Comparative Electro-Physiology: A Physico-Physiological Study

A leaf of Mimosa re- sponds to strong stimulus by complete collapse, and the recovery from this state is somewhat prolonged, taking from five to eighteen minutes, according to the Fic. 14. Photographic record of Elec- : trical Response by Galvanometric season. In order to obtain Negativity of Pulvinus of Mimosa, . : : when leaf is physically restrained from a series of FCSPONSe with falling. The first series in response to these recoveries, within a Series to stimuli twice as strong. Teasonable time, I find that it is necessary to apply

moderate stimulus. There is then a moderate fall, without complete collapse, and recovery under such circumstances is found to take place within a minute or so. In order to show that electrical response takes place, even when the leaf is prevented from giving mechanical expression, I held the petiole in a clamp, and obtained the set of electrical responses seen in fig. 14. The first series of this record were taken in answer to uniform stimuli of a given intensity, and the

second to stimuli twice as strong. We may here see how response is increased by increased intensity of stimulus. One peculiarity to be noticed in this figure is the trend of the base-line downwards, showing the increasing positivity of the pulvinus. In order to obtain a photographic record the experiment had to be carried out in a dark room, and under these circumstances the pulvinus undergoes an increase, or positive variation, of turgidity. And we shall see later that a positive turgidity variation is associated with galvano- metric positivity in the same way as the negative turgidity variation is found to be accompanied by galvanometric negativity. |

In consequence of the impression produced by the con- spicuous movements of the leaf of M/zmosa,it was assumed that only plants showing such movements were to be regarded as excitable. I have already shown elsewhere, however, that this test of lateral motile responses, as a sign of sensitiveness, is fallacious in the extreme. Such mechanical display is possible only when the two halves of an organ are unequally contractile, and there is consequently a greater expulsion of water from one, in response to stimulus, than from the other. If these conditions are not fulfilled, even the so-called ‘ sensi- tive’ Mimosa would appear to be insensitive. Thus, when we place a cut branch of J/zmosa in water, the pulvini of the leaves, on account of vigorous suction at the cut end, are rendered over-turgid, and the leaves become highly erected. On now applying stimulation, no responsive fall is found to take place ; this is due to the difficulty encountered in the expulsion of water from the gorged tissue. An intact plant, again, which in the light has been found highly sensitive, will often be found insensitive after a short time spent in a dark room. It will then be difficult to believe that the plant is of the sensitive class, for the hardest blow will fail to evoke any mechanical response. And not only does the Mimosa cease to show responsive movement under these cir- cumstances, which may perhaps be regarded as exceptional ; but under perfectly normal conditions also, its sensitiveness

varies so much that its motile response would seem at times almost to have disappeared. I have already pointed out that it is by the unequal excitabilities of the upper and lower halves of the pulvinus that that differential contrac- tion is induced which brings about the lateral response of the Mzmosa leaf. Now it is clear from this that if the differential excitability should be reduced or abolished, by any means whatsoever, there will then be a corresponding diminution or abolition of response. We shall see later that the excitability of a tissue depends upon its state of turgor, and in Mzmosa, from internal causes, a periodic variation is induced in the relative turgescence of the two halves of the pulvinus. We might then expect, in consequence of this, to find a periodic variation of motile sensibility. And certainly, whatever may be the cause, a long course of observation will convince the inquirer of the occurrence of great varia- tions in the sensibility of MW/zmosa at different times of the day. Thus, I had six specimens of this plant growing in pots in the open, and I found, watching them in the month of August, that at eight o’clock in the morning the pulvini of the leaves of all these plants were sensitive in the highest degree. Half an hour later, however, this sensitiveness had so far waned that they would give hardly any motile indi- cation. It is, perhaps, worth while to remark, in connection with this, that-a constant observer is able to judge, by a peculiar, though indescribable, attitude of the leaves, whether or not this condition of insensitiveness has supervened. Thus the mechanical movements of the belauded sensitive plants, such as MJzmosa,on which depended the arbitrary assumption that ‘ordinary’ plants were insensitive, rest on a basis which is itself extremely unreliable.

