Comparative Electro-Physiology: A Physico-Physiological Study

There is, however, a serious discrepancy in this view, _ inasmuch as, while local stimulation of the upper surface of the frog’s skin induces.a positive change, a similar stimula- tion of an unmistakably glandular surface is found to bring about a negative. If then the electrical effect on the skin of frog be the same as on a glandular surface, the dis- crepancy of their responsive reactions becomes inexplicable. As regards the excitatory change, very diverse results have been recorded when stimulus has been applied indi- rectly —that is to say, through the nerve. This fact is not to be wondered at, since the responsive effects are subject, as will be shown, to numerous modifying influences. It is generally

supposed, in the preparations made for these experiments, that it is one surface only which is electro-motive. I shall show, however, that the responsive effects are brought about by the differential excitability of the two. This response, again, is modified by relative changes induced in the two surfaces. And, in addition to these, still further compli- cations are introduced when the stimulus is indirect—that is to say, applied through the nerve. In this case, the relative excitations of the two surfaces will be determined by the particular distribution of the nerve-endings. Again, we shall see that, in an isolated preparation, the nerve itself is liable to undergo certain changes by which its trans- mitted effect may be modified even to reversal (p. 530). Thus, so long as it remains highly excitable, the transmitted effect is one of true excitatory galvanometric negativity. But with physiological depression, the conductivity of the nerve is very much lowered, and the effect transmitted becomes reversed to positivity.

For all these reasons, if we wish to study the specific reactions of skin, epithelium, and gland preparations, it is better to do so by observing them under direct stimulation. Engelmann, in studying the responsive reactions to direct stimulation of frog’s skin, found a negative variation of the current of rest. Since the latter was naturally ‘ingoing,’ as regards the upper or’ epidermal surface, this meant that the responsive current was ‘outgoing.’ Reid, again, work- ing on the skin of the eel, obtained ingoing response, or positive variations of the resting-current, by single induction shocks in either direction. Biedermann, in the mucous membranes of the tongue and stomach, obtained both positive and negative variations of the current of rest, Waller, using single induction shocks in either direction, found in the digestive mucosa both ingoing and outgoing responses, the former being much predominant.

Even under the simple conditions imposed by direct stimulation, then, the results obtained are seen to be in- consistent. They would appear to show both that the responsive effects in the different preparations are different and that, even in the same preparation, they may be reversed under unknown changes of circumstances. It appeared to me, as already said, that much light might be thrown on the questions thus raised by means of an investigation carried out on plants. The most perfect method of experiment here would consist in observing the

the preparation. Waller employed single induction shocks for this purpose, observing the after-effect. But in this case, the action of polarisation was not excluded. It would thus be more satisfactory, in order to eliminate this unknown element, to employ either a non-electrical mode of stimulus or an electrical form which would leave no resultant polarisation effect. The latter condition, as we have seen, was fulfilled by the employment of rapidly alternating currents, whose alternating components were absolutely equal.

As regards the application of a non-electrical form of stimulus, both thermal and mechanical forms may theoreti- cally be employed. Engelmann and others used heated metals in the proximity of one of the electrodes, for the production of thermal stimulus. This, however, has the disadvantage of thermo-electrical variation, due to unequal heating of the two contacts. Besides this, there is also the effect of a rising temperature, which, as we have seen, is opposite to that of sudden variation, the latter alone consti- tuting the excitatory effect. I have already explained how these difficulties may be overcome by using thermal shocks in which a sudden thermal variation is made to act on both contacts at once. The resultant response thus obtained was shown to be determined by the differential excitability of the two contacts under examination. As regards the mechanical mode of stimulation, previous observers have employed pressure or friction. Such stimulus, however, is at best merely qualitative. If it be applied at the contact itself, objections may be taken to the effect, as

due to, or modified by, the variation of contact resistance. ‘And if to avoid this the mechanical stimulus be applied, not at the electrode, but at a neighbouring point, the results will be quite different, according as the conductivity of the intervening tissue is great or slight. In the former case they will consist of the transmitted effect of true excitation ; in the latter of the indirect effect, whose electrical sign is the exact opposite. Specimen of skin pinned on hinged platform which is pressed against electrodes by elastic india-rubber. Electrodes rotated by cord, c c’. S, antagonistic spring, made of elastic. Enlarged view of electrode seen to the right. T, outer fixed brass tube ; T', inner rotating tube, holding non-polarisable electrode. P, pumice-stone.

