Comparative Electro-Physiology: A Physico-Physiological Study

arranged for three different methods of record. The first was without balance. Here the slope of the suction-curve | indicates the normal rate of suction, the enhanced rate under stimulus being indicated by flexure and increased steepness. Mode of Applica- Mode of Application tion of Stimulus of Stimulus A diminution in the rate of suction, or an expulsive effect, would, on the other hand, be indicated by a corresponding diminution of the slope, or reversal of the curve. The second method of record is carried out under exact

balance, and is, as already explained, an extremely delicate means of detecting variations in the rate of suction. If this rate be enhanced the curve rises suddenly from the balanced horizontal line, a depression inducing, on the contrary, a downward movement. The last method is that of Over- pensator at a uniform rate in larger quantity than necessary, a movement of the index is induced in a direction opposite to that of suction. Here the slope of the curve, due to over- balance, is down, and should the stimulus cause any enhance- ment of suction, the steep down-curve must be replaced by a less steep, or horizontal, or up-curve. The Method of Balance is, as I have said, the most delicate, but this Method of Over- balance enables us to detect the after-effect of stimulus in a striking manner.

As regards the form of this response by movement of water, reference has already been made to previous results, in which we saw that the excitatory effect in the plant, as studied by electrical response, was of two different types, according to certain phasic conditions. Highly excitable roots, we found, gave response by galvanometric negativity, indicating secretion or expulsion of water. Less excitable roots, on the other hand, gave response by positive variation, most probably indicating responsive absorption. These two opposite effects actually occur, as I find, under. different phasic conditions, in the suctional responses which I am about to describe. :

I shall deal first with the results which I invariably obtained in carrying out experiments on Crofom and certain other plants during the month of February. The Indian winter was just over, and the spring had not yet fully set in. The nights were still cold, though the days were growing warmer. The plants, therefore, owing to these peculiar climatic conditions, must be regarded as having been some- what sub-tonic. My first experiment related to the initiation of suctional activity in a specimen which, in this respect, had been previously at standstill. Stimulus might here be expected to renew that multiple activity on which suctional response, according to our theory, depends. We may here refer once more to the initiation of multiple activity by stimulus in a leaflet of Desmodium previously at standstill.

I have shown that when this plant is deprived of its store of latent energy by unfavourable conditions, then the multiple movement ofits leaflets is arrested. If the plant, for instance, be kept for some time in a dark room, the leaf- lets cease to pulsate. But if now an electrical shock of moderate intensity be given to the pulvinus, the incident stimulus, by its excitatory action, gives rise to a number of responsive movements, which again come to a stop as soon as the imparted energy is exhausted. Or we may, in such a case, employ the stimulus of light. A record of the subse- quent effect has already been given on a. previous page (cf. fig. 141).

It will there be noticed He that the quiescent leaflet is thrown into pulsatory movements after the lapse of a short latent period ; (2) that the increasing absorption of stimulus has the effect of augmenting the amplitude of response ; and lastly (3) that, owing to the presence of latent energy derived from the impinging stimulus, the activity of the leaflet con- tinues for some time, even on the cessation of stimulus itself. It is, in. fact, by that enhancement of the tonic condition, which comes about by the continuous absorption of energy from the’ environment, that the apparently autonomous response of the leaflet is maintained. There is, as has been said ‘before, no essential difference between multiple and

autonomously, comes to a state of standstill when its store of latent energy falls below par. Conversely, by the acces- sion of energy from external stimulus, activity is resumed, multiple passing into autonomous response. Returning, then, to the hydraulic mode of response, as observed in variations of suction, we might expect that the irapact of stimulus would initiate this in a tissue at suctional standstill. For this experiment I took a specimen of Croton and mounted it, with terminal electrodes, in the Shoshun- graph, At this time it showed a moderate rate of suction. It was then kept undisturbed in a dark room for forty-eight hours, at the end of which time, owing to the run-down of its latent energy, the suctional activity was found to be arrested. But on the application of electrical stimulation, the suctional

activity was again renewed, after a latent period of two minutes, and found to persist for a considerable time, even on the cessation of stimulation (fig. 226). We have: here, theny an exact parallel to the renewal of the so-called autonomous response of Desmodium leaflet referred to above, ee We have thus studied the phenomenon of the variation of suction by renewal when found at zero. We shall next con- Fic. 226. Renewal of Suction, Pre- viously at. Standstill, by Action of Stimulus This record was. taken

under balanced con- ditions. Vertical line represents moment of The half-shaded portion of figure repre- sents time of application. of stimulus. Suctional response is seen to be initiated after a latent.period of one minute, and to persist after the cessa- tion of stimulus, sider the case of a variation induced in the existing rate by the action of stimulus. The normal rate is exactly balanced, a condition which is represented in the photographic record by the straight line which results from the stationary position of the mercury index. Stimulus of 5 seconds’ duration was now applied, and the responsive acceleration is seen as a steep rise in the record (fig. 227). This responsive acceleration persists