For by this standard one identical plant ought to be classed as belonging to both the sensitive and insensitive groups, according to the time of day at which any particular observation is made. The fact that when the mechanical response of the leaf of Mimosa is physically restrained, excitatory electrical response takes place unimpeded, shows that we have a criterion by which to test the excitability of a plant, independently of any motile indication. On applying this test, I have found that not the so-called sensitive plants alone, but all plants and all organs of all plants, respond to stimulation. And from this I was led to the discovery that ordinary plants also, in spite of current misconceptions, exhibit motile response by mechanical contraction. The common error of regarding these plants as insensitive has arisen from the fact that in a radial organ, diffuse stimulation induces equal contractions on all sides. Hence those lateral movements, dependent on differential contraction, which are seen so conspicuously dis- played in Mimosa, cannot take place here. But that the organ as a whole undergoes a responsive contraction has been demonstrated by recording the consequent induced shortening of its length. Such longitudinal contraction is sometimes very considerable ; for instance, in the filamentous corona of Passiflora it may sometimes be as much as 20 per cent. of the original length.

Having thus shown that all plants are excitable, I shall proceed to demonstrate the fact by means of electrical response. In studying the excitatory effect on ordinary plants, we must bear in mind that there are two different ways of stimulating a given point: that is to say, locally or directly, and by transmission of excitation from a distance. Conducting tissues are capable of stimulation in either of these ways, but the feebly conducting must be subjected to local excitation, since the effect of stimulus applied at a distance cannot, in their case, reach the responding point. Organs containing fibro-vascular elements are fairly good conductors, and stimulus applied on them at one or two centimetres from the point to be stimulated will thus easily reach it. It must, however, be remembered that stimulus becomes enfeebled by transmission through a long tract, its effect at a great distance being negligible. Parenchymatous tissues are bad conductors of excitation, and in order to excite them, stimulus must therefore be applied directly. ©

we take a petiole or stem, and making suitable electrical connections (fig. 15), apply stimulus, say by contact of hot wire at the point marked x. After a short interval, necessary for the excitation to traverse the intervening distance, an electrical response is obtained, of galvanometric negativity. It is thus seen that the electrical response of ‘ ordinary’ is of the same sign as that of ‘sensitive’ plants, and that in both, again, it is like that of animal tissues.

proposition : namely, that all effective forms of stimulation induce an identical excitatory response of galvanometric negativity. Any sudden change of environmental conditions may constitute an efficient stimulus. Such are: sudden rise Stimulus applied to the right at x. Excitation reaches right contact first, causing galvanometric negativity of the point. of temperature ; any variation of pressure, whether of tension or compression ; mechanical blows or torsional vibration ; any prick or cut; the application of a chemical agent, such as acid ; the application of light ; the incidence or variation of electrical currents; and, lastly, the action of gravity. The stimulatory action of all these agents has already been demonstrated in my work on ‘ Plant Response,’! the excita- tion induced being there shown to find expression in appropriate mechanical movements. In the present volume I shall deal more particularly with the electrical reactions which they induce. The effects of the stimulating action of

1 Bose, Plant Response as a Means of Physiological Investigation, 1906. electrical currents, of light and of. gravity, will be taken up in special chapters devoted to their consideration, while here I shall demonstrate the exci- tatory effects of the other forms of stimulus enumerated. We have already observed the responsive effect which results from the sudden application of heat, by means of a hot wire. The effects of various forms of mechanical stimulation may now be subjected to demonstration, and first we have to observe the effect of the stimulus resulting from sudden tension. The specimen is clamped securely in the middle

(fig. 16), so that when a vertical pull Fic. 16. Excitation by Sudden Tension is given to the upper half, that Plant securely clamped. When half alone is subjected to a sud- suddenly pulled, tension in- . 4 duces galvanometric nega- denly increased tension, the lower tivity of A. Under these circumstances, there is an electrical response, A becoming galvanometrically negative. A is next subjected to mechanical compression, and for this purpose the piece of moistened cloth surrounding the specimen, and making the electrical connection at A, is placed between the two grooved halves of a cork. The enclosed plant tissue at A may now be made to undergo sudden compression, by squeezing the pieces of cork together. This gives rise to the same electrical response as before.

This fact, that both tension and compression will give rise to similar excitatory responses of galvanometric negativity, may receive independent demonstration by first making an electric connection at A with the upper side of the speci- men (fig. 17). When the tissue at A is now suddenly bent down, this upper side becomes convex: that is to say, it is subjected to tension. This gives rise to the excitatory response of negativity. The electrical connection at A is next removed to the lower side of the specimen, at a point A’. On now repeating the sudden flexure, A’ undergoes com-

pression instead of ten- sion. The result is a similar negative elec- trical response. Excitation may, again, be produced by means of a sudden blow at a point. This blow may be delivered by means of a spring-tapper (fig. 18), in which S is When E is connected with the upper point A x a sudden bending down causes tension of the spr ing proper and A. When connection is made with a’ the theattached rod Rcarries same flexure causes compression of A’. Both induce galvanometric negativity.

