Comparative Electro-Physiology: A Physico-Physiological Study

We have seen that the common effect of injury is to induce a galvanometric negativity of the point injured. We have further seen that in such a case the response to external stimulus is by a negative variation of the current of injury. We have next, then, to take up various instances which appear highly anomalous, cases, that is to say, in which the injured point, relatively to the uninjured, is, for some hitherto unknown reason, galvanometrically positive. As a result of this and other causes, there are, in addition to the cases already described in a previous chapter, instances in which response is found to take place, not by a negative, but by a positive, variation of the current of rest or of injury as the case may be.

The first point to be considered in connection with such abnormal responses is whether the experimental tissue is physiologically isotropic, that is to say, of equal excitability throughout, or anisotropic, possessed of unequal excitabilities at different points. The discussion of the first of these cases, the isotropic, I propose to defer to the following chapter. The anisotropic will be touched upon here, though its detailed consideration will be entered upon in the next.

As an example of the anisotropic organ, we may take the pulvinus of MWzmosa, in which the lower side is more excitable than the upper. In animal tissues also, such aniso- tropy is not uncommon. For example, we may have a _ muscular tissue terminating in a glandular. Owing to this anisotropy, the muscular and glandular surfaces are unequally excitable, and it will be shown in a later chapter that, generally speaking, it is the glandular which exhibits more intense excitatory galvanometric negativity. When such a

preparation is made, by cutting across the muscle, it is found that an electrical current flows from the uninjured gland to the injured muscle. From this it has been supposed that such a current was not the current of injury at all, but something of an unknown nature, essentially different. The consequent perplexity is the result of a failure to understand on the one hand that there is no such thing as a current of injury fer se, except as the after-effect of strong stimulation, and on the other, that the current induced in the tissue is always from the more excited to the less excited. In the present case of muscle-and-gland preparation, the excessive stimulation due to section becomes diffused all over the tissue, and since the glandular surface is the more excitable, its excitatory galvanometric negativity is greater than that of the sectioned muscle, which thus becomes relatively positive. We have here a striking demonstration of the necessity for regarding the electrical reaction as the sign, not of injury, but of the excitation caused by injury. In the case described, for instance, the physical injury is obviously incapable of transmission, and it is the consequent excitation which is con- ducted to the gland. _ The account of an experiment on a sensitive leaf of Mimosa will serve to elucidate the foregoing argument. If one contact, A, be made with the upper half of the pulvinus, and the other, C, with a distant and indifferent point, then, on giving a prick near A, we shall find that that contact, owing to excitation by injury, becomes galvanometrically negative. If, next, we make two contacts at diametrically opposite points of the pulvinus, A on the upper, and B on the lower, surfaces, it will then be found, on causing injury at the upper point A, that that point, relatively to B, becomes galvano- metrically positive.

This is because the stimulus caused bv the injury has become diffused throughout the pulvinus, witn the effect of causing greater excitation and consequent sreater galvanometric negativity at the more excitable B. It has been seen that a mechanical or thermal section acts as a strong stimulus. It has also been shown that recovery from a strong stimulus is very protracted. Hence, after such stimulation, there is persistent galvanometric negativity as an after-effect. As the intensity of this after- effect depends upon the intensity of stimulation, it will be seen that the galvanometric negativity near the section will be greater than at a distant point, where the transmitted effect of stimulation is feeble. From this it follows, that the so-called current of injury will flow in the tissue from the neighbourhood of the cut, to the distant and relatively un- excited end. The current of injury is thus an after-effect of strong stimulus. The peculiar electrical distribution which occurs in a muscle-cylinder is also found in a plant-cylinder, and both are equally explicable from the fact that the greatest excitatory after-effect occurs at the two sectioned ends, and that this decreases progressively towards the equator. The over-stimulated area of injury has its excit- ability depressed or abolished ; diffuse stimulation, causing sreater excitation of the uninjured contact, induces in it a greater excitatory effect of negativity, and this gives rise to a diminution of the existing difference of potential, as between the injured and uninjured. This is the explanation of response by the so-called negative variation.