A perfect method of direct mechanical stimulation has been described in Chapter III., the stimulus being vibrational. But for investigations on limp structures, such as skin, this method is not practicable, and the modification which I am now about to describe is necessary, in order to meet the difficulties of the case. The apparatus consists of a hinged platform, P (fig. 176), on which the specimen is securely pinned. The two electrodes, E and E’, rest with a definite pressure on the two points A and B, whose excitatory re- actions are to be studied. These electrodes have at their

lower ends projecting pumice-stone cylinders of equal sec- tions, soaked in normal saline. When the electrode E is rotated, the mechanical friction induces local excitation of the point A. B may also be subjected to similar isolated excitation in the same way. In order that such successive excitations may be quantitative and uniform, it is necessary first that a definite area at A or B should be stimulated. In. other words, there must be no lateral slip. For this reason |

the electrodes are passed through tubular holders which, from the description presently to be given, will be seen to allow rotation about a definite vertical axis. The extreme bases of the pumice-stone cylinders are, as has been said, of equal section. The glass electrode tube is tightly fixed, by means of a cork, inside a brass rotating tube. The latter, again, plays inside an outer brass tube, which is fixed. The inner brass tube is provided with two collars, one below and one above, by means of which rotation can take place without up or down movement. A string is also wound round it, by pulling which rotation is produced. The electrodes are perpendicular to the plane of the platform which carries the -specimen. It will thus be seen that any variation of the surface subjected to stimulation is prevented.

The next difficulty to be overcome is that of liability to variation in the pressure of the contact. It will be remembered that the platform is hinged. It is further held up against the electrodes by the tension of an elastic piece of india-rubber or a spiral spring of steel. This pressure can be regulated to a suitable value, and kept constant. The final difficulty is to apply successive stimuli of equal value, and to render them capable also of graduation from low to high values. This could be secured by rendering the successive rotations of the exciting electrodes equal in number and ‘in time of execution. The intensity of stimulus might then be increased by increasing the number of rotations or the pressure of the electrodes on the specimen.

In order to apply successive rotations of definite number, one end of the string wound round the ‘inner brass tube is attached to a piece of stretched india-rubber, which is fixed by its other end to the apparatus. The second end of the string is tasselled, after being passed through a fixed ring. This position of the string is adjusted by means of a knot, so that the india-rubber at its other end is already in a state of tension. When the tassel is now suddenly pulled and let go, it gives rise to a number of rotations in the positive, followed by an equal number of rotations in the opposite direction, the latter work being performed by the stretched antagonistic spring. It should be remembered that a mechanical rotation, whether p/us or mznus, gives rise to the same excitatory reaction. Next, to make the number of rotations definite, let us suppose the inner brass tube to have a circumference say of I cm. If the string be now pulled through a distance of 5 cm., and let go, there will be five rotations in the positive, followed by five rotations in the negative direction. A second knot in the string, at the distance of 5 cm. from the first, exactly limits the length of the pull; and increase or decrease in the intensity of the stimulus.can be brought about by a change in the distance of the regulating knot.

The distance between the two electrodes being always the same, the resistance of the interposed tissue remains approximately constant. To nullify any accidental variation, a high and constant external resistance is interposed in the galvanometer circuit. When the excitatory electro-motive variation of the specimen is very great, it is possible to use an external resistance as high as one million ohms. It should, however, be remembered that even if there be any unavoid- able variation of resistance, it will not in any way affect the discrimination of sign of the characteristic electro-motive response. For the excitatory effect at either electrode may be tested by repeating the experiment with the other. The experiments which will be described afforded definite and characteristic records, which were found capable of repetition. The physiological character of such responses was further demonstrated by repeating the experiment, after killing the

tissue with boiling water, when these electro-motive variations were found to disappear. How very reliable these responses can be rendered is shown by the photographic record in fig. 180, which is of very special interest, giving as it does the record of responses afforded by the intact human skin. Turning now to the nature of the response of. the skin, it has been found by Engelmann and others, as already said, that in the frog, while the natural resting current is from the —

outer surface to the inner, the responsive current is from inner to outer. Dr. Waller, again, undertook to analyse the constituent elements of this response, by passing induction shocks along each of the surfaces, first upper and then lower, and in both directions. He then observed the after-effect at one of the excited points, in relation to an indifferent point. In this way he found that an excited point on the upper surface becomes galvanometrically positive, the current being thus outgoing. The inner surface, however, he found to be ineffective. When an induction shock is passed across the tissue, the resultant response from lower to upper is thus, according to Dr. Waller, due to induced positivity of the upper surface.

