Comparative Electro-Physiology: A Physico-Physiological Study

to become effective by the summation of several. This is seen in fig. 24, where a single vibrational stimulus of 3°, (z) A single stimulus of 3° vibration pro- duced little or no effect, but the same stimulus when rapidly superposed thirty times pro- duced the large effect (4). (Leaf stalk of turnip.) alone ineffective, was found to evoke a large response when repeated with rapidity thirty times in succession. For the delivering of such equal and rapidly succeeding stimuli, I substitute for the single striker R an eight-spoked wheel, a complete rotation of which, by means of the handle, gives rise to a definite sum- mated effect: and a series of responses to such summated stimulations is found to be uniform. The galvanometer used for these experiments is a dead-beat instru- ment of D’Arsonval type. The sensitive- ness of this is such that a current of 10° ampere causes a deflection of I mm. at a distance of I metre. For a quick and accurate method of obtaining. records, I

devised the following form of response-recorder. The curves are obtained directly, by tracing the excursion of the galvanometer spot of light on a revolving drum (fig. 25). This drum, on which is wrapped the paper for receiving the record, is driven by clockwork. Different speeds of revolution can be given to it by adjustment of the clock- governor, or by changing the size of the driving-wheel. The galvanometer spot is thrown down on the drum by the inclined mirror M. The galvanometer deflection takes place at right angles to the motion of the paper; a stylographic pen attached to a carrier rests on the writing surface. The carrier slides over a rod parallel to the drum. As has been said before, the galvanometer deflection takes place parallel to the axis of the drum, and as long as the plant rests un- stimulated, the pen, remaining coincident with the stationary galvanometer spot on the revolving paper, describes a straight line. If, on stimulation, we trace the resulting excursion of the spot of light, by moving the carrier which holds the pen, the rising portion of the response curve will be obtained. The galvanometer spot will then return more or less gradually to its original position, and that part of the curve which is traced during this process constitutes the recovery. The ordinate in these curves represents the electro-motive variation, and the abscissa the time.

_ We can calibrate the value of the deflection by applying a small known E.M.¥., say of ‘1 volt, to the circuit, and noting the deflection which results. This gives us the value of the ordinate. The value of the abscissa which represents time is determined by the distance through which the recording surface moves, in unit time. In this simple manner accurate records are obtained. It has the additional advantage of enabling the observer to see at once whether the specimen is suitable for the purpose of investigation. A large number of records might be taken by this means, in a comparatively short time.

It is also easy to take the records photographically by wrapping a photographic film round the recording drum. I give in fig. 26 a series of responses taken from the root of radish (Raphanus sativus), in which the stimuli were applied at intervals of one minute. This shows how ex- tremely uniform the responses may be rendered, if proper precautions are taken, It may here be once more pointed out, that for convenience of inspection, the records in this book have been so taken that the normal electrical responses © of galvanometric negativity, unless specially stated to the contrary, are seen as up-curves, galvanometric positivity being represented by down-curves. These excitatory responses of

galvanometric negativity are obtained with all plants, and with every organ of the plant. I give here a table containing a list of specimens which will be found on stimulation to give fairly large electro-motive effects, occasionally as high as ‘rt volt. Geranium (Pelargonium) Stem : : . | Vine (V7tds vinifera) Amaranth (Amaranthus) Horse Chestnut (4 sceulus hippocastanum) Turnip (Brassica napus) These responses, being physiological, vary in intensity with the condition of the specimen. The same plant which gives strong electrical response in spring or summer, may exhibit but feeble responsiveness in autumn or winter. Again, we shall see in a subsequent chapter that any agent which depresses physiological activity will also depress the electri- cal response; and, lastly, when the specimen is killed, the normal response is abolished.

