Comparative Electro-Physiology: A Physico-Physiological Study

which sub-tonicity is manifested by arrest of growth, we may select a specimen in which, while the tissue is not fully tonic, there is still, nevertheless, a feeble rate of growth. In such a case we may expect the income from the absorption of stimulus to prove greater than the expenditure in the form of true excitatory response. Hence, if we subject such a tissue to the constant action of an external stimulus, we shall in the first stage obtain the predominant effect of the internal factor, with its positive turgidity-variation and enhanced rate of growth.

But by the continued action of this accumulating income, the tonic condition of the tissue will be raised to the normal, with a concomitant increase of excitability. It will now therefore be the excitatory component which becomes pre- dominant, resulting in the negative turgidity-variation, con- traction, and retardation of growth. In order to detect these variations of the normal rate of growth, under the action of stimulus, it is necessary to have at our disposal some very delicate means of record. This need I have, however, been able to meet, by devising the Balanced Crescograph, more fully described in my book on ‘ Plant Response.’ Here, the uniform rate of elongation of a growing organ causes a rotation of the recording Optic Lever. The spot of light from this lever falls upon a second mirror, which is subject to a compensating movement. When the balance is exact, the spot of light, reflected from the two mirrors, remains quiescent. When, however, the normal rate of growth, under the action of any agent, undergoes varia- tion, the balance is upset. Thus, when growth is accelerated, there is a movement of the recording spot of light in one ‘direction, say up, and when retarded, a movement in the opposite, say down.

In order to study the effect of external stimulus on a tissue in a slightly sub-tonic condition, I took a flower-bud of Crinum lily,and first obtained a balanced record, seen as a horizontal line (fig. 52). Stimulus of light was now applied, and it will be seen that after a short latent period the absorbed stimulus induced a positive turgidity-variation, with enhanced rate of growth, as seen by the up-curve. But by this absorption of the stimulus itself, the tonic condition of the specimen was raised, with consequent increase of ex- citability, and the response became normal. That is to say, it now consisted of contraction and retardation of growth as seen by the downward curve. The external stimulus was now cut off and the

dotted portion of the curve shows the after- effect. The after-effect is thus not a mere re- covery, but an enhanced rate of growth, due to the increased energy which remains latent. It was only when this was ex- hausted that the normal rate of growth was re- Fic. 52. Balanced Record of Variation of established, as seen in Growth in Flower-bud of Crinum Lily eo £ under Diffuse Stimulation of Light the horizontal part o the Continuous lines represent the effect during

curve. And as the tissue application of light, the dotted line on : f ‘ withdrawal of light. The plant was was now in full tonic originally in a sub-tonic condition, and condition the renewed application of light at x, after short aged ; latent period, induces preliminary ac- application of stimulus celeration of growth. After this follows . : . the normal retardation. On withdrawal of light did : “ gs hers of light, in the dotted portion of the curve induce a preliminary en- is seen the after-effect, followed by

be Sith ty f return to the normal rate of . growth. ancement of the rate o A second and long-continued application growth, but the normal of light induces retardation, followed by A oscillatory response. tion. Its long-continued application gave rise to the further Now owing to this fact that the response of growth gives us by means of the enhancement or depression of its rate, effects which correspond to the positive and negative, we are able clearly to perceive :

(1) That when the tonic condition or the excitability of the tissue is low, the predominant effect will be the positive. This has been shown during the course of the present chapter in the case of a very sub-tonic tissue of Zamarindus indicus, where the positive or growth-expansion effect was initiated by the action of stimulus. (2) That when the sub-tonicity of a tissue is not very great, incident stimulus will at first give the positive effect of an enhanced rate of growth. But with the absorption of the stimulus itself, the tonic condition of the tissue will be raised, and we shall then obtain the true excitatory reaction of con- traction and retardation of growth. Thus, in this intermediate case, the positive response will be seen to pass into normal negative. ,

(3) And, lastly, that when the tonic condition is already high, the excitatory negative response will predominate and we shall obtain normal contractile response. Both this and the previous intermediate cases are illustrated by the experi- ment described on Crinum lily. It was there seen that the first effect of incident light was positive, the tissue being sub-tonic ; subsequently, the tonic condition being raised, this response was converted into the excitatory negative. And on renewed application of stimulus thereafter the immediate response continued to be negative.

