Comparative Electro-Physiology: A Physico-Physiological Study

Fic. 103. Photographic Record of Response of Petiole of Cauliflower Fic. 102. Photographic Records. by the Diametric Method (a) Di-phasic response of petiole A contact was naturally more excitable, of Bryophyllum, the up compo- hence resultant ‘up’-response. Ex- nent being due to the excitation citability of A being depressed by of right side. Strong application local application of ice, the re- of KHO on the right abolished sultant response became converted this responsive component, giving to ‘down’; normal ‘up’-response rise in (4) to enhanced down was restored on allowing the tissue

purpose, I took a petiole of cauliflower. In this instance, the natural excitability of the upper contact, A, was greater than that of the lower, B. Hence the resultant response was not zero, but ‘up.’ The point A was now cooled locally by ice. This process so lowered its excitability that that of B was now relatively the greater, hence the resultant response was found to be reversed or ‘down. The point A was next allowed to return to the surrounding temperature of the room, records of the response being taken meanwhile, at intervals of one minute. It will be seen how, by means of the gradual restoration of the original excitability of A, the resultant response changes gradually from negative to zero, and then again from zero back to positive, indicating the restoration of the naturally greater excitability of A (fig. 103).

We have thus studied two different methods, both of which depend on interference, for the determination of the variations of excitability induced by different external agents. In a subsequent chapter we shall study a modification of this method, by means of which it is possible to demonstrate the variations not only of excitability but also of conductivity under various reagents. Different theories of current of injury—Pre-existence theory of Du Bois- Reymond—Electrical distribution in a muscle-cylinder—Electro-molecular theory of Bernstein—Hermann’s Alteration Theory—Experiments demon- strating that so-called current of injury is a persistent after-effect of over- stimulation—Residual galvanometric negativity of strongly excited tissue— Distribution of electrical potential in vegetable tissue with one end sectioned—Electrical distribution in plant-cylinder similar to that in muscle- cylinder—True significance of response by negative variation—Apparent abnormalities in so-called current of injury—‘ Positive’ current of injury.

IF a section be made of an uninjured nerve or muscle, the transverse contact will be found to be galvanometrically negative, as compared with an uninjured longitudinal contact. I shall have occasion in the present chapter to give a simple explanation of this phenomenon and of the excitatory nega- tive variation of the current of injury. It is, therefore, only necessary to recapitulate briefly the three theories which have hitherto been proposed on this subject.

The Pre-extstence Theory of Du Bois-Reymond supposed that the smallest particle had the same electro-motive characteristics as the entire tissue, each such electro-motive molecule consisting of two bi-polar portions, the positive poles of any two molecules being always face to face with each other. This theory was based upon the fact that a muscle-cylinder, for example, exhibited a peculiar distribu- tion of electrical tension. There are in such a cylinder, one longitudinal and two transverse surfaces. Midway in the cylinder is the equatorial zone of the longitudinal surface, and this zone is positive to all the rest. Thus the electro- motive difference between one electrode placed on the

equator, and the other, is increased as the latter is moved further and further away, say towards the right transverse section. The distribution of electrical tension on the left side of the equator is symmetrical with this (fig. 104). On these facts was based the theory of Du _ Bois- Reymond; but this has Fic. 104. Distribution of Electrical : me ‘nite hed Tension in Muscle-cylinder. since been found to be in- adequate. I skall later return to the explanation of the particular distribution of electrical tension involved.

According to the theory of Bernstein, known as the Electro-chemical Molecular Theory, the fundamental attribute of the molecule is chemical. Its poles are supposed to attach to themselves electro-negative groups of atoms, while its sides attach oxygen, and stimulation is supposed to be attended by explosive chemical changes. According to Hermann’s A/teration Theory, finally, all the _ electro-motive activities of living tissues are supposed to be due to chemical rather than molecular changes of the substance. In amplification of this theory, Hering attributes all electro- motive phenomena to the disturbance of equilibrium by up and down chemical changes.