I shall next describe a second and equally perfect method of stimulation, by means, namely, of thermal shocks. We have seen that a sudden thermal variation acts as an efficient stimulus. I have also shown in my ‘Plant Response’ that thermal radiation acts as a stimulating agent, in inducing excitatory contraction. Hence, if a tissue be surrounded by a platinum wire, through which an electrical heating-current can be sent, the enclosed tissue will be subjected to a sudden variation of temperature, and also to the thermal radiation proceeding from the heated wire. Now if in successive experiments the duration and intensity of the current flowing through the wire be maintained constant, the stimuli also will thereby be rendered constant. The thermal stimulator, as already said, surrounds the specimen, but is not in actual contact with it. This is to prevent any injury to the tissue, by scorching. The current is so adjusted as to make the platinum wire red-hot and this heating-current is closed for about half a second at a time. Should larger response be desired, it-can be obtained by the summated effect of a number of such shocks, or the thermal stimulator may be put in direct contact with the tissue, if care be taken that the rise of temperature is not so great as to injure it. :

The difficulty of ensuring similarity of duration to each individual shock is overcome by the use of a balanced key actuated by a metronome (fig. 27). A second rod is attached at right angles to the vibrating rod of the metronome, and carries a bent piece of brass in the form of two prongs. During the course of each vibration these prongs dip into two cups of mercury, thus closing the electrical circuit for a brief and definite time. When a second press-key, not shown in the figure, is open, the circuit is incomplete, and there is no thermal stimulation. The observer then presses this key, and counts, say, five strokes of the metronome,. after

is again opened. In this way, the sum- mated effect is ob- tained, of five equal thermal shocks. This Fic. 27. Stimulation by Thermal Shocks process is repeated as often as desired, at intervals of, say, one minute, by which time the tissue is generally found to have completely recovered from its ex- citatory electrical variation. : In the case of the experimental arrangements of which the diagram is given in fig. 27, stimulation is confined to one contact of the responding circuit. The method by which excitation was here prevented from reaching the distal contact is important. I shall, in the course of the present work, show that the parenchymatous tissue of the lamina of a leaf or leaflet is a bad conductor of excitation. Hence if the second contact of the circuit be made with this tissue, the stimulus does not reach the distal point. It is true that a certain small proportion might conceivably be conducted through the attenuated fibro-vascular channel of the midrib. But even so remote a contingency is provided against by a transverse cut across the midrib on the hither side of the contact.

The arrangements, then, being made in the manner de- scribed, the tissue may be subjected to the action of successive uniform stimuli. How regular the resulting responses may be rendered will be seen from fig. 28, in which is given a series of responses obtained from the petiole of a fern (fig, 28) under successive thermal shocks, imparted at intervals of one minute. We have hitherto studied the responses caused by uniform stimuli. We shall next observe the increase of responsive effects brought about by increase of stimulus. In

Fic, 28. Photographic Record of Uniform Response in Petiole of Fern to transmitted excitation animal tissues it is found, speaking generally, that increasing stimuli induce increasing effects, but that this process has a limit ; and in plant tissues the same is found to be the case. In order to obtain effects of the simplest type, not compli- cated by any secondary phenomena, it is necessary to choose specimens which exhibit little fatigue. In the first of these the stimulus was ap- plied by means of the spring-tapper. The first stimulus was given by a fall of the striking-lever from the height h; the second from 2h; and

; Fic. the third from 3h. The response- ee F Taps of increasing strength curves (fig. 29) clearly show the in- 1:2:3:4 producing in- crease of effect due to this increasing acheter eaiinat ees stimulus. In the second series, the stimulus applied was vibrational, and increased from 2°5° to 12°5° by steps of 2°5° at a time. Fig. 30 shows how the intensity of response tends under these conditions to approach a limit. The following table gives the absolute values of the responsive electro-motive variations. ;