The fact that by means of growth-response, it is possible to obtain indications of the external and internal work per- formed by absorbed stimulus, enables us to demonstrate a proposition of great importance, that, namely, under certain conditions, the sum of the work done, internally and externally, by a given stimulus, is constant. This will be the case where there is little or no dissipation of energy in the course of transformation. In considering the question of the relative proportions of the incident stimulus utilised for in- ternal and external work respectively, we find it clear, from considerations already adduced, that the lower the tonic con- dition the greater will be the proportion of stimulus held latent for the performance of internal work. The nearer is

the tonic condition, on the other hand, to the critical level, the greater will be the excitatory overflow, and the smaller the latent component. The internal and external factors will thus be complementary to each other. pia ‘On subjecting this inference to experimental demon- stration by means of growth-response, I fully succeeded in verifying it. According to this method of growth-response, it will be remembered, the true excitatory effect is measured by retardation of the normal rate of growth, the internal factor of increased latent energy being represented, on the other hand, by a corresponding enhancement of the rate of growth. This being understood, it was found that in a particular specimen of growing tissue, whose tonic condition was somewhat low, the external and internal effects caused by a given stimulus were in the proportion of 32 to 13'5. When the tonic condition of the specimen was raised, how- ever, and the same stimulus was applied, the external effect was found to be enhanced to 38 at the expense of the internal, which was now found to be lowered to 8°5. The sum of the work done, both internally and externally, is seen to be in both these cases approximately the same, being in the former experiment 455, and in the latter 46's. . ~ We have seen that, of the two antagonistic factors of response, the positive will predominate if the excitability of the tissue be in any way diminished. Such a loss of excitability may occur in either of two ways: (1) by the sub-tonicity of the tissue itself; (2) by the depression con- sequent on fatigue. Under either of these conditions then we may expect to obtain the exhibition of the positive effect. The exhibition of the positive effect. under fatigue will be described in the course of the next chapter.

We shall here consider instances in addition to those already given, of the occurrence of the positive effect in a tissue which is sub-tonic. We have to bear in mind that the work which incident stimulus is called upon to perform is two-fold, both internal and external, and that there is a certain critical excitatory level, above which only is the normal responsive expression possible. The actual potential or excitatory level of a tissue depends on its tonic condition and the intensity of the incident stimulus. Now this existing potential of the tissue - may be anything within a wide range, S T, when sub-tonic, N when normal, or H T when hyper-tonic or above the ordinary normal degree (fig. 53). Since wb Roes Ph FeRAt ara it is necessary that the incident stimulus should cause the critical level C to be slightly exceeded, if there is to be an excitatory overflow, we can see that the intensity of the stimulus re- oh. ee. quisite to evoke response will be greater eg Poe in proportion as the tonicity of the tissue itself is low. Thus when the tissue is extremely sub-tonic, a stimulus of or- dinary intensity could never avail to raise the energy of the system above See rad fa se the critical point, and the response Tonic Level must then therefore be positive. Under N,normal;sT,sub-tonic; these circumstances it will only be by HT, hyper-tonic ; and » : ; z c, the critical level | the impact of excessively strong sti- mulus, or by the cumulative action of a series of moderate stimuli, that the critical point can be reached and passed, and the normal negative response evoked.

