Comparative Electro-Physiology: A Physico-Physiological Study

arrangement, the galvanometer is kept open, and closed only during ‘or of a second at the required increasing intervals of time. Fe . There may be a pre-existing difference of potential in the plant, as between the two points of galvanometric contact, N, and N, In order that this may not be a source of disturbance, a compensating potentiometer arrangement, C, is employed. The slider of the compensator is so adjusted that it exactly balances the resting difference of potential in the specimen. Under these circumstances, neither make nor break of the galvanometer occasions any deflection. And this balance is readjusted for each experiment of the series.

I give below two tables which. summarise rheotomic observations on specimens of the petiole of cauliflower. By successive intervals of a given length should always be under- stood the mean interval: that is to say, the period between-the application of stimulus and the mid-point in the removal of the short circuit. Thus, for the mean interval of ‘02 of a second, the middle of the .striking-rod 8B, whose breadth is I cm., placed at a distance of 2 cm. from A. The galvano- meter is therefore acted on for ‘oI of a second, throughout ‘the period from ‘o15 to ‘025 of a second, after stimulation. In the first two sets of results here given, the observations will -be seen to have been taken at the somewhat long intervals of ‘1 of asecond. The stimulus applied in these cases was moderate. In the case of a third specimen, the observations on which will be given subsequently, the results were recorded at intervals of ‘o1 of a second.

It will be seen from the observations made on the first of these two specimens that the maximum electro-motive effect was attained in three-tenths of a second after excitation. In the second case, the maximum was reached in two-tenths of a second. The curve given in fig. 39 shows how quickly the electro-motive variation attains a maximum, and how rapid is its decline after reaching this point. There apppears to be practically no latent period, the induction of the electro- motive effect being apparently immediate. This will be made evident by the results given in the next series.

Fic. 39. Curve showing Rise and Fall of Responsive E.M. Change, under moderate stimulation Ordinate represents galvanometric deflection ; abscissa, time. Large division = 1 second. (Petiole of cauliflower. ) For the next experiment, I took the stem of Amaranth, which I find to be more excitable and more quickly reacting - than the petiole of cauliflower. The intensity of stimulus was here greater than in the last case. I may state now what will be demonstrated in full later, that a strong stimulus often gives rise, not to a single, but to multiple responses. I had previously detected these multiple responses by means of both mechanical and galvanometric indications, and found them to have periodicities varying from some I5 seconds to several minutes. . Indeed, had they been much*quicker than they were, they could not have been detected, owing to the

inertia of the motile leaflets or of the galvanometer needle. By the employment of the rheotomic method, however, I was able to detect multiple responses having periodicities of the order of one-tenth or so of a second. All the experiments carried out on Amaranth gave two or three waves of electro- motive variation, the character of which will be understood from the following table and curve (fig. 40). It will here be seen that even after so short an interval as ‘ol of a second, a considerable electromotive change had

Fic. 40. Response Curve from Rheotomic Observation in Stem of Amaranth under strong stimulation Note occurrence of multiple response. Large division of abscissa = *1 second. already been induced. The first maximum occurred after y5, the second after *3, and the third after ‘6 of a second. We thus see that there is a rhythm in these multiple responses, the successive maxima being here found to occur at periods which constitute multiples of ‘15 second. The third of these rhythms may be presumed to be missing, owing to the fact that no observation was taken at ‘45 second, as, at the time when these experiments were carried out, I was unaware of the existence of such rhythmicities. It was by the curves plotted from these data that my attention was first drawn to their occurrence.

It will thus be seen that the electro-motive variation is initiated practically simultaneously with the impact of stimulus on the organ. With moderate stimulus, the maxi- mum variation is reached within two-tenths of a second, or this period may be made still shorter by the employment either of a more quickly reacting tissue, or of a greater _intensity of stimulus. Strong stimulation is apt to give rise Motile responses of opposite signs, characteristic of positive and negative turgidity-variations—Indirect hydrostatic effect of stimulus causes expansion and erection of leaf—Dositive and negative work—Wave of increased hydro- static tension transmitted with relatively greater velocity than wave of true excitation — Method of separating hydro-positive and excitatory effects—In- . direct effect of stimulus, causing positive turgidity-variation’ induces galvano- ‘metric positivity—Antagonistic elements in the electrical response —Separation of hydro-positive from true excitatory effect by means of physiological block.

~HAVING now described that fundamental electrical response of galvanometric negativity which is characteristic of excita- tion, I shall next proceed to deal with an opposite type of response—namely, that of galvanometric positivity. The combination of these two factors, in varying degrees of each, in the electrical response of plants, has been a source in the past of the greatest perplexity, leading investigators to con- tradictory results. And it can only be by disentangling them, and by ascertaining the conditions under which each invari- ably occurs, that precision will be arrived at in the field of electrical response.