TABLE SHOWING THE INCREASED ELECTRO-MOTIVE VARIATION INDUCED In such normal cases an inerease of response is always induced with increasing stimulation. A diminution of response may, however, sometimes appear, with increasing Fic. 30. Increased Response with Increasing Vibrational Stimuli (Cauliflower-stalk) Vertical line = +1 volt. Stimuli applied at intervals of three minutes. stimulus. But this is merely a secondary effect, due to fatigue. The following records (fig. 31) will show in what manner this may be brought about. They were taken with specimens of the petiole of cauliflower, in one of which (A) fatigue was absent, while in the other (B) it was present. In the first specimen the recovery from each stimulus was

complete. Every response in this series starts, therefore, from a position of equilibrium, and the height of each single response increases with increasing stimulation. In the second case, however, the molecular derangement consequent on stimulation is not completely removed after any single FIG. 31. Responses to Increasing Stimulus obtained with Two Specimens of Stalk of Cauliflower stimulus of the series. That the recovery is only partial is seen in the gradual shifting of the base-line upwards. In the former case the base-liné had been horizontal, represent- ing a condition of complete equilibrium. Now, however, the base-line, or line of modified equilibrium, is tilted upwards. Thus, even here, if we measure the heights of successive

responses from the line of absolute equilibrium, they will be found to increase with increasing stimulus. Ordinarily, how- ever, no allowance is made for the shifting of the base-line, the responses being measured instead from the -place of its previous recovery, or point of modified equilibrium. In this way these responses undergo an apparent diminution. I have occasionally observed another curious phenomenon in connection with the subject of response under increasing

stimulus. During the gradual increase of the stimulus from — a low value, there would at first be no response. But on reaching a certain critical value, a response would suddenly be evoked which was maximum—that is to say, would not be exceeded, even when the stimulus was further increased. We have here a parallel case to what is known in animal physiology as the ‘all or none’ principle. In the case of cardiac muscle, for example, there is a certain minimal intensity of stimulus which is effective in inducing response. ‘But further increase of stimulation causes no concomitant increase of effect.

When a tissue is subjected to rapidly succeeding stimuli, the excitatory effects are superposed upon each other. In stimulus is added to that of the first, before that has time to disappear. The result is a summation of effects more or less complete ; and these attain a maximum. With moderate frequency of stimulation, such a tetanic effect is incomplete, tending to become more and more complete, with the progressive increase of frequency (fig. 52). I have obtained results in every way similar to these, with the mechanical response of ordinary plants. In fig. 33 is given a photo- graphic record of tetanus, taken from the longitudinal motile responses of the style of Datura alba. Similar tetanic effects are also obtained in the electric response of plants, of which the records seen in fig. 34 form an example.

The difficulties in the quantitative observation of electrical response have thus been overcome by the employment of two different methods of stimulation—namely, torsional vibration, and stimulation by thermal shocks. In the case of the former, the intensity of stimulation was seen to depend on the amplitude of vibration. In the latter, stimulus intensity was determined by that of the thermal variation, which again was regulated by the intensity and duration of the electrical heating-current. It was also seen to be important that the

Record to left shows incomplete tetanus, with moderate frequency of stimulation. Record to right shows tetanus more complete, with greater frequency of stimulation (Brodie). excitation of one contact should be prevented from reaching the other, and this was provided against in two different ways. In the first of these, a physical block was interposed between the two contacts. In the second, the distal contact was made with the non-conducting tissue of a lateral leaf.

a es (b) Fic. 33. Photographic Record Fic. 34. Fusion of Effect ot of Genesis of Tetanus in Rapidly Succeeding Stimuli i scle ; (4) i t. Plants (Style of Datura alba) wi stg aati | let tin When these precautions were observed, it was found that uniform stimuli induced uniform response. Stimuli which were singly ineffective, were found on repetition to become effective. A tetanic effect was obtained by the rapid super- position of stimuli. Response - curve showing general time - relations — Instantaneous mechanical stimulation by electro-magnetic release—Arrangement of the rheotome— Tabular statement of results of rheotomic observations—Rhythmic multiple responses.