In an anisotropic tissue the excitation caused by injury, when diffused, induces greater galvanometric negativity of the more excitable part. If this be the distal end, the re- sultant persistent current will be from the distal uninjured to the proximal injured. An apparently anomalous case: will thus arise of a ‘ positive’ current of injury, so-called. Anomalous case of response by positive variation—Inquiry into the cause— Electric exploration of dying and dead tissue : death being natural—Determi- nation of electric distribution in tissue with one, end killed—Dying tissue shows maximum negativity, and dead tissue, positivity to living —Explanation of this peculiar distribution—Response by negative or positive variation, depending on degree of injury—Three typical cases—Explanation by theory of assimilation and dissimilation misleading— All response finally traceable to simple fundamental reactions.

WE have seen in the last chapter that, in order to obtain response by negative variation, it is customary among investigators on animal physiology to kill one end of the experimental tissue, say by scalding. It is generally sup- posed also that dead tissue is negative to living. On stimu- lation, the induced negativity of the living contact, now superposed on the existing P.D. of the unilaterally killed tissue, causes a negative variation of it. This mode of investi- gation, by means of the negative variation, is one which has hitherto, as we have seen, been universally regarded as reliable.

In the course of my investigations on the response of vegetable tissues, by this mode of negative variation, however, I have sometimes found response to take place by the positive variation. Taking, for example, a stem of Lalsam, I killed one end by immersion in boiling water. On now subjecting this to diffuse vibrational stimulus, the responsive action was- found to induce a positive variation of the existing current. On further investigation, I found that the excitatory electrical variation at the living contact had remained normal; that is to say, the direction of the responsive current was away from the excited living, and towards the killed end. I next

found that the so-called ‘current of injury’ had in this case, owing to some hitherto unknown cause, undergone a reversal, and was now from the living to the dead, the latter being galvanometrically positive to the former, to the extent of ‘08 volt. The abnormality of the response lay, then, in this fact, that the current of reference had become reversed, and that the responsive current, due to excitation, was now con- cordant with it, instead of antagonistic, thus constituting a positive variation (fig. 112). Later on, I discovered many instances in which the killed end was positive to the unkilled. Since, then, it is possible for the current of reference itself to undergo such obscure and spontaneous reversals, from un- known causes, it is easy to see how uncertain the study of re- sponsive phenomena must become, if we are to depend upon the negative variation as our only Fic, 112. Response by Positive reliable means for their investiga- Variation of Resting Current tion. I next, therefore, turned my acti a aaeys howe Py attention towards an inquiry into reversed, the killed end having the causes of these anomalous Dans cathe a aise si reversals. »

The subject therefore resolved itself into an investigation as to what conditions determined the negativity or positivity of a tissue at the onset of death. My first attempt, then, was to study a case in which the approach of death was natural, and not the result of any sudden or violent change, such as might conceivably give rise to abnormal reactions. And in ‘ my search for suitable specimens, I noticed that often, owing to local mal-nutrition or other causes, the leaves of plants exhibited spots or areas, from which, as centres, death pro- ceeded in constantly widening circles. Thus, in the leaves of Colocasia, for example, we find such dead and dying areas in otherwise fairly healthy leaves. The innermost of these

patches may be quite dark and discoloured, while, as the living tissue is approached, this dark passes imperceptibly into yellow colour. And beyond this, again, we find the discolouration of yellow passing into the vivid green of living tissue. Proceeding thus in a radial direction inwards, towards the centre of such a patch from the living green, we shall find all possible stages of death, from its initiation, somewhere on the border-line between green and yellow, to its phase of completion, in the dark central area. On testing the electrical conditions of these different parts, I found that the border between green and yellow was negative to the living green surface. But the same point was also negative to the dead central area, and more negative to this than to the living tissue. Hence the dead was relatively positive to the living. Or if we make one fixed contact on the living tissue, and if the second exploring contact be made with various points