We have seen that excitation of the pulvinus of Mimosa induces negative turgidity-variation, with fall of the leaf, and galvanometric negativity. What, then, would be the out- ward expression of an increase of turgidity—that is to say, of the positive turgidity-variation ? With regard, first, to the mechanical expression, we may subject the question to an experimental test. The cut end of a branch of Mimosa, bearing leaves, is fixed watertight in one end of a U-tube, filled with water, and the other

end is connected alternately with a vacuum and a force pump, by means of which a diminution or increase of hydro- static pressure may be induced at will. In this way it is possible to suck water away from, or force it into, the plant and its organs, thus producing negative and positive turgidity- variations at will. When turgidity is thus diminished the indicating leaf is seen to fall. This is what happens also under the ordinary negative turgidity-variation induced by excitation. When turgidity is increased, on the other hand, the leaf is erected (fig. 41). In the case of the negative variation of turgidity, the pulvinus as a whole loses water,

The plant was subjected to diminished pressure up to a, and to normal pressure to 4, after which the pressure was increased. The effect of diminished pressure, in the depression of the leaf, continues for a while. The ordinate represents movement of tip of leaf in cm., abscissa represents time. but more from the lower and more excitable half than from the upper. In the case of the positive turgidity- variation, also, it is again the more excitable lower half which absorbs the greater quantity of water. Thus, in Mzmosa, and in Bzophytum, the mechanical indication of increased turgidity is an erection of the leaf or leaflet. The characteristic electrical indication of this will be observed presently.

It has already been mentioned that the direct application of stimulus at a point causes a negative turgidity-variation of that point. We shall now see whether, under any circumstances, stimulus will induce a positive turgidity- variation. If we now apply moderate stimulus on the stem of J/zmosa, at a certain distance below the pulvinus, an excitatory expulsion of water will occur at the point directly stimulated. Such an expulsion of water will then cause, it is clear, a hydrostatic disturbance of increased pressure. And this hydro-mechanical disturbance will be transmitted with relatively great velocity. Now such an increase of pressure, as we have seen, causes an increase of turgidity at the puivinus, in consequence of which the leaf ought to be erected. And although this hydrostatic disturbance is transmitted very quickly, yet a certain time is con- sumed in the process of forcing water into the pulvinus, by which to bring about the erection of the leaf. After the passage of the hydrostatic wave, there follows Fic. 42. Experimental Arrange- the wave of true excitation, passing sea arn ares from cell to cell, and inducing the given to Direct and In- Characteristic reaction, of negative eee eee by Leaf of tyroidity - variation. And when Thermal stimulator at s produces this excitatory wave reaches the trey smlstion my gene pulvinus, the previous erectile stimulation, at a distant point, Movement should give place ~ to Sys eterna to indirect effect excitatory depression, or fall of the leaf. In fig. 42 are seen the

arrangements for an ptoknehiueiit on Mimosa by which these inductions may be verified. Moderate thermal stimulus is applied at S, at a certain distance below the indicating leaf. This latter is attached by a thread to a writing-lever, which traces the response-record on a smoked revolving drum. When the stimulus is applied at a point S very near the pulvinus, the response takes place by a negative turgidity- variation, with a concomitant fall of the leaf, seen in fig. 43 (a) as an up-curve. When a moderate stimulus is applied

at a greater distance S,,, the hydrostatic wave causing the positive turgidity-variation brings about an erectile twitch. This is followed by a responsive fall, when the true excitation reaches the organ (fig. 43, 0). It has thus been shown that by separating the responding point from the point stimulated, or the receptive point, it is possible to discriminate two different effects which are both brought about by stimulus. It is most important, moreover, to distinguish between these two factors : namely, the direct effect of stimulus causing contraction, and its indirect effect, causing expansion. We have seen that direct excitation and transmitted excitation both induce contrac- tion, negative turgidity-variation and fall of the leaf. Unfor- tunately, in animal physiology, Fic. 43. Mechanical Responses

it has been customary to apply Reet eaaesmesa eee (a) record of responsive fall when the term zuzdirect to that form stimulus applied near the re- of stimulation which is applied sponding organ; (4) response . : * when stimulus is applied on at a distance. And it has not same side, but at greater dis- . . tance, s,,. A preliminary erectile been noticed that such stimulus seuliocie ins bere: fallowed.” bey is capable of inducing diametri- the true excitatory depression. : ' This is due to the indirect effect cally opposite results, according first transmitted being succeeded as the true excitatory effect by the direct. Had the stimulus applied been feebler, or more reaches, or does not reach, the distant, there would have been responding organ. When the ons first, or indirect erectile

