Comparative Electro-Physiology: A Physico-Physiological Study

with gravity. Thus, a shoot laid horizontally will curve up-. wards till the free end becomes vertical. In connection with this subject there are two different points to be elucidated. First, is the question as to the mode in which gravity exer- cises stimulation ; and second, that of the method by which, in answer to this stimulus, a definite responsive curvature takes place. As regards the first of these, it may be said that the only conceivable way in which gravity could produce stimulation is by some differential effect of weight acting on the responding cells. According to this, the necessary dif- ferential weight-effect may be due to the weight of cell-

contents, whether of the sap itself or of those heavy particles like starch-grains, which are contained in it. The former, or Theory of Hydrostatic Pressure, was suggested by Pfeffer and supported by Czapek ; the other, or Theory of Stato- liths, has been advocated by Noll, Haberlandt, and Nemec. In the case of a multicellular plant laid horizontally (fig. 264), E and E’ may be regarded as areas in which stimu- lation is caused by the weight of the particles. It is obvious that the effects produced on the E Pe ee a eee upper and lower sides of the shoot are antago- nistic;. yet, in spite of this, we obtaina resultant &'

curvature upwards. This P a - shows that the excitation Fic. 264. Diagrammatic Representation of of one side must be a Multicellular Organ laid Horizontally and Exposed to Geotropic Stimulus. greater than that of the On the upper side the statoliths act on the other, the particular direc- inner, and on the lower side on the tioneditapedeaieal a eee, age poe wall (after Francis being due to this fact. It is clear, then, that the induced curvature upwards of the horizontally-laid shoot is due to the effective action of the weight particles on either the upper or the lower side of the shoot. We can see that if it is the upper which is the more effective, then curvature must take place by excitatory con- traction ; if, on the contrary, the lower be the more effective, the curvature is then to be regarded as the result of respon- sive expansion. There has been considerable uncertainty as to which of these is actually the case, the prevailing view being that it is the expansion of the lower surface which is the active factor.'

That it is, however, the excitatory contraction of the upper side which is the active factor in this curvature I have already demonstrated by alternate unilateral applications of 1 For more detailed account see Plant Response, pp. 495 to 511. cold. It is known that the continuous application of cold diminishes or abolishes the excitability of a tissue. If, then, it is by excitatory action on the upper side that curvature is induced, it will be found that the local application of cold on that side will retard or arrest this responsive curvature, the application of cold on the lower side producing practically no effect. If, on the contrary, this curvature should be mainly due to some excitatory action on the lower side, we may then expect to find that the application of cold on that side has

FIG. 265. Effect'on Apogeotropic Movement of Temporary Application of Cold on Upper and Lower Surfaces respectively Application above is seen to produce arrest of movement, while application below has no perceptible effect. Ordinate of curve represents up-move- ment of tip of organ in mm ; abscissa represents time. the effect of arresting the growing curvature, its application on the upper being more or less ineffective. On carrying out these experiments it was found that cold had the effect of arresting curvature only when it was applied on the upper side of the shoot (fig. 265). This conclusively proved that gravitational stimulus, acting on the horizontally-laid shoot, induced response by excitatory contraction of the upper side. This fact, of response by contraction, is fully concordant with what we know of the effect on the plant of other forms of stimulation. This question I intend, however, in the course

of the present chapter, to submit to independent examina- tion by means of electrical response. __ I have already shown that . F the unilateral pressure of par- ticles on the growing organ is effective in inducing curvature set | in such a way that the side acted upon becomes concave. This experiment was carried out by subjecting one side of a growing organ to the pres- sure of iron particles, which were pressed against it by the pig 266. action of an electro-magnet on sentation of Experiment showing ‘ ‘ Curvature Induced by Unilateral the opposite side (fig. 266). Pressure Exerted by Particles