' Ifa cut branch of any plant be kept in water for several days, its suctional activity, as is well known, disappears. This is commonly attributed to the blocking of the cut end by mucilage and bacterial growths, since the making of a fresh section is found to renew the activity. This making of a fresh section, during a period that depends on the intensity of stimulus and the condition of the tissue, after which it declines slowly. After recovery, however, the rate is generally somewhat higher than at first. This is due to the persistent after- effect of stimulus absorbed, If the enhanced suctional rate be now again balanced, and stimulus. applied once more, there will be a still further enhancement of the rate. In this way, owing to the succes- sive increase of latent energy, the suctional activity is enhanced till it reaches a limit, after which there is but little additional effect to be induced by stimulus. There is another and _ in- teresting effect which is often observed, in consequence of

The record was taken under balanced conditions. Half-shaded portion represents application of stimulus for 30 seconds. Lower record shows latent period to be 45 seconds. After _re-balance, stimulus of 30 seconds was once more applied. -Upper record now shows reduction of latent energy absorbed from previous stimulation. This is the diminu- tion of the latent period, after which response takes place. This will be seen in the following | record (fig. 228). The initial rate of suction was in this case balanced, as usual, and stimulus of thirty seconds’ duration was applied. The responsive acceleration is seen to take place forty-five seconds after the cessation of stimulus. The enhanced rate was balanced, and a stimulus of thirty seconds’ duration

however, does not decide the question; for in making it, is involved the strong mechanical stimulus of a cut. The outgrowths may, no doubt, obstruct the passage of water, and yet the total abolition of suction not be due to this cause alone. The more effective cause is, in fact, the run-down of energy, as proved by the experiment described above. In another experiment I took a cut stem in which suction had come to a standstill, and, without disturbing the mucilaginous end, I supplied it with water somewhat above the ordinary tem- perature. This thermal stimulation at once initiated renewed suctional_ activity with great vigour.

was applied once more. It is here seen that this stimulus induces a further acceleration. The latent period, however, of this second response is reduced from forty-five seconds, which was its value in the first case, to thirty seconds. This variation of latent period is brought out still more clearly by the application of a stimulus of shorter duration, in which case the latent period is more prolonged, and its variations, therefore, more easily observed. In order to show this, I took a fresh specimen of Cyvof¢on, and, after the initial balance, applied stimulus of five seconds’ duration. It will be seen from the photographic record (fig. 229) that the

Fic, 229. Photographic Record showing Variation of Latent Period as After-effect of Stimulus Stimulus applied was for 5 seconds. Moment of application represented by vertical line. Lower record shows latent period to be 25 minutes. After re-balance, stimulus of 5 seconds was again applied. Upper record now shows reduction of latent period to 20 minutes, latent period was here very long, being as much as twenty- five minutes. After re-balance stimulus was once more applied, lasting, as before, for five seconds. The latent period in the second case is seen to be reduced to twenty minutes. |

As an interesting and independent verification of the enhancement of suction by stimulus, I now took a number of response-curves, using the Method of Over-balance. Here, it will be remembered, the normal over-balance is indicated by a down-curve, and acceleration of suction by diminution of the slope, or even by reversal, of this curve. In fig. 230 is seen a record obtained in this manner the first down part of which shows the curve of over-balance. Stimulus of halt a minute’s duration was now applied, and it will be noticed

Stimulus of 30 seconds neutralises over-balance and reverses curve, that on account of the re- sponsive acceleration the slope becomes increasingly diminished, till,- after an interval of one minute and a half, the curve becomes horizontal. After this it is reversed to the upward direc- tion. It will thus be seen that the responsive accelera- tion has here, induced a rate of suction which is not merely sufficient to compensate the over-balance, but greatly ex- ceeds it. .In the next photo-

eraphic record (fig. 231) I have been successful in showing the immediate and persistent after-effects. In order to do Fic. 231. Photographic Record of Effect of Stimulus on Over-balance First stimulus for 2 seconds, represented by first vertical line, neutralises and reverses over-balance. Horizontal record after reversal represents persistent after-effect. Second stimulus for 2 seconds, represented by second vertical line, gives risejto up-record, the persistent after- effect being represented by a curve of diminished slope.

this within the limited range of a photographic plate, I employed stimulus of the short duration of two seconds. The first part of the record shows the normal down-curve of over-balance. Stimulus of two seconds’ duration was now applied, at the place marked in the record with a vertical line. This is seen to induce a growing diminution, in the slope of the curve culminating in reversal; and afterwards, owing to the persistence of the after-effect of stimulus, the record becomes horizontal. A second stimulus of two seconds’ duration was now applied. ‘This is seen to induce a further enhancement of the rate, which is shown by the up- curve. The slope of this curve undergoes a slow decline with the waning of the immediate effect of stimulus. But on | account of that component of the stimulus which remains latent in the tissue, there is induced a more or less persistent after-effect, which is greater than the after-effect due to the first stimulus. For while the after-effect of the first stimulus was seen to make the record horizontal, the second after- effect renders the curve slightly ascending. From these and other facts previously enumerated it will be under- stood that the effect ot latent stimulus derived from external sources is to increase suctional activity up to a certain limit.