intervening tissue is highly conducting, the transmitted effect induces exactly the same result as if stimulus were applied directly. But we shall see that when the intervening tissue is non-conducting or feebly conducting, true excitation is not transmitted, and the effect which makes its appearance at the responding point is then due to increase of hydrostatic tension, causing positive turgidity-variation, with the concomitant effect of expansion, and, in the case of J/zmosa, of erection of the leaf. This latter effect of positive turgidity-variation and expansion, I shall therefore distinguish as the INDIRECT EFFECT of stimulus, in contradistinction to the term INDIRECT STIMU- LATION, as it is generally used. The last-named, however, I shall myself always refer to under the title of TRANS- MITTED STIMULATION. If the intervening tissue be of

Fic. 44. Mechanical Response of Biophy/um to Thermal Stimulation Stimulus was applied at some distance from the responding leaflet. And the preliminary erectile twitch is due to the prior arrival of the hydrostatic disturbance. Thick dot represents moment of application of stimulus. moderate conducting power, we shall, as in the case of the experiments on J/zmosa, obtain a preliminary erectile twitch, due to the indirect effect of stimulus, followed by a fall, in consequence of the transmission of the true excitatory effect. In fig. 44 is seen this twofold expression of the indirect and transmitted effects of stimulus, given by the leaflet of Biophytum.

and of true excitation, induce, as we have now seen, opposite responsive reactions. But of these, that due to true excita- tion is, generally. speaking, greatly predominant. Hence, when these two waves reach the responding organ in close succession, as is the case when the point of stimulation is very near, the excitatory effect masks the hydrostatic. In order, then; to ‘separate them, we may employ various methods. First, in the case of highly conducting tissues, the stimulus must be applied at a sufficient distance to make the slow excitatory wave lag adequately behind the quickly travelling hydrostatic wave. Or we may choose a direction of transmission of excitation which will be relatively slow. Thus I have found that transmission across a stem, for example, is very much slower than along its length. Hence, on applying moderate stimulus at S, (fig. 42) ata point on the stem diametrically opposite the pulvinus, of the given leaf, it is found that the excitation reaches the pulvinus only after a measurable interval, the hydrostatic effect inducing erectile response much earlier. Thus in a given experiment, whose record was taken on a fast-moving drum (fig. 45), the erectile response took place ‘6 second after the application of stimu- lus, whereas the true excitatory fall did not occur till 3:45 seconds had elapsed—that is to say, 2°85 seconds later. It is to be borne in mind that’ a certain interval of time passes, even after the arrival of the respective waves, before the tur- gidity-variation is able to give rise to the motile indication. Let us next examine the results at the responding tissue of the indirect effect of stimulus. The distant receptive point contracts on stimulation, and sends to the responding organ a wave of increased hydrostatic tension. This, as we have seen, forces water in, and expands the tissue..

Work is thus done ox the tissue which increases its store of energy. In this the indirect is unlike the typical direct effect of stimulus. For the latter causes the impulsive fall of the leaf, which represents work done dy the tissue, and an expenditure of energy. We must, therefore, recognise two distinct respon- sive effects, according as the work done is fosétive—done on the tissue—or negative: that is to say, done by the tissue. The outward manifestations of these two processes are respectively expansion and contraction. The positive, as we shall see, is not the result of hydrostatic disturbance as such, but is the effect of energy transmitted hydraulically.

The indirect effect of stimulus, then, gives rise to positive turgidity-variation, and increases the internal or -potential Fic. 45. Record of Response of A/zmosa Leaf, taken on a fast- moving drum Stimulus applied at moment @, on point of stem diametrically opposite to responding leaf. Hydro-positive erectile response occurs at 4, *6 second after application of stimulus. True excitatory response of fall takes place at c, 3°45 seconds after application of stimulus. Time-marks represent fifths of a second.

energy of the organ. A positive turgidity-variation is thus concomitant with an increase of internal energy and a negative turgidity-variation with the reverse. In observing the mechanical response, we saw that the expression of positive turgidity-variation, due to the indirect effect of stimulus, consisted of an erection, and was therefore of opposite sign to that of the negative turgidity-variation, caused by true excitation, and expressing itself in a fall, of

the leaf. We shall now see whether a similar difference exists between the electrical expressions of the positive and negative turgidity-variations. In carrying out this experiment, I took a specimen of Biophytum and applied stimulus at a distance from the par- ticular leaflet whose responses were to be observed, arranging, at the same time, for a simultaneous record of the mechanical and electrical responses. It will be seen from fig. 46 that the preliminary erectile twitch, due to the positive turgidity- variation, has, as its concomitant, galvanometric positivity. And this is followed in both records by its opposite: namely, the contractile fall and the galvanometric negativity of true excitation.