The magnetic particles in this. F, flower-bud of Crinum; s, india- f ti d ; | rubber strip studded with iron case tunctione as virtua particles attracted by electro- statoliths, and a curvature was magnet, M, causing palate : . ° pressure on growing region ; 1, induced by the excited side iodux attached to Hower There is, however, one difficulty in connection with the statolithic theory of stimulation. When the stem is held erect the particles rest on the bases of the cells, and their general distribution on the two sides of the organ is symmetrical. Asymmetry of distri- bution is induced, how- ever, when the shoot is laid . horizontally, and stimulation might beex- Fic. 267. Record of Responsive Curvature

-.4.-- -Induced in Bud of Crinum Lily by Uni- pected to follow within lateral Pressure of Particles a very short time, since the. displacement of particles from base to side cannot take long. This being so, the geotropic curvature of the shoot upwards should take place within a short time. But, instead of this, we find that the curvature is at first downwards; and | it is not till after the lapse of a period, which is sometimes as much as an hour in duration, that there is a reversal of this downward movement into the normal apo-geotropic move- ment. Fig. 268 gives a curve which exhibits this prelimi- nary movement persisting for nearly forty minutes before its ultimate reversal into the usual normal apogeotropic curva- ture upwards.

This delay in the appearance of the characteristic response may, however, be explained from the known fact that steady tension increases, whereas compression retards, the rate of growth. Thus, in a horizontally-laid shoot, there is a ten- dency to curve down by its own weight, the upper side being Fic, 268.. Record of Apogeotropic Response in Scape of Uriclis Lily’ The up-curvature due to apogeotropic action proper commenced forty minutes after the specimen was laid horizontally.

in this way subjected to tension, and the lower to compression. The effect of these is an increased rate of growth on the upper, associated with a decreased rate on the lower, sides of the specimen, giving rise to a downward curvature. The fact that geotropic stimulus has to overcome this action before its own characteristic effect can be exhibited may account for the observed delay in its appearance. A crucial experiment in support of this explanation will be given presently.

We have seen in the last chapter that the presence of internal excitation is capable of detection by electrical indi- cations, and by taking advantage of this fact we have an independent means of coping with the obscurities of the present problem. I have demonstrated, by means of experi- ments already referred to, that it is the upper side which, under the action of gravity, undergoes excitatory contraction. It follows that, as an indirect effect of such contraction, the water expelled from the upper will reach the lower side of the shoot, and cause an increased turgidity and expansion of that side. The apogeotropic curvature is thus brought about directly by the contraction of the upper, and indirectly by the expansion of the lower, side of the horizontally-laid shoot. This being so, we may expect, in accordance with our pre- vious investigations on the true excitatory and hydrostatic effects respectively, that that side will become galvano- metrically negative which is excited by geotropic stimulus. And it is found that it is in fact the upper side of the hori- zontally-laid shoot which exhibits galvanometric negativity ; the lower, in which the positive turgidity-effect had been indirectly induced, being found to show galvanometric posi- tivity. 3 But this result is only obtained simultaneously with the induction of the normal apogeotropic curvature. And, before this, other disturbing effects may occur. We take an erect stem and make two galvanometric contacts, one on each side of its growing region. The stem is next laid horizontally. As regards the electrical effects which are now exhibited three distinct phases are to be distinguished. First, in con- sequence of the mechanical disturbance due to the laying of the organ on one side, there will be an excitatory electrical effect on both upper and lower sides, and the resultant re- sponsive current will be determined by the difference of excitability which may happen to exist, in this particular individual, between the two. This variable stage is termi- nated in the course of about ten minutes. We next have a steady condition, brought about by the effects of tension and compression already described as acting on the upper and lower sides respectively.