It is my intention to show in the course of the present chapter that the current of injury is an after-effect of over-stimulation. And since excitation is fundamentally due to molecular upset, we shali best understand the electro-motive changes concomitant with it, if we first study it and its after-effect under the simplest conditions, namely those of inorganic substances. For here the action of such complicating factors as assimilation and dissimilation is clearly out of the question.

We have found for example that a piece of well-annealed wire was iso-electric throughout its length. In the first place, when a portion of it was subjected to any molecular disturbance, an electro-motive difference was induced, as between the molecularly disturbed or excited and the un- disturbed areas. The intensity of this electro-motive change, in the second place, was seen to increase with intensity of excitation. And, thirdly, the recovery from excitation was seen to be delayed, where the intensity of stimulus was strong (fig. 105). This is shown in the electrical

Fic. 105. Photographic Record showing Persistent Electrical After-Effect in Inorganic Substance under Strong Stimulation. Note the tilt of base-line upwards response of tin as a persistent after-effect, the sign of which is the same as that of the excitatory electro-motive change. A similar state of things is exhibited mechanically in a torsioned wire. When the torsion is moderate, and the molecular distortion slight, the released wire quickly re- covers its original position of equilibrium. But when the torsion is excessive and the wire strained beyond a certain limit, it remains for a long time in a torsioned condition,

even after it has been set free. Recovery is thus, in such a case, indefinitely delayed. In other words, a molecularly over-strained substance exhibits a persistent after-effect. Turning next to plant response, we find a similar per- sistence of the after-effect to occur in consequence of over- stimulation. And first we shall take the simplest case— that in which the tissue is directly stimulated. Here the specimen was petiole of cauliflower, and increasing stimuli

Fic. 106. Photographic Record exhibiting Persistent Galvanometric Negativity in Plant Tissue after Strong Stimulation Stimuli applied at intervals of three minutes. Vertical line = ‘1 volt. were applied, at intervals of three minutes, by means of a gradually increasing angle of torsional vibration. It will be noticed that whereas the electrical recovery from moderate stimulation—as seen in the first of the series—is complete, it becomes, with increasing stimulus, more and more in- complete (fig. 106). In other words, the tissue, after strong stimulation, is seen to exhibit an after-effect of residual galvanometric negativity, which is really due to incomplete molecular recovery, in consequence of over-strain.

We shall now, however, take an instance in which excitation is transmitted and observe the persistent negative after-effect, due to strong stimulation. We know that a cut (mechanical section) or the application of a hot wire (thermal section) acts as a strong stimulus, and the effective intensity of such stimulation will obviously decrease with increasing distance from the point of stimulation. Hence, if we observe the persistent excitatory change of galvanometric negativity, which is induced as between an indifferent point—say, the surface of a leaf—and points increasingly near to the zone of section, we shall find that the electro-motive change is greatest at the point of section, and is progressively lessened as we recede from it. This induction may be verified experimentally by taking readings of the persistent negativity, as between an indifferent point, B, and points such as the contacts a, 0, c, d, A (fig. 107), which are further and further removed from the point of section. For this purpose we may employ a capillary electrometer, whose indica- tions are independent of the varying resistance of the interposed tissue. The magnifying power of the observing microscope was so adjusted that ‘1 volt gave a reading of 100 divisions of the micrometer. In carrying out an experiment on the leaf of Colocasza I found the electrical distribution, as between an indifferent point on the lamina and points on the sectioned petiole, at increasing distances from the section, to be as shown in the following table :

TABLE SHOWING ELECTRICAL DISTRIBUTION IN SPECIMEN OF Co/ocasia. Tiintaie thom section E.M. difference between indifferent and given points It will thus be seen that points near the sectioned end are more negative than others further away. method of investigation. The resistance is here maintained constant by having the permanent. contacts at A and the indifferent point B (fig. 107). The specimen is a stem of Calotropis gigantea. A thermal section is made at first, say, at a distance of 3 cm. from A. The persistent galvanometric

negativity of A will now be due to the after-effect of stimula- tion by section. The thermal injury is now repeatcd at | Fic. 107. Experimental Arrange- Fic. 108. Records showing in- ment for determining Electrical creasing Persistent Galvano- Effect due to Section metric Negativity, according as injury is caused nearer to proximal contact A, z.e. moved from 3 to'5 cm, distance decreasing distances from A. I give a series of records (fig. 108), from which it will be seen that when the stimulus of thermal section occurs at some distance, there is no

persistent after-effect, recovery being complete. But as the effect becomes greater and greater. From observations made . in the course of a similar experiment, I obtained the following results, given in tabular form, which show the increasing value, with lessening distance, of this persistent galvano- metric negativity. In fig. 109 we have a curve which illustrates these results, and explains why the maximum negativity is at the zone of section, diminishing rapidly as we recede from it. It is obvious that if these sections had been made to the right as