_ successively on a radial line passing from this to the centre of the dead area, these contacts will pass in succession through the living, the dying, and the dead. The variation of elec- trical potential will be found to be at its greatest along this line. The electro-motive difference between the point which has been fixed on the living tissue, and the exploring second contact, will at first be found to increase. The maximum difference is attained on reaching the border-line between green and yellow, or very little beyond this, this point being galvanometrically the most negative. On now passing further inward from this point, the maximum difference is found to decrease, till we come to a point in the dead tissue which is iso-electric with the living. On now again passing inwards, to the still more completely dead tissue of the central area, we find that we are approaching points which are more and more galvanometrically positive, as compared with the living tissue. The dying point on the border-line between green and yellow is thus the most negative, and points to the right or left of this are positive in comparison with it, the dead, however, being more positive than the living. It has been said that the electro-motive variation is most rapid along the

radial line. On the other hand, we obtain series of equi- potential surfaces whose outlines closely follow those of the boundaries of the different degrees of discolouration. [shall next proceed to give quantitative measurements. The first point to be considered is that of the choice of a definite electrical level, which is to be used as a standard. If this point be selected in the living tissue, we shall find that our standard of comparison is extremely variable, since the tonic condition, on which its electrical level depends, is itself subject to change. The only condition which cannot be modified in any way is that of complete death. This may be taken, then, as the standard level. The method of experi- ment will thus consist in selecting a series of equidistant points, abcd,and so on, 5 mm. apart, along a radial line, passing outwards from the central area, which is completely dead, to the green tissue. The non-polarisable contacts E and E’ are first placed on a and 4, then on @ and ¢, ¢c and d, and so forth. The external circuit contains a high resistance, compared with which any difference of resistance, as between any 5 mm. of interposed tissue, becomes negligible. Hence, the successive deflections of the galvanometer indicate the electro-motive difference that exists between a and @, 0 and «¢, and so on.

One difficulty which is experienced, in these measure- ments of small electro-motive differences, lies in securing the iso-electric condition of the non-polarisable electrodes them- selves. Whatever precautions are taken in the construction of these, a small electro-motive difference will sometimes be found to exist between them. The existence of such a difference is easily tested by bringing the kaolin ends of the two electrodes in contact, or by dipping both of them close together in a vessel of normal saline solution. Any electro- motive difference of the electrodes, however small, will now give rise to a large galvanometric deflection.

This difficulty may be overcome by first taking special precautions as to the purity of zinc rods and the chemicals employed, and secondly, by keeping the electrodes for a long time short-circuited, with their ends dipped in normal saline. In very obstinate cases, however, I succeeded in eliminating all differences by subjecting the electrodes to cyclic variations of alternating electro-motive force. By means of a Pohl’s commutator, without cross-bars, the electrodes were put in connection with an alternating source of E.M.F., and with the galvanometer intended to test the resulting variation in the E.M. difference, by turns. — A small hand-driven alternating-current generator was used for this purpose. The speed of rotation of this machine was gradually raised to a maximum, and afterwards as gradually slowed down. Thus at each cycle the electrodes were subjected to ascending and descending intensities of alternating electro-motive variations. The effect of such cyclic changes, in diminishing the existing electro-motive difference between a pair of electrodes, specially selected for carelessness of preparation, will be clearly seen from the following tabular

Condition at starting Galvanometric deflection E.M. difference Original difference . , 360 divisions 009 volt After first cycle : F 40 Js 52?) After second cycle . ; fe) ie One, After third cycle. ; fe) - ee It will thus be seen that, after a very short time of this treat- ment, the two electrodes were rendered iso-electric. I next proceeded to determine the distribution of elec- trical potential in the various portions, living and dead, of the leaf, In order to remove any accidental strain, the leaf was placed in tepid water, and kept there for about half an hour, till the water was cooled to the surrounding temperature. The experiment was then carried out, in the manner already described, and the following tabular statement shows the results obtained. The electrodes, it will be remembered, were placed successively at points 5 mm. apart from each other, along a radial line proceeding from the dead tissue to the living, the first point being taken as zero;