This gives rise, as we have seen, to increased growth, with its attendant positive turgidity, on the upper as compared with the lower side. | Hence, during this stage, when the curvature is proceed- ing downwards, we may expect that the upper side will be galvanometrically positive in relation to the lower, the direction of the current in the horizontal shoot being from below to above. And finally, in the third stage, we have the geotropic stimulation effectively overpowering and reversing this downward movement. And since it is the upper side that is now geotropically excited to an effective extent, we find that that side becomes galvanometrically negative. Thus, the electrical indication, like the mechanical, gives in this ultimate stage the characteristic response of the

Fic. 269. Photographic Record of Geo-electric Response in the Scape of Uriclis Lily laid horizontally In the first phase of response, the current is from the upper surface to the lower, the upper being galvanometrically positive. After fifty minutes, the excitatory geotropic effect reverses the current, which is now ascending, or from below to above, the excited upper-surface being galvanometrically negative. _ (Compare corresponding mechanical record, fig. 268.)

plant-tissue to gravitational stimulus. The excitatory effect is now exhibited mechanically by the contraction and concavity, and electrically by the galvanometric negativity of the upper side of the horizontally laid shoot (fig. 269). These similarities between the mechanical and electrical records will be seen on comparing this figure with fig. 268. We have thus seen that, owing to secondary mechanical disturbances, the proper exhibition of the true geotropic response is delayed. And from this it is difficult to say how quickly the geotropic response follows the dis-

placement of the hypothetical statolithic particles. I have been able to overcome this difficulty, which at first appears very great, in the following way. It has been shown that the state of excitation, even when all mechanical expression of it is restrained, may be detected by galvanometric nega- tivity. Those secondary effects, due to mechanical dis- turbance, which mask for a time the excitatory effect of gravitational stimulus, may thus be eliminated completely by restraining all movement of the shoot. The problem thus resolves itself into the fixing of an experimental shoot, say the peduncle of Uviclis lily—in such a way that mechanical

Fic, 270, Experimental Arrangement for Subjecting Organ to Geotropic Stimulus, Mechanical Response being Restrained response is completely restrained. The next point is to subject the specimen, at a given moment, to the stimulus of gravity, and record the consequent electric response. I shall now describe the experimental method by which these conditions were successfully met. It is clear that when any two points are acted on symmetrically by the force of gravity, there is no resultant geotropic action. This is the case in regard to two diametrically opposite points, A and B, situated laterally on an erect shoot. When the shoot is laid horizontally, two lateral points are again acted on sym- metrically by. the force of gravity, and there is thus xo differ- ential action as. between the two. But if the shoot be now rotated on itself,.so that one of these points is diametrically

above, and the other below, a differential action will be induced as between the upper and lower sides, the upper being relatively the more excited. In the following ex- periment I took a specimen of Uvzclzs lily, and fixed the entire plant horizontally, as seen in the figure (fig. 270). The pivoted support allowed the responsive points A and B to be at first lateral. Owing to symmetry there was now no Fic. 271. Geo-electric Response of the Physically Restrained Scape of Uriclis Lily

Up-curve represents responsive current from upper to lower surface during action of geotropic stimulus, Down-curve represents recovery on cessation of stimulus. Response commenced after latent period of one minute ; after-effect persisted for two minutes. Breaks in curve are due to obscuration of recording spot of light at brief intervals. . differential action of gravity, nor consequent electrical varia- tion, as between the two. The galvanometric record was now a horizontal line. The specimen, on its support, was next quickly rotated through 90°. The statolithic particles were thus displaced, falling on the inner tangential wall of the upper side, and outer tangential wall of the lower. An electrical response was perceived in about one minute, which went on augmenting with time, the upper side being in- creasingly galvanometrically negative (fig. 271). The fact

already demonstrated by the alternate cooling of the upper and lower surfaces is again seen here: namely, that it is the upper surface which exhibits the true excitatory effect, by induced concavity in the case of mechanical and by galvano- metric negativity in that of electrical response. It will also be seen that when secondary disturbing causes are removed, the response to gravity is immediate, showing that there is no anomalous delay between the displacement of the hypo- thetical particles and the consequent response. By now rotating the specimen back through 90°, the action of gravity is virtually removed. The after-effect persists for two minutes, and after this the response curve shows the usual recovery,