rod—the lifter L—passes through SR. It is provided with a screw-thread, by means of which its length, projecting downwards, is regulated. By means of this the height or intensity of the stroke may be varied. As one of the spokes of the cog-wheel c is rotated past L, the spring is lifted and released, and T delivers a sharp tap. The height of the lift, and therefore the intensity of the stroke, is measured by a graduated scale _ not shown in the figure. We can increase the intensity

of this stroke through a wide range, first, by augmenting the projecting length of the spring by a sliding catch. We may give isolated single taps, or superpose a series in rapid succession according as the wheel is rotated slowly or quickly. Stimulation, again, may be effected by the prick of a needle or pin in the neighbourhood of A. Response to this also occurs by the normal galvanometric negativity. Successive pricks may thus give rise to successive responses.

Or the specimen may be subjected to torsional vibration. It is here held in the middle by a clamp, and stimulus of torsional vibration is applied (2) @) at one end. - The stimulation a of A makes that end gal- vanometrically negative, the direction of the current outside the circuit being towards, and in the tissue away from, A. Vibration of Blah op is 2 or es 3 j Current of response when B induces responsive nega- 2B is stimulated > tivity of B (fig. 19), the current of response being

Fic. 19.. The Torsional Vibrator (a) The plant is clamped at c, between now reversed. In the cases 7 A pee B. Pig tee" ; : . : esponses obtained by alternately just described, it will be stimulating the two ends. Stimula- noticed that stimulus is ap- tion of A produces upward response ; is, therefore, specially applicable when we wish to study the excitability of such tissues as are not good conductors of excitation, the method of transmitted stimulation being here, therefore, inapplicable.

In order to observe the effect of chemical sijinailabion, the given agent—sulphuric or hydrochloric acid—is applied at x at ashort distance from the proximal contact. The trans- mitted excitation is now again demonstrated by the induced galvanometric negativity of that contact. It will thus be seen that, whatever be the effective form of stimulus employed, it gives rise to a definite and invariable electrical response whose sign is always one of galvanometric negativity.

It was shown, then, in the course of this chapter that the excitatory change in ‘sensitive’ plants is characterised by contraction, negative turgidity variation, mechanical depression of the leaf, and by the electricals response of galvanometric negativity, all these effects being concomitant. It was further shown that electrical response is independent of the mechanical, being unimpeded in its occurrence when the leaf is physically restrained. The same electrical response of galvanometric negativity is also obtained from the tissues of the so called ‘ ordinary’ plants. And these electrical responses of plant tissues, it was further noted, are identical in sign with the corresponding responses given by animal tissues. |

All forms of stimulus, moreover—mechanical, thermal, photic, chemical, and electrical—induce the same excitatory response of galvanometric negativity. Conditions of obtaining uniform response—Torsional vibration as a form of stimulus—Method of block—Effective intensity of stimulus dependent on period of vibration—Additive action of feeble stimuli—Response recorder— Uniform electric responses—List of suitable specimens—Effect of season on excitability—Stimulation by thermal shocks—Thermal stimulator —Second method of confining excitation to one contact—-Increasing response to increas- ing stimulus— Effect of fatigue—Tetanus.

A QUALITATIVE demonstration has been given in the last chapter of the induction of galvanometric negativity in plant tissues, in response to the excitation caused by various forms of stimulus. This galvanometric response is thus a sign or indication of the state of excitation ; and under normal con- ditions it will be of uniform extent, provided only that the stimuli are also uniform. Assuming this ideal condition to be secured, it is clear that the physiological modifications induced by various agents will be manifested by a corre- sponding modification of response. The conditions essential to such application of stimulus are, then, (1) that it should be capable of uniform repetition ; (2) that it should be capable of increase or decrease by definite amounts; and (3) that it should be of such a nature as to cause no injury, by which the excitability of the tissue might be changed in some unknown degree. These conditions, on which the success of the electro-physiological investigation depends, are very difficult to meet. Chemical stimulation, for example, cannot be uniformly repeated. Electrical stimulation, again, which has the advantage of being easy to render quantitative, is open to the objection that by escape of current it may induce

galvanometric disturbance. Indeed, as the response is electrical, it is obvious that if we are to obtain unimpugnable results, a non-electrical form of stimulus is almost a necessity. But it is only after providing against various sources of error that the electrical form of stimulation can be used with con- fidence. The stimulation caused by mechanical blows can be repeated, it is true, with uniform intensity. But the point struck is subjected to increasing injury, and its excitability thus undergoes an unknown variation. | I have, however, been able to devise two different modes of stimulation, in which all these difficulties have been —