It will be observed that as we proceed from the dead to the dying, the negativity of the latter rapidly increases, the maximum being at 30 mm. from the zero-point taken on the Fic. 113. Distribution of Electric Potential in Lamina of Co/ocaséa along a radial line from dead to living through intermediate stages. Ab- scissa gives distance in mm. from chosen centre in dead tissue, ordinate represents galvanometric negativity in divisions. Dead tissue repre- sented dark, dying shaded, and living white.

dead tissue. This point of maximum-negativity almost coincides with the visible border-line between the yellow and the green. Beyond this, however, there is an electrical reversal, the living becoming increasingly positive, as com- pared with the dying. An inspection of the curve (fig. 113) shows that while there is a point in the tissue between the dying and the dead, which is equipotential with the living, the completely dead tissue is positive to the living.

I next carried out an experiment in which death was artificially induced, by immersing a portion of the tissue in boiling water. In connection with this, I may say that it is extremely difficult to ensure the complete death of a thick — tissue. It is only the outside layers which undergo death easily, but the interior tissues, from their protected position, are extremely resistant, and it is only after prolonged immersion in boiling water that death can really be ensured throughout. In the present experiment, however, where only a part of the tissue is to be killed, such prolonged immersion would cause death to encroach upon those portions of the tissue which were intended to be kept alive. This difficulty was met by choosing a specimen, the inside of which was accessible to boiling water. The peduncle of the water-lily (Nymphea alba) in transverse section appears extremely reticulated, and there is thus no difficulty in exposing all its parts to the direct action of the hot water.

The upper end of the peduncle was kept surrounded by a cloth moistened in ice-cold water, the lower end being immersed in boiling water for ten minutes. The specimen was then placed.in tepid water, and allowed to cool down slowly. In this way a length of the peduncle was ob- tained, in which one end was completely killed, whereas the other remained fully alive, the intermediate portions showing all stages of the transition from the living to the dead condition. In order to determine the electrical distribution in its different parts, I now employed the potentiometer method of balance. One electrode was per- manently connected with that dying point which by a previous test had been found to exhibit maximum nega- tivity. The second electrode was placed at successive points,

each of which was nearer than the last by 5 mm. to the dead end, which was to the left. The same process was now re- peated, the successive | readings however being taken towards the right or living end. At each point, the electro-motive difference was balanced by the potentiometer. This straight form of potentiometer had a Fic. 114. Straight Form Potentiometer scale divided into one x8 isa stretched wire with added resistances, Rand R’. Sis a storage cell. When the thousand parts (fig. 114), key, K, is turned to the right, one scale

and when its terminal division = ‘ooI volt, when turned to the . left one scale division = ‘or volt. P is the electro-motive force was plant. adjusted to 1 volt, each } division of the potentiometer was equal to ‘oor volt. The following table gives the results obtained : Towards left or dead end, : : Towards right or living end ; ; t t = ; ; , E.M. difference in yo55 volt “ee ee, iene C + (—) or E.M. difference in y;55 volt Here, also, as in the case of natural death, we find a point in the dying tissue which is most negative. From the curve given in fig. 115, it will also be seen that as we pass away from this point in either direction towards the living or dead area, we find an increasing positivity ; the curve for the dead portion is, however, much steeper than that for the living. Thus two points, one 1'5 cm. to the left in the dead tissue, and another 5 cm. to the right in the living tissue, are iso-electric. But while the maximum positivity of the living

is ‘O18 volt, that of the dead is 0233 volt. Hence the dead tissue is here positive to the living, to the extent of 0053 volt. We have seen that the prevailing idea is that the dead is negative to the living. But from the results here shown, we can see that this is not a complete statement of the case. Since then the electro-motive variation, instead of showing a 2 3 4 +e) Fic. 115. Distribution of Electric Potential in Petiole of Vymphea alba, one end of which has been killed.