Transmission of excitation in plants not due to hydromechanical disturbance, but instance of transmission of protoplasmic changes—Difficulties in accurate determination of velocity of transmission—A perfect method—Diminution of conductivity by fatigue—Increased velocity of transmission with increasing stimulus—Effect of cold in diminishing conductivity—Effect of rise of tem- - perature in enhancing conductivity—Excitatory concomitant of mechanical and electrical response—Electrical methods of determining velocity of trans- mission—Method of comparison of longitudinal and transverse conductivities —Tables of comparative velocities in animal and plant— Existence of two distinct nervous impulses, positive and negative.

WHEN a point in the tissue is stimulated the state of excita- tion is often found to be transmitted to a distance. This is well seen in the case of sensitive plants, where the excitation applied at one point is found to give rise to motile responses of the distant leaf or leaflets. In this transmission of excitatory impulses to a distance in the plant we have a phenomenon which would seem to be analogous to nervous transmission in the animal. For certain reasons to be given presently, however, it has usually been supposed that there is actually nothing in common between the two. ‘The nervous system belongs exclusively to the animal organisation, and, indeed, to the more highly de- veloped Metazoa only. Plants, unicellular animals, and the lower Metazoa have no nerves, and if in exceptional cases (as in the excitatory movements of many plants) there are forms of activity which resemble the vital manifestations of the animal organisation, as effected by nerves, it is easy to prove that the resemblance is merely superficial.’ *

This assumption, that there could be nothing in common between the transmission of excitatory impulses in plant and animal, was thought to derive support from Pfeffer’s experi- ment on the effect of anesthetics on conduction in J/zmosa. The anzsthetisation of the pulvinus is found to abolish its motile excitability. The effect of strong stimulus was never- theless found by Pfeffer to be transmitted across the anzsthe- tised area, giving rise to the depression of leaves beyond. It was natural to infer from this that, as the motile excitability of the pulvinus was abolished by the anesthetic, so must the protoplasmic conductivity also have been abolished. It was therefore inferred that—unlike the conduction of stimulus in animal tissues, where such transmission is known to take place by the propagation of protoplasmic changes—the ap- parent conduction of excitation in a plant was purely hydro- mechanical.

But I have shown elsewhere, and shal]! demonstrate again in Chapter XX XIII. by different means, that though excita- bility and conductivity are related phenomena, yet the varia- tion of the one is not necessarily identical with that of the other. Thus a certain degree of anzsthetisation may be sufficient to induce arrest of motile excitability, and yet may not always abolish conductivity.! Another objection which has been urged against the theory of the transmission of protoplasmic changes through the plant is based on certain experiments of Haberlandt. In these the excitation in J/zmosa is said to have been propa- gated over dead tracts of the petiole, these portions having been killed by scalding. But it is extremely difficult to ensure the death of interior tissues by such means as super- ficial scalding. I have found that a portion of a plant tissue which had been subjected locally to the action of boiling water afterwards exhibited signs of true excitatory electrical response. It is only by prolonged immersion in boiling water that the electrical response is. totally abolished. Only after such treatment, therefore, can one be quite sure that the

interior tissue is really killed by scalding, and unless this is thoroughly done it is easy to see that the inner cells may continue to conduct excitation. There is, moreover, another possibility, that of pseudo- conduction, by which the effect of stimulus might appear to be transmitted across dead areas. In Haberlandt’s experi- ment, even if the intervening tissues had been killed, there would still be two masses of tissue, separated from each other | by an intervening area of dead tissue. A strong stimulus applied to either of these might then cause an excitatory expulsion of water, capable, when transmitted across the dead. area, of imparting a mechanical blow to the second living tissue, sufficient to set up excitation de novo in that portion of the petiole. ,

