Comparative Electro-Physiology: A Physico-Physiological Study

I give below (figs. 90, 91), two sets of records, one of which shows the effect of chloral and the other formalin. Fic. 90. Photographic Record showing Action of Chloral Hydrate on the Responses of Leaf-stalk of Cauliflower normal responses, shown in fig. QI, is seen a very interesting instance of alternating fatigue. In order to bring out clearly the main phenomena, I have postponed till now the consideration of a point of some difficulty. To determine the influence of a reagent in modifying the excitability of a tissue, we rely upon its effect in exalting or depressing the responsive E.M. Variation, and we read this effect by means of changes induced in the galvanometric deflection. Now as long as the resistance of the circuit remains constant, an increase or decrease of galvanometric deflection will accurately indicate a heightened or depressed E.M. Variation, due to augmented or lowered

Before t After Fic. 91. Photographic Record showing Action of Formalin (Radish) excitability, induced by the reagent in the tissue. But by the introduction of the chemical reagent the resistance of the tissue may undergo a change, and, owing to this cause, modification of response, as read by the galvanometer, may be induced without any E.M. Variation; The observed variation of response may thus be partly owing to some unknown change of resistance, as well as to that of the E.M. Variation.

This difficulty may, however, be obviated by interposing a very large and constant resistance in the external circuit. The variation in the tissue then becomes negligible, the galvanometric deflections being now proportional to the electro-motive variation, An actual experiment will make this point clear. Taking a carrot as a specimen, I found its resistance f/us the resistance of the non-polarisable electrodes to be 20,000 ohms. The application of a chemical reagent reduced this to 19,000 ohms. The resistance of the galva- nometer used was 1,000 ohms, and the high constant external resistance interposed was I million ohms. The variation of resistance induced in the circuit by the application of the reagent was thus 1,000 in 1,020,000, or less than one part in a thousand. :

In studying the variation of excitability in animal tissues, the method of negative variation is employed. But I may here draw attention to the advantage which is afforded by the employment of the Method of Block instead. For, in the method of negative variation, one contact being injured, the chemical reagents act on injured and uninjured unequally. It thus happens that by this unequal action the resting difference of potential is indefinitely altered. But the intensity of response in this method of injury may to a certain extent be dependent on the resting difference. It is thus seen that, when this method is employed, a factor is introduced which may give rise to complications.

According to the Block Method, however, the two contacts are made with uninjured surfaces, and the effect of the reagents on both is similar. Thus no advantage is given to either contact over the other. The changes now detected in the response are therefore due to no adventitious circum- stance, but to the reagent itself. If further proof be desired of the effect ascribed to the action of the reagent, we can now obtain it by the alternate stimulation of the two ends A and B. I give below (fig. 92) a record of responses obtained in this way from the petiole of turnip. This petiole was somewhat conical in form, and owing to this difference between the A and B ends, the responses given by one were slightly smaller than those given by the other, though the stimuli were equal in the two cases. A few drops of a 10 per cent. solution of NaOH were applied at both ends. The record shows how quickly this reagent abolished the

response of both. In the next figure (fig. 93) is given a photo- graphic record, showing the marked depression of response induced by a strong solution of KOH, and in order to show that under the given experimental conditions, the variation of resistance does not in any way affect the responses, the deflection produced in the galvanometer by the application of an E.M.F. of ‘1 volt to the circuit is shown at the beginning and end of the record. The equality of these two deflections shows that the resistance in the circuit has remained practically the same throughout the experiment.

Fic. 92. Abolition of Response at both A and B Ends by the Action of NaOH Stimuli of 30° vibration were applied at intervals of one minute to A and B alternately. Response was completely abolished twenty-four Therefore, the change in the amplitude of the E. M. responses recorded may be taken as due entirely to the variation in the excitability of the tissue. In the experiments just described, the stimulus was applied directly at the responding point. By the application of a chemical reagent, not only was the responsive excitability of the tissue modified, but its receptivity, or power of receiving stimulus, also underwent a change. It will be shown later that the receptive excitability and the responsive excitability are not necessarily the same. The records which have just been given show what is, strictly speaking, the

effect of the reagent on both receptivity and responsivity jointly. If, however, we wish to study the effect of the reagent on responsive excitability alone, it will be necessary to separate the receptive from the responding point, and apply the reagent on the latter. This may be done by the method of trans- mitted stimulation described previously. Successive uniform Fic. 93. Photographic Record showing the nearly complete Abolition of Response by strong KOH The two vertical lines are galvanometer deflections due to ‘1 volt, before

and after the application of reagent. It will be noticed that the total resistance remains unchanged. stimuli applied at a given point cause excitatory response at the separate responding point, the record of which is taken ; after this, the chemical reagent is applied locally at the responding point. It will be seen that the receptive excitability and the conductivity of the intervening tissue remain unaffected, changes being induced at the responding area alone.