The point of maximum negativity is taken as zero, distances to the left or towards the dead taken as mznws, to the right or living, as Aplus. Ordinate represents potential difference in thousandths of a volt. progressive change from the living to the dead, exhibits a maximum difference, followed by a reversal, it may be asked, what is the reason of this anomaly ? Much light is thrown on this subject from the results given by another line of inquiry, to be explained in detail in Chapter XVI. _ It is there shown that the plant-tissue on the first onset of death exhibits a sudden contraction, indicative

of a strong excitatory reaction. This corresponds with the rigor mortts of the animal, and by means of suitable apparatus, the concomitant mechanical response can be recorded. An electrical record of the same phenomenon may also be obtained, in the form of an electrical spasm of galvanometric negativity. Succeeding to this rigor of the dying tissue, a post-mortem relaxation takes place, with a concomitant change from galvanometric negativity to positivity.

Now in a tissue which has been killed unilaterally only, it will be understood that all possible gradations are to be expected. Passing from the completely dead to the fully _alive, we must necessarily pass through various zones, beginning with the abnormally relaxed, through the inter- mediate highly contracted. and rigored tissue on the death- frontier, to the living, which is not so contracted as the dying, and not so relaxed as the dead. At the point where the onset of death-.is recent, the rigor, or excitatory contraction and galvanometric negativity, are at their maximum, Compared with:this, the slightly tonically con- tracted living is positive, but not so positive as the abnormally relaxed dead. :

The death-frontier, however, is not fixed. It is con- tinually encroaching on-the living. The line of maximum rigor and galvanometric negativity is thus also shifting in the same direction. Along with this, however, the opposite process of post-mortem relaxation is proceeding; so that a point which was, in consequence of rigor, maximally negative, becomes gradually converted to positive. This positivity of dead tissue as compared with living, which has here been demonstrated in the case of the plant, I find to be also true of animal tissue, in those cases which I have investigated. ‘Thus, while an injured.and dying area in a frog’s nerve is negative, an already dead area is positive, relatively to the living nerve. There is, moreover, an intermediate area, between the dying and dead portions of the nerve, which is iso-electric to the living,

Hence, having one contact fixed on a living area and the other on (1) the dying, (2) the intermediate, and (3) the dead tissue, we shall obtain three different types of what is known as the ‘injury-current.’ In the first of these the second contact will be negative, a condition which has hitherto been assumed to be the sole characteristic of the current of injury. But there are two other cases to be considered. Of these, when the second contact is made at a point intermediate between the dying and dead tissues, we shall find it to be iso-electric with the first, or living contact. And thirdly, when the second contact is on a dead area, the latter will be positive to the first, or living contact. We thus find three cases of the current of injury— the first being negative, the second zero, and the third positive.

Taking the first of these—that in which the injured contact is negative—the action-current, in response to stimulus, will bring about a negative variation of the so- called current of injury. In the second, the result will be indeterminate, since the injury-current is zero. In the third, the response will be by a positive variation of the current of injury. I give below three photographic records in illustration of these three cases, obtained with vegetable nerve. I may state here that I have often observed results precisely similar in the case of frog’s nerve also. In the first record, in fig. 116, the thermal injury was moderate. The injured point was thus negative, and the current of injury is represented here by an up-line. The responses are seen to be by negative variation. In the second record the injury was greater, and the injured point was almost neutral; that is to say, on making contact there was a slight up-twitch, which subsided to zero. There is here, then, no current of injury. The subsequent responses are, however, down, the action-current being away from the living contact. In the third record the injury was so great as completely to kill the injured point, which thus became positive to the living. The

reversed injury-current is represented as down, the subse- quent excitatory responses are also down, and constitute a positive variation of the current of injury. It will thus be seen that an identical excitatory reaction of the living tissue appears to give rise to directly opposite Fic. 116. Photographic Records of Responses of Vegetable Nerve, one end of which has been injured In the first injury was slight; current of injury represented up, response by negative variation. In the second, injury greater; injured point neutral, response down. In the third, injured point killed; injury current reversed down, response by positive variation.