Having now described the various effects induced in a slightly sub-tonic tissue, under the simplest mode of stimulation, namely, the terminal, we shall proceed to inquire as to what are the effects induced under a somewhat more com- plex mode of stimulation. This is the case with sub-terminal stimulus, where the point stimulated is not on the external extremity, but within the tissue, though near the lower end. Under these conditions the excitatory wave will proceed in two opposite directions, upwards and downwards. The short terminal zone, however, being close to the directly stimulated area, will be more intensely affected than the extended upper region. For this reason there is likely to be a predominant expulsion of water from the lower end. Under continued strong stimulation, however, the more intensely excited lower zone may become fatigued. The

stimulus, may now be expected to reassert itself, thus con- verting expulsion into renewed suction. In any case, whatever the explanation, I find that the result of this mode of stimulus is, first, a movement of expulsion, followed, under continued intense stimulus, by renewal of the upward suction. These various effects are seen in the following photographic record, where the up-curve represents the normal unbalanced -suction (fig. 232). Continuous stimulation was now applied at the point marked with a verticalline. It will be seen that normal suction is here diminished, and afterwards reversed into expulsion. This expulsive movement continues, as I already knew from previous experiments, for a considerable length of time, before the second re- versal to suction is brought Fic. 232. Photographic Record of about by fatigue of the lower

. 4 i b- i l . cat ee Continuous Sub-termina zone. In order to expedite Response was taken without balance the reversal, so that the Continuous stimulation applied from cyrye might remain within . moment represented by vertical line. This induced diminution, arrest, and the plate, I applied a still reversal of response to expulsion. : : Stronger stimulation applied at second SLrenges stimulation, at the vertical line. This induced a second point marked by the second

reversal to suction. Thin white line . ‘ A shows duration of application of vertical line. This was done stimulus of moderate intensity; and by increasing the voltage subsequent thick line, of greater ; 4a ae intensity. which worked the primary of the induction coil from six to eight volts. It will be seen how this reversed the expulsion, converting it into renewed suction. We have seen, as already stated in the electrical response of roots, that while less excitable old roots will generally

negative. We saw, further, that there was reason to associate this positive response with the process of absorp- tion, and the negative, conversely, with that of expulsion or secretion. With various kinds of tissues, moreover, we have found, and shall see further, that as a general rule positive response is obtained, either when the tissue is sub-tonic, and very slightly. excitable, or when the stimulus- intensity is feeble, and when the tissue is fatigued by over- stimulation. Negative response, on the other hand, is characteristic of highly excitable tissues. Under natural conditions, then, when the roots are subjected to the moderate stimulation of such factors as contact with soil, water, and food, we might expect their response to be positive or absorptive. In cut branches, also, in which the tissue is not extremely excitable, a simple terminal applica- tion of stimulus induces, as we saw, the absorpto-positive effect, either by initiating suction, or by enhancing that which was already taking place.

But in highly excitable young roots, in contact with a stimulating supply of inorganic food, the characteristic response, as we have seen, is by secretion. The electrical response also of young roots we found to be negative. Between these two extremes of positive and negative, then, there must be an intermediate case in which the responsive action to external stimulus will be zero. Applying this to the parallel case of suctional response in cut branches, we should expect to meet with. two different cases besides that already given. In one, where the tissue is very highly excitable, the response under simple terminal stimulation will be negative or expulsive. In tissues, however, which are not so highly excitable, but more excitable than those sub-tonic specimens whose characteristic responses I have already described, it might be possible to find cases in which the suctional response to stimulus will be zero. In the sub-tonic tissues referred to, we have already seen that in consequence of increase of internal energy by external stimulus, the suctional response tends to reach a

limit, after which further stimulation would produce little or no effect. 7 Thus we see that if a tissue from any cause be sub-tonic, even moderately strong stimulus will induce positive or absorptive response. If it, on the other hand, be highly excitable, the response may be expected to be negative or expulsive. Between these two, in the intermediate state of excitability, the effect will be neither one nor the other, that is to say, zero. These results concern the application of somewhat strong stimulus, such as that of electrical shocks. Feeble stimulus will generally evoke response by absorption. We can also clearly see that the tonic condition, and there- fore the excitability, of a tissue will vary with the seasons, being low at the end of winter, and high in spring or summer. Thus the same strong stimulus which in the one season will induce absorption, might be expected in the other to provoke expulsion. In experimenting on suction during the period of seasonal variation, I obtained results which verified these inferences,