IN taking records of the electric response of plants, a galvanometer of fairly high sensitiveness is required. One which gives a deflection of I mm. at a scale-distance of I-metre, under a current of 10°° ampere is found, as already said, to be suitable for practical purposes. I used for most of the experiments in this work a dead-beat galvanometer of the D’Arsonval type. The natural period of swing in these galvanometers is somewhat long, however, and the response- record thus lags behind the electro-motive changes induced by stimulus. In order, therefore, to investigate the time-relations of a growing electro-motive reaction in a plant, after the recep- tion of the stimulating shock, it is necessary to employ a rheotomic mode of investigation. An account of this, and of the results obtained, will be given in the course of the present chapter. The after-effect of stimulus is found to be somewhat persistent and to vary in duration in different specimens. In some cases, recovery is complete in a very short time; in others it takes very much longer. For the purpose of forming a general idea of this difference two response-records are given here, one of which was taken from a stem of the quickly-reacting Amaranth (fig. 35), and the other from the more sluggish Colocasza. It will be seen that

while in the first of these the recovery was completed in fifteen seconds, in the latter, even after the lapse of forty seconds, it was still far from complete. Indeed, in this case it was not altogether accomplished till after several minutes. The character of the tissue again is an important factor in determining the time required for recovery. Thus it will be shown that in a vegetable structure functioning as nerve, recovery is much more rapid than in ordinary tissue. The physiological modification induced by season, moreover, is seen in the fact that response and recovery are quicker in

Fic. 35- Response of (a) quickly reacting Amaranth ; (4) of sluggish Colocasia summer than in winter. This difference is demonstrated by mechanical response also, for in that of the leaf of Mzmosa, as already stated, it is found that, whereas in summer the period is six minutes, in winter it is as long as eighteen, or three times as much. In the study of the time-relations of response, we may overcome the difficulty of the galvanometer-inertia by using, as already said, some modification of the rheotome, originally devised by Bernstein. The relative values of electrical variation induced at various periods after the impact of the excitatory shock may here be found by making brief

galvanometric contacts of equal period at the required intervals. : 3 The difficulty in this investigation lies in the instan- taneous application of a stimulus at a definite moment, and in the successful adjustment of the subsequent interval at which the resulting responsive current is to be led to the gsalvanometer and recorded. Instantaneous stimulation can, it is true, be effected by electrical shock. But polarisation, and other disturbances caused by it, might give rise to unknown variations in the responsive effect. Hence, it is advisable when recording the electrical response to employ, if possible, a non-electrical form of stimulus. And it is only after the successful employment of such an unimpeachable method, that we can feel any confidence in the use, after due precautions have been taken, of the electrical stimulus itself, as will be described ina later chapter. Another obstacle to be overcome is the elimination of the unknown element of time required for transmission when stimulus is applied at a distance from the responding point. This uncertainty can only be removed by applying the stimulus directly on the responding point itself. All these difficulties I have successfully met by employing the mechanical form of stimulation, which I am now about to describe. We have seen that a stimulus of definite intensity may be imparted by a quick torsional vibration of either twist or un-twist, or of the one followed by the other. The intensity of this stimulus, as we saw, depends on the angle of torsion, and remains constant as long as that angle is maintained the same. For this-purpose I use the vibrational apparatus already described, successive excitations being produced on one side only, say the right. The torsion-head is set by pulling a vertical thread by which the index is made to rest against the stop P. This pull of the vertical thread is against the antagonistic action of the spiral spring S (fig. 36).