Thus the intensity of the minimally-effective stimulus in evoking normal response will afford us a measure of the tonicity of the tissue. If the latter be high, then the feeblest stimulus will precipitate outward response, and indeed, if excessive, response will occur on little or no provocation, and such movements we call‘autonomous.’ It must be remem- bered, however, that it was by the previous absorption of stimuli that the tissue was brought to this point of unstable equilibrium at which the added impact of an infinitesimal stimulus causes it to bubble over, as it were, into apparently spontaneous activity.

being thus negative, we must go to the other extreme of great sub-tonicity if we are to be successful in demonstrating the occurrence of the unmixed positive response. This considera- tion leads us to expect that positive response will be evoked on moderate stimulation from tissues that are either not highly tonic or protoplasmically defective. I shall show in Chapter XXII. that in cells of epidermis, where the proto- plasmic contents have been reduced to a minimum, response to moderate stimulus tends in general to be positive, Even highly excitable tissues like nerve, as will be shown later, when cut off from their supply of energy, often become so sub-tonic as to give positive response. I shall here show how ordinary tissues exhibit this effect, when the tonic con- dition is allowed to fall to such an extent as to render the tissue extremely sub-tonic. For this purpose I took a cut specimen of petiole of cauliflower, and kept it without water for a couple of days. By this process the specimen, became somewhat withered. I next proceeded to take records of its electrical responses under increasing stimuli. The intensity of these stimuli rose from I to 10 units. It will be seen from the record (fig. 54) that each stimulus up to 9 evoked positive response, and that it was the strong stimulus of | 10 which gave rise to the normal response of negativity. This constitutes the first instance of a phenomenon which I shall show later to be of very extended occurrence—the induction, namely, of one effect under moderate, and its opposite under very feeble stimulation. It is not so easy to demonstrate this fact with a highly excitable, as with a some- what sub-tonic tissue, where the critical intensity of stimulus for the evoking of normal response need not be impracticably low.

A point to be taken into account here is the after-effect of sub-minimal stimulus in enhancing subsequent normal excitability. Thus it is found in taking the record of responses to a succession of feeble stimuli, that though they are at first abnormal positive, they are afterwards converted into ‘normal negative. That it is the after-effect of the. previous stimulation which thus enhances previous excitability. may also be demonstrated by subjecting the tissue to con- tinuous stimulation or tetanisation, when the abnormal positive is found to pass into normal negative.

From the experiments that have been described, it would appear that the several kinds of response characteristic of various tissues are relatively rather than absolutely different. The true excitatory reaction of an excitable tissue, is one of galvanometric negativity. Any diminution of the ex-— citability—whether by lowering of tonic condition or other Fic. 54. Photographic Record of Abnormal Positive passing into Normal Nega- tive Response in a Withered Specimen of Leaf-stalk of Cauliflower

Stimulus was gradually increased from I to 10, by means of spring-tapper. When the stimulus intensity was 10, the response became reversed into normal negative. (Parts of 8 and 9 are out of the plate.) This record is to be read from right to left. _Down-records stand for positive, and up-curves for negative responses. causes—will bring about a decrease of this negativity, which may culminate in actual positivity. Thus negative is not separated from positive response by any break of continuity ; but we are able, on the contrary, to trace a gradual transition from one to the other. Moreover, in every response we have the two antagonistic elements, positive and negative, either actually or potentially present. The form taken by the resultant response is entirely determined by the question of what proportion of the stimulus impinging upon the tissue becomes latent ; and this in its turn depends upon the tonic

condition of the tissue. When the absorbed stimulus is wholly retained, response is positive, but by this absorption the tonicity of the tissue and its excitability are both raised. When the tonic condition of the tissue, on the other hand, is already high, and its excitability great, a large proportion of the energy finds outward expression, and we obtain the normal negative response. Between these two extremes, we may observe many effects of interference, due to the play of the two antagonistic elements. If, then, the time-relations be not coincident, variations will be induced which will find expression in different types, diphasic response, positive followed by negative, and vzce versa.