Fic. 109. Curve showing the Electrical Distribution in Stem with one Sectioned End Ordinate represents galvanometric negativity ; abscissa, the distance from sectioned end. well as to the left of A, the result would have been a duplicate series of changes of galvanometric negativity in reference to A, on. that side also. Such a series is represented in fig. 110, by means of dotted lines. It will also be seen from this figure that the greatest electro-motive difference exists

as between the equatorial point A and the two terminal sections a and a’; that symmetrical points cc’, 0 0’, a a’, are equipotential ; and that a point relatively nearer the terminal section is galvanometrically negative, in reference to one further away from it. It will also be seen that this electrical distribution is exactly the same as that seen in a muscle- cylinder, with terminal sections, as given in fig. 104. Thus, without the postulation of any electro-motive mole- cules so-called, these experimental results afford a simple and direct explanation of the so-called current of injury, as the excitatory after-effect of strong stimulation.

Fic. 110. Electrical Distribution in Plant-cylinder with Opposite Ends Sectioned The Current of Injury is simply therefore an excitatory after-effect, due to incomplete recovery from over-strain. But even after strong stimulation a slow recovery may occur, and the Current of Injury will thus undergo a progressive diminution. This will probably account for Engelmann’s observation that in medullated nerves the E.M.F. of the artificial cross-section fell, by as much as from 25 to 60 per cent., in the first two hours after section, and disappeared altogether within twenty-four. The renewal of the cross- section he found to renew the original difference. This is an obvious case of renewal of the effect by re-stimulation.

takes place when the injury has not been excessive. If, however this has been too great, the injured tissue will then pass gradually into a condition of permanent death. But the electrical change concomitant with death is one of positivity, as I shall show in the next chapter. Thus the subsidence of the galvanometric negativity of an injured point may be brought about by either of two processes, which are exactly opposite—namely, recovery or death.

Turning next to the subject of the Negative Variation of an existing current of rest, as a reliable index to the state of excitation, two different questions arise. First: why, in order to obtain response to diffuse stimulation, is it necessary previously to subject one of the contacts to injury? And secondly: why is the responsive action-current opposite in direction to the resting-current, thus constituting a negative variation of it? With reference to the first of these questions, we have already seen that when two points, A and B, are simul- taneously excited, the resultant electro-motive response is equal to E,—E,. If, then, the excitabilities of these two points are the same, it is clear that the resultant response will be zero. From this we can see that, in order to obtain a resultant response, we must depress or abolish the excita- bility of one of the two contacts.

This inference may be verified by the employment of the Method of Block and of longitudinal balance. Two equal and opposite responses are first obtained at A and B. Then one end, say B, is injured by thermal section. The specimen being now replaced in the vibratory apparatus, it is found that, whereas the A half gives strong response, the end B gives none. Or the B end of the specimen may be injured by a few drops of strong potash, the other end remaining uninjured. The end A is then stimulated, and a strong response is obtained. The end B is next stimulated, and there is little or no response. The block between A and B is now removed, and the specimen stimulated throughout its length. Though the stimulus now acts on both contacts,

yet, owing to the irresponsive condition of B, there is a resultant response, and the direction of this action-current is found to be from A to B. We have thus experimentally verified the assumption that in the same tissue an uninjured portion will be thrown into a greater excitatory state than an injured, by the action of the same stimulus. ; When the point B is injured, there is generally speaking a more or less persistent current set up which flows from | Bto A. But we saw that the direction of the action-current was opposite—that is to say, from Ato B. This will explain the reason why the action-current causes a diminution or negative variation of the current of injury, so called. One method of doing this is to cause injury to one of the two points. If this be such as to kill the tissue, then its excitability is permanently abolished. Or by causing the excessive stimulation of injury, we may simply depress the excitability of the tissue for a longer or shorter time.