During the process of the setting, which is carried out slowly, there is a slight excitatory disturbance. But this is allowed to subside, The vertical thread by which the torsion-head is ‘set,’ is kept pulled up by an electro-magnetic arrangement shown in fig. 37, where the electro-magnet is seen to hold a soft iron armature at the end of the thread. At the moment when stimulation is to be effected the current which energises the electro- magnet is interrupted by an automatic arrangement which will be described later. By the break of the current the armature is released and a semi-vibration of the torsion-

Fic. 36. Arrangement for In- stantaneous Stimulation Torsion-head set by string against stop Q is suddenly let go by electro-magnetic head is suddenly produced, the amplitude of which has been predetermined by suitable adjustment of the stop P. Suc- A, B, striking rods attached to revolving rheotomic disc; K,, key for electro-magnetic release of torsional stimulator ; K,, for unshunting the galvanometer, G; E, electro-magnet with its armature by which the vibration-head, Vv, is set at a definite torsion-angle; N,, N,, non- polarisable electrodes making electric contacts with specimen ;?c, com-

cessive stimuli of equal intensity may thus be applied on the experimental tissue at whatever time may be desired. The next point is to secure an automatic arrangement by which galvanometric connections can be made with the experimental tissue, for a short period of time, say, ‘o1 of a second. In order to study the growing electro-motive changes, these short-lived contacts are to be effected in successive experiments at gradually increasing intervals of ‘oI, ‘02, ‘03 seconds, and so on after stimulation. It should be remem- bered in connection with this subject that the reactions in plant tissues are much more sluggish than those in the. animal. The time-intervals here provided for, therefore, are even smaller than would have been strictly necessary,

The general plan of the apparatus for carrying out this investigation is seen in fig. 37. The revolving rheotome-disc — carries two striking-bars, A and B, of which A is fixed, and B capable of an increasing angular adjustment behind A. The bar A, striking against the key K,, interrupts the electro- magnetic circuit E, thus causing stimulation. All this time, the galvanometer G is short-circuited by key K,, and it is -only when the striker B unshunts the galvanometer, by striking against K,, that the responsive current can act on the galvanometer. C is the compensating potentiometer, the object of which will be described presently. The rheotome- disc is rotated by means of a motor, provided with a perfect governor, the period of asingle rotation being adjusted to one second. The circumference of the disc is 100 cm. One centimetre of this circumference therefore represents an in- terval of time of ‘o1 second. The breadth of the striker B is also 1 cm. and it will therefore pass over a given point in the course of ‘or second. These striking-rods attached to the disc, impinge, as already said, against two electrical keys K,, and kK, which are adjusted along the same radius of the disc. K, is a balanced key, one end of which carries a two-pronged brass fork, both prongs of which are normally dipped in cups of mercury, thus completing the particular electric circuit. By the blow given by the striker A on a projecting rod attached to K,, this fork is tilted upwards, and the circuit is broken. The striker B then impinges on the second key, K,.

Here, the prong is kept down by a spring S, the circuit being re-made, as soon as the breadth of the striker B has passed over the projecting rod—that is to say, in ‘oI second (fig. 38). The interval of time be- tween the actions on the two keys, by which the two different electrical cir- cuits are broken in suc- cession, can be gradually increased, by increasing the angle between A and B. The key K,, as already said, controls the electro- magnet, which, on _ its

“stimulation of the tissue. *'* 38. en Sian £3 The rotation of the rheo- K,, actuated by rod A, K, by B. become uniform, on the Starting of the motor, but attains this when one revolution has been completed. Therefore the experimental observations are not made till the speed has become steady. By pressing the key K, during the first revolution (fig. 37), the break-action of A on K, is postponed. K, is then opened, and during the next revolution, stimulation is effected.

In order to obtain the galvanometric effect of excita- tion at definite short intervals of time after the stimulus has been applied, the galvanometer short circuit, as stated before, is removed at those definite intervals. The adjust- ment of the striking-rod: B, in relation to A, enables us to open the short circuit, for ‘ol of a second, at increasing intervals. When the rod B is at a distance of I cm. from 4, the short circuit is removed after ‘ol second, when at 2 cm. after ‘02 second, and so on. Thus, in the arrangement just described, the galvanometer is short-circuited, except at those definite intervals required for observation. In a second