The question considered in the course of the present chapter has been that of the energy received and given out by the tissue, and the molecular work, positive and negative, performed during these processes. Such work, however, is itself the result of molecular distortions brought about by stimulus, and the question of the amplitude of response, as related to the degree of distortion, will be discussed in the following chapter. Chemical theory of response—Insufficiency of the theory of assimilation and dis- |

similation—Similar responsive effects seen in inorganic matter—Modifying in- fluence of molecular condition on response— Five molecular stages, A, B, C, D, E —Staircase effect, uniform response, fatigue—No sharp line of demarcation between physical and chemical phenomena—Volta-chemical effect and by- products—Phasic alternation—Alternating fatigue—Rapid fatigue under con- tinuous stimulation—In sub-tonic tissue summated effect of latent components raises tonicity and excitability—Response not always disproportionately greater than stimulus—Instances of stimulus partially held latent : staircase and ad-

ditive effects, multiple response, renewed growth— Bifurcated responsive ex- pression. ACCORDING to current theories, living matter is maintained in a state of equilibrium by the two opposed chemical pro- cesses of assimilation and dissimilation. It is supposed that stimulus causes a down or dissimilatory change, which is again compensated during recovery by the building-up or assimilative change. In the case of uniform responses, again, these two processes are regarded as balancing each other. On this theory, when the down change is the greater of the two, the potential energy of the system falls below par ; for the building-up process cannot then sufficiently repair the chemical depreciation caused by it. Hence occurs dimi- nution of response, or fatigue, which is supposed to be further accentuated by the accumulation of deleterious fatigue-stuffs. The disappearance of fatigue after a period of rest is ex- plained by the renovating action of the blood-supply, which is also regarded as the means of carrying away the fatigue- stuffs.

A serious objection to these explanations lies, however, in the fact, that even excised and bloodless muscles exhibit recovery from fatigue after a period of rest. In isolated vegetable tissues, again, where there is no active circulation of renovating material, the same effect, and its removal after a period of rest, are observed. Thus the difficulties en- countered in explaining fatigue, on purely chemical ‘con- siderations, are great enough; but still greater are those difficulties which arise when we come to deal with the stair- case effect—typically shown in cardiac muscle—in which successive responses to uniform stimuli exhibit a gradual enhancement of amplitude. The results obtained here are in direct opposition to the theory described; for in this particular case we have to assume that the same stimulus which is usually supposed to cause a chemical breakdown, has become efficient to induce. an effect exactly the reverse.

Of the two antagonistic elements in the electrical response, moreover, it is the positive which is supposed to be associated with the assimilative, and the negative with the dissimilative change. If this supposition were correct, however, it would be natural to expect that the positive response would be manifested predominantly in vigorously growing tissues, in which assimilation must be at its greatest. Fatigued tissues on the other hand, in which dissimilatory changes are sup-— posed to be predominant, should manifest negativity as their characteristic response ; moribund tissues, in contrast with the actively growing, might also be expected to exhibit respon- sive negativity. In actual fact, however, the very reverse is the case. For in vigorous tissues, normal response is by galvanometric negativity ; and it is the over-fatigued or - moribund which characteristically exhibit the positive re- sponse.

It would be difficult again to conceive of assimilation and dissimilation in the case of inorganic matter. Yet even in inorganic matter we find reproduced all the various types met with in the response of living tissues: namely, uniform response, the staircase effect, and fatigue. Response being really due to molecular upset from a condition of equilibrium, we can see how different forms of responsive expression will occur, according to the various molecular conditions of the

substance at the time being. One of the most important factors, then, in determining the character of response is the molecular condition of the substance itself. The numerous anomalies hitherto encountered in our interpretation of responsive phenomena are all traceable to our failure to take this factor of molecular condition into account. For a full exposition of the modifying influence which it exercises on response, however, though I shall here state some of the principal conclusions which I have arrived at, the reader is — referred to Chapter XLII.

