nerve slowly compressed may be without effect, but if quickly pinched, a twitch of the muscle results.

Of all artificial stimuli, the electrical is not only the most effective, but as before stated, the least injurious to nerve. Me¬ chanical stimulation produces more or less injury. Thermal change which is great enough to have an effect is likewise detrimental. Chemical stimulation if not harmful, is at least difficult to control. Moderate electrical changes have none of these objections.

Although we are most interested in the physiology of mam¬ malian muscle, we employ muscles from cold-blooded animals because, while similar in function, they survive for a much longer period when deprived of their circulation.

Experiment 2. — Nerve-Muscle Preparation. — Kill a frog in the following manner: By bending the frog’s head down, a depres¬ sion between the skull and first vertebra can be felt on sliding a blunt-pointed seeker back along the mid-line of the top of the skull. Plunge either the seeker or a scalpel into this de¬ pression and by moving crosswise sever the cord. Turn the seeker forward into the skull cavity and by transverse move¬ ment destroy the brain. Next destroy the cord by passing the seeker down into the spinal cord. If the frog is properly pithed it becomes limp. A frog thus prepared is dead as an organism although the heart and other tissues may live for a few hours. There can be no pain whatsoever. This method of killing a frog is called "pithing”.

In a pithed frog, cut across the body just behind the fore limbs, with strong scissors. Remove the viscera, being careful to avoid injury of the nerves lying along the dorsal wall of the body cavity. Now divide the preparation by cutting longi¬ tudinally through the middle of the vertebrae and pelvis. Lay one leg aside until needed. Strip the skin from the other leg, being sure to keep the outer surface of the skin from coming in contact with the muscle.

Voluntary Muscle.

Lay the preparation on a clean glass plate and keep it moistened with isotonic salt solution. Carefully dissect out the sciatic nerve by separating the muscles on the dorsal aspect of the thigh and freeing the nerve from the surrounding tissues. Always lift the nerve with a glass hook and avoid stretching it. By means of the vertebrae raise the roots of the sciatic nerve and clip beneath it until it is free down to the knee. Cut away the other muscles and the bone below the knee and free the femur from all muscle except the gastrocnemius.

In making a nerve-muscle preparation one must always remember that slight injury to muscle or nerve may ruin it for experimental purposes. Such injury may be caused by stretching, pressing, drying or chemical agents. NaCl dis¬ solved in water to the extent of 0.7% produces an isotonic solution somewhat similar to the tissue fluid of the frog, there¬ fore useful in keeping the tissues moist without the injurious effect of pure water. Apply this solution to both the nerve and muscle frequently.

NERVE-MUSCLE PREPARATION.

Experiment 3. — Different Kinds of Stimuli. — Fix the femur of a nerve-muscle preparation in the flat-jawed clamp, after it has been fastened to the stand. Clamp the nerve holder to the stand just below and then lay the whole sciatic nerve on the glass plate. Fasten the muscle lever underneath. Attach the tendon, by means of a hooked pin to the lever. Now adjust the after-loading screw so that the muscle supports no weight but is fully extended when at rest. Place a ten gram weight upon the scale pan (Fig. 2). Arrange the lever so that it will write on the smoked paper of the kymograph and obtain records with the following types of stimuli:

Mechanical. — Pinch the nerve near its end with forceps. Can you get a response the second time from the same place? Chemical. — Place a few salt crystals on the fresh end of the nerve, the injured portion having been cut away. After a few contractions of the muscle thoroughly remove the salt with isotonic salt solution.

Experimental Physiology.

Thermal.— Touch the fresh end of the nerve with a hot glass rod.

Electrical —Connect the stimulating electrodes directly to the current supply on the table but with a simple key in the circuit. Try the effect of make and break beginning with 2 volts and then increasing the current.

Fig. 2. Apparatus for recording muscle contraction: (a) femur clamp; ( b ) nerve holder (c) electrodes; ( d ) muscle lever; ( e ) signal magnet; (J) after-loading screw; (g) wires connected in the primary circuit.

Next connect the electrodes to the secondary coil of the inductorium using a primary current of 2 volts; stimulate with make and break shocks of different intensities.

Compare the effects of make and break in direct and indirect currents.

\ Minimal and Maximal Stimulation.— Beginning with a stimulus too weak to cause contraction, then very gradually increasing its strength, a point is reached where a slight contraction

Voluntary Muscle.

results. This is called the minimal stimulus. In other words it is the least stimulus which will cause a contraction. On the other hand the maximal stimulus is the least stimulus which will produce maximal contraction. When a series of stimuli of increasing strength, beginning with the minimal, is sent into a nerve or muscle, one obtains a graded response (Fig. 3). This is true no matter how finely graded is the increase of the stimulus. (See all-or-none principle, p. 55).

^Experiment 4. — Response to Stimuli of Different Intensities,

— For this experiment employ a muscle which has been cut away from its nerve. Instead of using ordinary stimulating electrodes connect one terminal of the secondary coil to the

Fig. 3. Contractions produced by graded stimulation. Secondary coil moved 5 mm. at each step. Both make and break shocks employed. Make contraction begins at m.

femur end of the muscle by wire and in a similar way connect the other terminal to ihe binding post on the muscle lever. Then a bit of fine wire is wound around the tendon and finally fastened to the coarser wire at the binding post on the muscle lever. In this manner a current can be passed through the muscle.

Starting with the secondary as far as possible from the primary, cautiously move it forward by stages, each time stimulating the muscle with a make and break shqck. When the first perceptible contraction is produced you have reached the threshold. Between each stimulation move the drum a

Experimental Physiology.

short distance by hand, not more than 1 mm. Now carefully move the coil toward the primary so that at each position there is produced a slightly greater contraction than before. Distinguish the “make” and “break” contractions on the record by the letters m and b placed beneath. Also designate the position of the secondary coil. It is possible by carefully increasing the stimulus to obtain a graduated series of con¬ tractions from both the make and break shocks (Fig. 3). When the series is complete, indicate the minimal and maximal stimuli.

Discuss the application of the all-or-none principle to your records.

INDEPENDENT EXCITABILITY OF MUSCLE.

Ordinarily the question, as to whether a muscle could respond if its nerves were absent, is of academic interest. But if we wish to test the power of a muscle which has lost its voluntary function by injury to the nerve, we find long after the nerve has degenerated, that the muscle responds to direct electrical stimulation. It can also be shown that muscle is directly excitable by paralyzing the motor end-plates, which are structures connecting motor nerve fibres with muscle fibres. This can be done by the injection of the poison “curare” into the circulation. After a few minutes has elapsed, stimulation of the nerve no longer affects the muscle, while stimulation of the muscle causes it to contract. From such experiments as these it appears that muscle can contract without the mediation of either nerve fibres or motor end plates.

The various types of stimuli can affect a muscle directly, perhaps not so readily as through the nerve fibres however. Amongst these stimuli there is one type which should be dwelt upon par¬ ticularly in a certain connection. That is chemical change. Sup¬ pose you permit a fresh muscle, which has been dissected from the body, to be exposed to dry air. Moisture quickly evaporates from its surface. The solutions within the cells become more con¬ centrated. Chemical stimulation may result, producing twitches. In any case the cells are injured if the water loss goes far.