It will thus be seen that the increase of internal energy, with its positive turgidity-variation, has, as its electrical expression, galvanometric positivity. Besides this, the mere physical movement of water in the tissue gives rise to a certain electrical varia- tion of positivity, and this can still be detected, even after the tissue is killed. The question of how to discriminate what proportion of the electro- positivity was due to this mere water - movement, and what to the increase of turgidity, associated with the. increase of in-

Fic. 46. The Abnormal Positive preceding the Normal Negative in Mechanical and ternal energy, I at first Electrical Responses in Biophytuni found it very difficult to * represents the moment of application of ; , stimulus. The upper is the mechanical decide. But I ultimately and the lower the’ electrical record. succeeded in doing this of the leaf or galvanometric positivity. by bringing a plant to a condition just short of death, and thus abolishing its true excitatory reaction. In this condition, the responsive

indication was found to be one of considerable electro- positivity. On finally killing the-plant, however, the positive change due to water-movement was found to represent so insignificant a proportion of the whole as to be negligible, In order to exhibit the electrical expression of the excitatory and hydro-positive effects of stimulation, in ordinary plants, I took a petiole of cauliflower and made one connection, the proximal, with a point on it, and the other with an indifferent point on the surface of the lamina. In order to obtain the unmistakable hydro- static effect, the petiole was sud- denly squeezed, at a distance of 6.cm. from the proximal contact, and this gave rise, as will be seen (fig. 47, a), to a positive response, represented downwards. This was repeated once more, and the same effect observed. I next applied thermal stimulus at a dis- tance of 4 cm. from the respond- ing point. In this case hydro- static and excitatory disturbances

PIG, ty vical Reponse of Peticle Static shortly followed by the a, hydro-positive ; 2, di-phasic; experiment on Mimosa. This phasic response, the hydrostatic positive being followed by the excitatory negative (fig. 47, 0). Reference has already been made to the observation of Burdon Sanderson, that in the lamina of the Dzonea leaf the immediate response was one of galvanometric positivity. Mis- taking this for the true excitatory effect, he concluded that the response of the plant was of opposite sign to that of the

animal. From the experiment just described, however, it will be seen that the effect observed by him was in reality due, not to true excitation, but to the hydrostatic disturbance, or indirect effect of stimulus. In the next record (fig. 47, ¢) we see the effect of stimulus applied nearer: that is to say, at a distance of 2 cm. from the proximal contact. Owing to the propinquity of the point of stimulation, the two disturbances are not now sufficiently separated, and the excitatory negative reaction completely masks the hydrostatic positive effect.

It is thus seen that, as has been said, one method of exhibiting these two effects separately is to apply stimulus at a point so distant from the proximal contact that there is an interval between the arrival of the two waves of hydro- static and excitatory disturbance respectively. It is obvious, then, that if the tissue under experiment be a good con- ductor of excitation, we must place the point of stimulation at a long distance from the first electrode, in order that the effect of excitation may lag sufficiently behind the hydrostatic wave. Similarly, in a bad conductor of excita-— tion, it will be the indirect effect alone which will reach the proximal contact, unless the stimulus applied be very near, and very strong.

In order to distinguish these two opposite effects from each other, I shall in future refer to that hydrostatic effect which causes expansion and galvanometric positivity as ‘the hydro-positive effect, by way of differentiating it from ‘the true excitatory. effect, of negative turgidity- variation and galvanometric negativity. It has already been said that tissues which exhibit a high degree of. conduction are characterised by more or less of protoplasmic continuity. Hence, fibro-vascular elements are relatively good, and parenchymatous. tissues bad, conductors of excitation. The cells of the potato tuber for this reason exhibit very little power of transmitting excitation. When, therefore, in experimenting with this

tissue, stimulation was caused by application of a hot wire so near as I cm. to the proximal contact, it was the hydro- positive effect alone which reached it, giving rise to posi- tive response. It was only, indeed, by applying the stimulus very near, at a distance of 3 mm., that the true excitatory From what has been said, it will be seen that when a given point is excited by transmitted stimulation, two antagonistic elec- trical effects are induced—one of positivity, due to hydro-positive action, and the other of negativity, due to true excitation. When the stimulator is near to, or co- incides with, the responding point, the tissue is subjected to rapidly succeeding positive and negative turgidity-variations, and the elec- trical indication of the latter being the more intense, it masks the