I shall now give a few instances of response in plants by negative variation, Taking the petiole of turnip, we injure an area on its surface, say B. A current is now observed to flow in the petiole from the injured B to the uninjured A. The induced difference of potential depends on the condition of the plant, and the season. In the experiment here described, its value was ‘13 volt. A sharp mechanical tap was now given to the petiole, between A and B, and a sudden diminution, or negative variation, of current occurred, the resting potential difference being decreased by ‘026 volt. A second and stronger tap induced a second response, causing a greater diminution of potential difference by ‘047 volt.

In another experiment, the specimen employed was a petiole of cauliflower (Brassica oleracea). ‘The first up-line to the right indicates the current of injury. The three re- sponses which succeed are induced by a given intensity of stimulus, the next series of six, being in response to stimulus nearly twice as strong, exhibit signs of fatigue (fig. 111). The current of injury generally undergoes a diminution with time. This is often, as has been explained, on account

of slow recovery from the excessive stimulation of injury. Response by negative variation is then found to undergo a decline. It is in general vaguely accepted that, in order to obtain a response by negative variation an‘antecedent current of injury is necessary, by whose induced variation we may be able to record responsive effects. In cases of the dis- appearance of the current of injury, it is supposed that response must necessarily vanish, since its antecedent con- dition no longer exists. But I have already shown, and shall

Fic. 111. Record of Responses in Plant (Leaf-stalk of Cauliflower) by Method of Negative Variation The first three records are for stimulus intensity I ; the next six are for in- tensity twice as strong; the successive responses exhibit fatigue. The vertical line to the left represents ‘I volt. The record is to be read from right to left. have occasion again to show in the next chapter, that these suppositions are altogether erroneous. For we may obtain the usual response when the current of. injury is zero, or even positive. In fact, the only essential condition for the obtaining of resultant response is that at one contact the excitability should be in a state of relative depression.

In that case in which response becomes enfeebled, with the gradual decline and vanishing of the current of injury, a simple explanation is often applicable. When the tissue is injured, it does not necessarily die. In fact, I have often found that, in order to ensure death—in the case for instance of thermal section—a prolonged application of the fatal temperature is necessary. In ordinary cases of injury caused by the application of heat, I find that we have merely exces- sive stimulation of the point, with depression of excitability. But after a long interval, excitability is more or less restored, with the gradual passing away of the effect of injury. The subsidence of the current of injury thus also denotes the restoration of excitability to a greater or less extent. Hence that differential action between the uninjured and injured contacts, which determines the amplitude of the resultant response, will become correspondingly diminished. And when response has undergone diminution from this cause, a fresh injury is found to renew its amplitude. This is due to the reduction of excitability now freshly brought about at one of the contacts. €

There are, however, two other additional factors which may further contribute to the enhancement of response after a recent injury. We have seen that, as a general rule, the resultant response will be E,—£E, where E, means the excitatory electrical change induced at A, and E, that induced at B. It would therefore appear that this value will be at its maximum when the excitability of B is totally abolished by reason of injury, the resultant effect being due to the unopposed electrical excitation at A. But we have seen that when the true excitatory negative variation of a point is abolished, it may nevertheless exhibit a positive electrical variation, due to hydro-positive action. When this happens to be the case, this positive effect at B, conspiring with the true excitatory effect at A, may bring about a response larger than we should have supposed to be maximum.

Again, though over-stimulation of a point diminishes its excitability, yet moderate stimulation often enhances it. The effect of this, in enhancing resultant response, is well seen in the case of conducting nerves. Thus, when the point B is injured, the excitation caused by injury reaches A, and causes moderate stimulation of that point. As an after-effect of this moderate stimulation, A often becomes more than normally excitable.! It is thus seen how, after a recent injury, these two factors—of a hydro-positive effect at the injured, and of increased excitability at the uninjured contact, in consequence of moderate transmitted stimulation—may act to enhance the response.