From the fact, that every type of response is to be obtained from inorganic matter, where chemical assimilation and dissimilation are obviously out of the question, it is clear that the fundamental phenomenon must be dependent on physical or molecular, and not on such hypothetical chemical changes. It must, however, be remembered that though re- sponse phenomena and their modifications are undoubtedly in the first place physical or molecular, yet in the borderland between physics and chemistry there is no sharp line of demarcation. For example, yellow phosphorus becomes converted, under the stimulus of light, into the red, or allotropic, variety. This molecular change, however,cis also attended by a concomitant change in the chemical activity, phosphorus in its allotropic condition being less active than in the yellow. Under certain circumstances, further, it is possible to have a secondary series of chemical events follow- ing upon a condition of unequal molecular strain. A homo- geneous living tissue, when unstimulated, is iso-electric. When stimulated, however, an electro-motive difference is induced, as between the stimulated and unstimulated parts of the tissue. . The result is an electrical current attended by electro-chemical changes. As a consequence of such volta-chemical action, when prolonged, by-products (fatigue stuffs?) may be accumulated, and these may have a de- pressing effect on the activity of the tissue. Hence, just as, after very prolonged activity of a voltaic combination, it is necessary to renew the active element and change the

electrolyte, surcharged with by-products, so after sustained activity of a living tissue, the process of renewal, or renova- tion, will be necessary. It is thus seen how upon the funda- mental molecular derangement, a chain of very various chemical events may follow, as its after-effect. And it is only by going in this way to the very root of the pheno- menon that we can avoid the many contradictions with which we are confronted by the chemical theory.

In studying various response phenomena, our conclusions are necessarily based upon the observation of the amplitude of responses. It is therefore important at this point to draw attention to the danger of hasty inferences. On finding, for instance, that the amplitude of response in a given case is diminished, we are apt to infer that the responding tissue has undergone depreciation. But this is not invariably the case. In the entire process of response, while stimulus induces molecular upset, we must remember that there is also an internal factor, which brings about molecular restitu- tion. Now, if this force of restitution be inany way enhanced, it is easy to see that the responsive distortion of the mole- cules will find itself opposed, with consequent diminution of amplitude. We shall thus often find that a rise of tem- perature, by enhancing the force of recovery, actually causes a diminution of response. That this is not due, however, to any depreciation of the tissue is seen from the fact that the same rise of temperature enhances another excitatory pro- perty of the tissue—namely, the speed of its conduction.

I shall now give a brief account of the modifying influence exercised on response by the molecular condition. It will be shown, in the Chapter (XLII) on the Modification of Response under Cyclic Molecular Variation, that a given response is not determined merely by the nature of the responding substance, but also by the amount of the energy which it possesses. Starting from the lowest condition of sub-tonicity, a substance undergoes progressive molecular transformation by the action of the impinging stimulus itself. Five stages may be roughly distinguished in this transformation. In the

first, or A, stage of extreme sub-tonicity, we have absorption without excitatory response. By this absorption the sub- stance passes into the next, or B, stage, which is the stage of transition, where response is converted from the abnormal to normal. Above this stage the rate of molecular transforma- tion is very rapid. From the residual after-effect of stimulus, the substance now passes from the stage B to the stage 0, which is a condition of more or less stability. Further | stimulation carries the substance to stages D and E. Here the molecular distortion from the normal equilibrium is very great. Stimulation applied in this condition has little further effect in inducing response. That is to say, excit- ability is here reduced to a minimum. In this extremely distorted position, moreover, the substance has a strong tendency to revert to the position of normal equilibrium.

In the A condition of extreme sub-tonicity, since there ‘is absorption without excitation, the response which we obtain is abnormal positive. Intense or long-continued stimulation carries the substance into the B stage, with its normal negative response often preceded by diphasic. An example of this has already been given in fig. 54, obtained from the sub-tonic petiole of cauliflower. We shall meet, however, with numerous other examples in a great variety of tissues. Arriving at the B stage, the substance is still somewhat sub-tonic, and the rate of molecular transformation here is rapid. From the after-effect of stimulus the mole- cules of the somewhat inert substance become incipiently distorted in the same direction as that of normal response. A proportion of the incident stimulus is thus utilised in inducing a favourable molecular disposition. A repetition of the original stimulus will now give rise to a greater excitatory reaction than before. Thus at the B stage we obtain a stair- case increase of response. This fact—that by the after-effect of previous stimulation the molecules may be incipiently dis- torted in a direction favourable to excitatory response—finds ‘illustration in stimuli individually ineffective being made effective by repetition, The result here is evidently made