I shall, however, be able to show that conduction is brought about in the plant by the same transmission of excitatory protoplasmic changes which occurs in the animal ; and that those agencies—such as cold, anesthetics, fatigue, and the polar effects of currents—which induce its variation in the one case, have the same identical effect in the other also. The electrical responses, again, afford us a crucial ‘method of distinguishing between hydrostatic and excitatory effects. For, had the transmission of excitation taken place in plants by means solely of the propagation of hydrostatic disturbance, its electrical sign would then have been one of galvanometric positivity alone. But we have found, on the contrary, that the sign of the true excitatory reaction is always of galvanometric negativity. The distinct characters of these true excitatory and hydrostatic waves have already been demonstrated in various experiments, in some of which the hydrostatic has been seen as a positive twitch preceding the true excitatory negative, while in others the transmission of the negative was abolished by the application of a selective block, such as chloroform, thus bringing about the exhibition of the positive alone (p. 66). So true indeed is it that excitatory changes are propagated in the plant as in the animal, that I have actually been able to isolate certain

tissues whose responsive peculiarities are indistinguishable from those of animal nerves. These must therefore be regarded as vegetable nerves (Chapter XX XII.). The determination of the velocity of transmission of excitation in sensitive plants may be made by applying a stimulus at any point and observing the interval which elapses before motile effects are visible at a given distance. It is, however, impossible by such means to obtain accurate and consistent results until certain factors of variation are successfully eliminated. These are (1) indefinite changes of excitability owing to in- jury caused by stimulus at the point of application ; (2) changes of conductivity caused by fatigue; and (3) the unknown effects of varying intensities of stimulus on velocity of transmission.

As a result of investi- gations on this subject I found that the velocity of Fic. 272. Diagrammatic Representation of Electrical Connections for Deter- transmission can only be mination of Velocities of Centrifugal ‘ and Centripetal Transmissions regarded as a determinate Fe : ‘ X A and B are exciting’ electrodes, and L the quantity when the intensity indicating leaflet. value. Excessive stimulation, again, is found, by causing injury, to modify the excitability and conductivity of the tissue. These difficulties, however, are overcome by the employment of a stimulus which does not cause injury, and which is capable of repetition at uniform intensity. One such form of stimulation is obtained by the use of discharge from a condenser previously charged to a known,voltage (fig. 272). As regards those changes of conductivity which are due to fatigue, I have found that fatigue is removed, and conductivity fully restored, after the lapse of a definite period of rest, varying in duration in different plants from four to

ten minutes. In the case of Azophytum, for instance, the required interval was found to. be about five minutes. | Taking a specimen of Szophytum, and employing the method of determination which has been described, I found successive values which were very consistent ; and, having thus secured conditions which made it possible to obtain exact results, I proceeded next to investigate the effects of Before proceeding to describe these results in detail, how- ever, it should be mentioned that, though the velocity of transmission of excitation is constant in the same plant under uniform conditions, yet this is not necessarily the case,

if the direction of conduction be reversed. In the petiole of Biophytum, for example, the centrifugal velocity is always higher than the centripetal, being about fifty per cent. _ greater. . In order to study the effect on velocity of progressive fatigue, we may gradually shorten the interval of rest. The velocity of transmission in the petiole of Bzophytum when fresh was found to be 1°88 mm. per second in the centripetal direction. Before making a second experiment on the same specimen, an intervening period of rest of three minutes was allowed. This was found to reduce the velocity slightly, it being now 1°86 mm. per second. The following table shows the results obtained by a series of five experiments on the

same specimen. The distance through which the transmis- sion was observed was 27 mm. TABLE SHOWING VARIATIONS OF VELOCITY OF TRANSMISSION AND. OF It will thus be seen that the fatigue due to having only half a minute’s rest reduced the normal velocity of the specimen by 18 per cent. The effect of intensity of stimulus on velocity of trans- mission was next studied. The stimulus employed was that of condenser discharge, increased intensity being obtained by an increasing voltage of charge. In this way it was found that velocity increased with increasing intensity of stimulus. This fact is shown in the following table, which gives the results of an experiment on a petiole of Liophytum.