Plant P is securely held byavicev. The twoends are clamped by holders cc’. By means of handles H H’, torsional vibration may be imparted to either the end A or end B of the plant. The end view (4) shows how the amplitude of vibration is predetermined by means of movable stops, Ss’. successfully overcome. rendering the results as perfect as possible. These are (1) torsional vibration, and (2) the application of thermal shocks. For the obtaining of perfect responses, it must be said here that there is still another condition to be fulfilled. If we wish to obtain the pure effect of stimulus at one contact, say A, special care must be taken that excitation does not reach the second contact, B; for otherwise, unknown effects of interference will occur. This may, it is true, be obviated by means of the method of relative depression or method of negative variation, so called, to be described in a subsequent chapter. But the experi- mental mode which I am about to describe, in which a block

is interposed between A and B, is much more perfect. According to this arrangement, the specimen is tightly clamped in the middle, by which device the excitation’ of either end is practically precluded from affecting the other. Stimulation is brought about by means of torsional vibration. The stem or petiole is fixed, at its middle, in a vice, V, the free ends being held in tubes, C C’, each provided Fic, 21. Complete Apparatus for Method of Block and Vibratory Stimulation

Amplitude of vibration which determines the intensity of stimulus is measured by the graduated circle seen to the right. Temperature is regulated by the electric heating coil R. For experiments on action of anzesthetics, vapour of chloroform is blown in through the side tube. with three clamping jaws. A torsional vibration may now be imparted to the specimen at either end by means of the handles H and H’ (fig. 20). The amplitude of vibration which determines the intensity of stimulus can be accurately measured by the graduated circle, and may be predeter- mined by means of the sliding stops s Ss... The complete vibrational apparatus, by means of which various experi- mental investigations may be carried out, is given in fig. 21,

Moistened cotton threads in connection with the non-polari- sable electrodes, E E, make secure electrical contacts with A and B. For experimenting on the effects of temperature, there is an electrical heating coil, R, inside the chamber. For the study of the effects of different gases, there are inlet and outlet tubes, which enable a stream of the required gas or vapour to be circulated through the chamber. If the A end of the specimen be now suddenly torsioned through a given number of degrees, a responsive electro-

wards subsides gradually. If next the torsioned end be suddenly brought back to the original position, a second electro- motive response is obtained, similar to the first. Hence, in the case of a to-and-fro ab e ad_ vibration, the responsive effects are addi- a tive, and we have the further advantage 7 aa 22. Influence of that the tissue at the end of the operation is uddenness on the F im ‘ a Efficiency of Stimu- returned to its original physical condition. his In order that successive stimuli may The curves a, 4, ¢, d, 2 : are responses to be equally effective, another factor besides

same amplitude, , : 2 30°. Ina the vi- tion has to be considered. It is to be bration was. Very borne in mind that the effectiveness of the stimulus in evoking response depends also on the rapidity of the onset of the dis- turbance. In the application of vibratory stimulation to plants, I find the extent of response to depend to some degree on the quickness with which the vibration is effected. I give below records of responses to successive stimuli, induced by vibration through the same amplitude, which were delivered with increasing rapidity (fig. 22). It will be noticed that an increasing quickness of vibration increased the response, but that this reached a limit. If we wish, then, to maintain the effective intensity of stimulus constant, we must meet two conditions. First, the amplitude of vibration must be kept the same. This is done by means

of the graduated circle and movable stops: and, second, the vibration period must be uniform. This last condition is effected by an arrangement shown in fig. 23. The torsion- head is kept tense by means of a stretched spiral spring, s, made of steel. From this torsion-head there projects an elastic brass piece, B. R is a striker which can be made to give a quick stroke to B, by the rotation of the handle. A quick to-and-fro vibration is thus produced, by the blow given to B, acting against the tension of the antagonistic spring S. The amplitude of the angular vibration is at the

Fic. 23. Spring Attachment for obtaining Vibration of Uniform Rapidity same time predetermined by means of the stops P and qQ. The arrangements described are as used in ordinary work. But for certain experiments on differential excitability, a second striker, R’, may be attached to the other end of the apparatus, and by this means the opposite contacts in con- nection with E and E’ may be excited simultaneously. In order to obtain responses of great amplitude, it is now necessary to increase the amplitude of vibration. But this may give rise to fatigue. By way of avoiding this, therefore, it is still possible to obtain enlarged response by the additive effect of repeated feeble stimuli. In the electrical response of plants a sub-minimal stimulus, singly ineffective, is found