These experiments, as will be remembered, were begun _ in February, when the spring had scarcely commenced, and the plants were in a sub-tonic condition. Under these circumstances, we saw that the terminal application ot stimulus uniformly evoked positive response, by enhance- ment of suction. By the end of February, however, when warmer weather prevailed, and the vigour of the plants was evidently greater, I was surprised to find that the same stimulus, applied in the same way, to similar specimens of Croton evoked little or no response. A week later—that is to say, in the beginning of March, when the Indian spring was well advanced, and the physiological activity of the plants high—I found that the response which had thus seemed to disappear was renewed, but had become reversed in sign. Strong terminal stimulation now, as a rule, evoked responsive expulsion.

From the considerations enumerated at the beginning of these investigations, it was seen that the various physical theories brought forward to account for the ascent of sap were admittedly inadequate. The further objections, urged against the fundamentally excitatory nature of the processes involved, on the ground of the important part played in the ascent by sap-wood, generally regarded as dead, I have also shown to be untenable. The sap-wood I have shown to be not dead, but living, and to exhibit the normal response of living tissues to excitation. The long persistence of: suction, when the roots are killed with hot water, or the cut specimen placed in poison, was shown to be accounted for by the fact that the death of any individual zone does not arrest suction in those above. I have shown, moreover, that those agents, such as rising temperature, which exalt the general physiological activity of the tissue, enhance suctional activity also. Those which, like cold or anzsthetics, act, on the other hand, to depress the general physiological activity, will depress and arrest its suctional activity also. And, finally, the fact. that the water-movement is a form of excitatory response has been fully demonstrated by the experiments described and the records given in the course of the present chapter. The physiological theory of the ascent of sap may thus be regarded as established.

Heliotropic plant movements reducible to fundamental reaction of contraction or expansion—Various mechanical effects of light in pulvinated and growing organs—Electrical response induced by light not specific, but cencomitant to excitatory effects—Electrical response of plant to light not determined by presence or absence of chloroplasts—Effect of unilateral application of stimu- lus on transversely distal point—Positive response due to indirect effect and negative to transmission of true excitation—Mechanical response of leaf of Mimosa to light applied on upper half of pulvinus—Mechanical response consists of erection or positive ovement, followed by fall or negative move- ment—Electrical response of leaf of A/¢mosa to light applied on upper halt of pulvinus ; induction in lower half of pulvinus of positivity followed by negativity—Longitudinal transmission of excitatory effect, with concomitant galvanometric negativity—Direct effect of light and positive after-effect— Circumstances which are effective in reversing normal response—Plants in slightly sub-tonic condition give positive followed by negative response— Exemplified by (a) electrical and (6) growth response—Examples of positive response to light—Periodic variation of excitability—Multiple mechanical response under light—Direct and after effect—Multiple electrical response under light, with phasic alternations of (— + — +) or (+ — + —)—After effects; unmasking of antagonistic elements, either A/ws or minus—Three types of after-effects.

THE first important point that arises, in connection with the response of living tissues to light, is the question whether such response is peculiar in its character, or fundamentally similar to that which is evoked by other forms of stimulus. The mechanical movements of plants under light are apparently so diverse that it would at first sight appear almost impossible to derive them all from any common fundamental reaction. Thus, some plant organs are found to turn towards the light, others away from it, and others again to remain perpendicular to it. Thus three different typical effects—positive, negative, and dia-helio-

tropic—are induced in different cases by the same stimulus. These effects, moreover, are found to occur in growing as well as in pulvinated organs. This incon- sistency of effects has been a source of great perplexity, inclining observers to the belief that the action of any given plant organ under light is determined, not by some definite reaction, but by its own power to decide what is for its individual advantage. I have shown elsewhere, however,! that as regards mechanical response, the reaction of plant organs to the stimulus of light is extremely definite. This, like other forms of stimuius, induces negative turgidity variation and contraction, as well as consequent retardation of growth in growing organs. Such excitatory effects, moreover, if the tissue be of fair conducting power, may be transmitted in either a transverse or a longitudinal direction. The intensity of this transmitted excitatory effect is thus dependent, as I have shown, on the intensity and duration of stimulus and on the conductivity of the tissue. If neither the intensity of the stimulus nor the conductivity of the tissue be great, it will be the indirect or hydro-positive effect which will reach the distant point, there to induce a positive turgidity variation and expansion. The foregoing observations relate to tissues in a normal condition of excitability. When the tissue is sub-tonic, however, the absorbed stimulus, as we have seen, increases the internal energy and brings about a re- sponsive expansion.