With vegetable specimens, however, such as the outer skin of apple, the results obtained by him were opposite to those of the frog’s skin. ‘The responsive currents were here found to be ingoing, the excited point being galvanometrically negative. The explanation offered, in regard to these results, is that living tissues have the peculiarity of responding by ‘blaze currents’ to electrical shocks. The use of this phrase, however, as already said, offers no real explanation ; but even apart from this point, the question remains, Why should the blaze currents, so called, be directly opposed in the cases of frog’s skin and of the particular vegetable skins which are mentioned, respectively? In answer to this, the hypothesis put forward by Dr. Waller is, that the difference arises from the different natures of animal and vegetable protoplasms.'

' ¢ Vegetable protoplasm is in major degree an instrument of synthesis and accumulation, in minor degree the seat of analysis and emission, Animal I shall, however, be able to adduce facts and considera- tions from which it will be possible to arrive at a simpler and more conclusive explanation of these phenomena, on the basis of the differential physiological excitability of the two surfaces. I shall show, moreover, that the difference between animal and vegetable protoplasm, thus assumed to exist, has nothing to do with the question.

We have seen that when the physiological activity of a tissue is in any way impaired, its normal excitatory re-action of galvanometric negativity is depressed. This may even go so far as to cause an actual reversal of the response, to galvanometric positivity, as we found in the case of depressed tissues (p. 84). Taking a vegetable specimen, then, say a hollow petiole or peduncle, we find that the outer surface, which is habitually exposed to the manifold influences of the environment, becomes histologically modified, being much more cuticularised than-the inner. Thus these outer and exposed cells generally become reduced in size, and thick- walled, with little protoplasmic contents. Hence, as regards functional activity, these epidermal cells are in a_physio- logical sense very much degraded. We should then expect their excitability to be proportionately lowered in comparison with, say, the inner surface of the same tube, protected as that has been from outside influences. And the variation of physiological excitability thus induced may involve not only the surface, but also the subjacent layers to a certain extent.

Theoretically, then, the induced galvanometric negativity of the outer would be less than that of the inner surface,! and simultaneous excitation of both, by whatever means produced, should give rise to a resultant responsive current protoplasm is in major degree an instrument of analysis and emission, in minor degree the seat of synthesis and accumulation. The vegetable, in most immediate contact with inert things, combines, organises, and accumulates, The animal, in less immediate contact with inert matter, disrupts, utilises, and dissipates in their fragments organic compounds that it has received ready made from other animals and from plants.’—Waller, Signs of Life, p. 85.

1 This refers to normal skin, and not to that in which the surface is typically glandular. from inner to outer. The degree of this diminution of excitatory negativity in the outer surface, moreover, cul- minating as this may in actual positivity, will depend upon the extent of its transformation. In connection with this it should be remembered that, in order to bring out the differential excitabilities of the two surfaces, it is necessary to apply localised stimuli of an intensity not too excessive. For, if the stimulus be very strong, there is always a possibility of its affecting deeper layers of the tissue and thus causing complications in the resultant excitatory changes. The intensity of stimulus which may be safely

3 y such complications will depend b- a zl on the conductivity of the Pd A creme tissue. Epidermal cells are, ee Gar pas generally speaking, feeble con- os | ductors, but in this matter it ee must be understood that the ; differences in this respect be- Fic. 177. Diagram Representing ‘ : Different Levels of Excitability, tween different tissues are not Pika: sels ease eee ' absolute, but a question of Diagram to right of figure shows ; degree, and may to a certain

how resultant up response (inner to outer) may be obtained when extent be modified under dif- induced change at A is plus, and at B minus, or when induced ferent circumstances. Thus a eee less negative than feebly conducting tissue, under a favourable condition of tem- perature and strong intensity of stimulation, will become to a certain extent conducting. Highly conducting tissues like nerve, on the other hand, under unfavourable circumstances conductors. Returning now to the question of the responsive reactions

of skin, we see the theoretical possibility of the following typical reactions. Let the scale of excitabilities be repre- sented by diagram to the left of fig. 177. Now, if the trans- formation of the outer epidermal surface, A, be maximum, the sign of its reaction will exhibit the greatest extent of deviation from the normal negativity. That is to say, its response will become absolutely positive, as represented by a above the zero line. The response of the inner surface, B, may be normal and strongly negative, as represented by e below the zero line. When both these surfaces, then, are simultaneously excited, the excitatory positive variation, or ‘outgoing’ current at A, will conspire with the ‘ingoing’ current at B and the resultant electro-motive difference will be B, + A,, the direction of the responsive current being thus from inner to outer. But the same resultant up-response will also be induced, even if the reaction of both surfaces be negative, provided only that that of the outer, A,, be less negative than that of the inner, B,, as explained by the diagram to the right of fig. 177. The responsive current will then be represented as B,—A,, that is to say, as proceeding from the more negative B to the less negative A. We have thus examined the two extreme cases possible under the following formula, in which the arrows show the direction of the responsive current, from the more to the less induced negative:

I shall next proceed to demonstrate the existence of these two extreme types, taking vegetable skins as the experimental specimen. It was supposed by Dr. Waller, as will be recalled here, that owing to characteristic differences between animal and vegetable protoplasm the response of vegetable skin was opposite to that of animal skin: that is to say, the former was ‘ingoing’ and the latter ‘outgoing.’ That this generalisation is not, however, justified, will be seen from the experiments which I am about to describe, carried out on the skin of grape.

These results, it should here be pointed out, are not dependent upon any one method of inquiry, for each problem was subjected to attack and analysis by four different modes of experiment. The first of these (1) was by the Rotary Method of Mechanical Stimulation. This method has the great advantage that by it the absolute response of each surface is displayed separately, without either being affected by the other. There is here, moreover, no complication due to the polarisation factor, inevitable when uni-directioned induction shocks are employed for excitation. Thus, after the individual responses of each surface have been analysed, we are able to arrive at.a definite conclusion as to what would be the character of the resultant

response if both the surfaces were simultaneously excited. | This conclusion is then submitted to three other tests. Thus, (2) the two surfaces of the specimen are subjected simul- taneously to the same thermal shocks, according to the method already described. Again, (3) the Method of the After Effect under equi-alternating shocks is employed. And, finally, (4) the Direct Effect of these equi-alternating shocks of moderate intensity is recorded. The results obtained by all these diverse methods are in complete concordance with each other, and fully support the theo- retical inferences which have already been made.

I took the skin of a ripe muscatel grape, such as are available in Calcutta. On making the galvanometric con- nections with the outer and inner surfaces, a resting current, -C, was found to flow in the skin from the outer to the inner, just as in the skin of the frog (right-hand diagram, fig. 178). The grape skin was now mounted in the rotary stimulating apparatus, first, for the stimulation of the outer or epidermal surface, with the outer layer placed upwards. The distance between the two electrodes was always the same—namely, 2 cm. On now stimulating one of the two contacts, response took place by the induced galvanometric positivity of that point. That is to say, the current was ‘outgoing, into the galvanometer circuit, from the surface of the skin. When the second point was now stimulated the deflection previously obtained was reversed, the second contact thus also exhibiting galvanometric positivity on excitation.

The position of the skin in the apparatus was now changed, the inner surface being placed upwards. In this way points diametrically opposite to those in the last case were subjected to excitation. It was now found that the responses of the inner surface were normal—that is to say, of galvanometric negativity. I give here (fig. 178, a) records of two successive sets of responses obtained from the external and internal points A and B. These records clearly demon- strate that the resultant up-response, on simultaneous exci- tation of the outer and inner surfaces, is brought about by the induced galvanometric positivity of the outer added to the induced negativity of the inner surface. As the resist- ance of the circuit in the two successive experiments was maintained approxi-

mately the same, the . + bs amplitude of these re- Pile F sponses gives a fairly el t a . One sf \ induced on the two sur- Fic. 178. Electrical Response of Grape- faces. The positive or skin to Rotary Mechanical Stimulation (a) A, positive response of outer surface ; B, negative response of inner surface ; outer surface is here (6) c, current of rest, from outer to inner ; ; R, excitatory response from inner to outer, slightly greater than the consisting of summated results of positive ‘ ingoing’ effect. The pig aes of outer with negative response

diagram in fig. 178, 3, shows how the individual effects conspire to give rise toa responsive current from the inner to the outer. In order to test the reality of the correspondence between this response of the grape skin and that of the frog, I now repeated these experiments, employing the same apparatus for mechanical stimulation, on the skin of the frog. From the records given in fig. 179 it will be seen that the isolated response of the outer surface is positive, or ‘outgoing,’ that of the inner being negative, or ‘ingoing. The amplitude of the former was, however, much greater than that of the latter. These responses disappeared altogether when the tissue had been killed by immersion in boiling water. From isolated responses obtained by means of induction shocks, Dr. Waller had been led to regard the outer surface of frog’s