The specimen employed was the petiole offern. The thermal stimulator was at a distance of 1°5 cm. from the proximal electrode. In fig. 94 is shown the stimulating action of a Fic. 94. Photographic Record showing the Stimulatory Action of Solution of Sugar 2 per cent. solution of sugar, inducing a continuous enhance- ment of response for some time. Another stimulating agent is a dilute solution of Na,Co,. Fic. 95. Photographic Record showing Continuous Action of 2 per cent. Na,CO, Solution

enhancement of amplitude of response, but when given in strong solution, induces depression. An intermediate strength of solution shows preliminary enhancement followed by de- While pursuing another line of inquiry on the effect of various strengths of solution of Na,Co, on the natural current, I obtained results which were parallel (p. 122). It was there shown that dilute solution of Na,Co, induced a positive variation of the natural current ; a strong solution, a negative variation, and that a solution of intermediate strength induced a preliminary positive followed by a negative variation. Thus the positive variation in the last-named experiments, already shown to be indicative of increased excitability, was here seen to correspond with heightened amplitude of response, while the negative variation on the other hand is seen to coincide with depression of excitability. The application of a strong solution inducing excitation, carries the molecular condition of the tissue to the stage E, where, as we know, the excitability is depressed.

Another fact elucidated by this and similar inquiries, which I have pursued elsewhere,' lies in the fact that the difference between stimulants and poisons, so called, is often one merely of degree. Thus a stimulatory reagent, if given in large quantities, will be found to induce a profound depression, whereas a poisonous reagent in minute quantities may be found to act as a stimulant. In carrying out a similar investigation with regard to growth response, | found that sugar, for instance, which is stimulating in solutions of, say, I to 5 per cent., becomes depressing when the solution is very strong. Copper sulphate again, which is regarded as a poison, is only so at 1 per cent. and upwards, a solution of ‘2 per cent. being actually a stimulant. The difference between sugar and copper sulphate is here seen to lie in the fact that in the latter case the range of safety is very narrow. Another fact, which must be borne in mind in this connec- tion, is that a substance like sugar is used by the plant for general metabolic processes, and thus removed from the sphere of action. Thus continuous absorption of sugar could not for a long time bring about sufficient accumulation to cause depression. With copper sulphate, however, the case

is different. Here, the constant absorption of the minimal stimulatory dose would cause accumulation in the system, and thus ultimately bring about the death of the plant. Fic. 96. Photographic Record showing the Depressing Action of 5 per cent. HCl Acid The effect of very dilute acids is often to induce an enhancement of excitability, while strong solutions induce depression and abolition. In fig. 96 is shown the depression Fic. 97, Photographic Record showing Effect of I per cent. KHO

Note the preliminary positive twitch at the fourth response after application. and abolition induced by the rene. of a 5 per cent. solution of hydrochloric acid. In dealing with the question of electrical response, we have seen that two opposed electrical effects occur in the tissue subjected to stimulation. One of these is the positive effect, and the other, the true excitatory change of galvano- metric negativity. As the latter is, under normal conditions, predominant, the simultaneous effect of both is a resultant negativity. The positive effect may, however, be unmasked, as we have seen, by abolishing the true excitatory effect of negativity (p. 66). This positivity may also be un- masked, if, by the action of a chemical reagent, the time-

Note the complete reversal of response to positive at the beginning, and its subsequent abolition. relations of the two responses are changed, so that instead of occurring simultaneously, the one is made to lag behind the other. This case will be seen very strikingly illustrated in fig. 97, which exhibits the effect of a 1 per cent. solution of KHO, on response to transmitted stimulation in the petiole of fern. In the normal responses here given, we observe the resultant response of galvanometric negativity. The application of KHO is first seen to reduce the excit- ability, as indicated by the reduced height of the responses. Later, we observe that the true excitatory effect is delayed.

Hence the positive effect is no longer completely masked. Its existence is now seen as a preliminary downward twitch in a di-phasic response, in the case of the fourth and succeeding records, after the application of KHO. In fig. 98, a stronger, namely a 5 per cent. solution of KOH, was used. And here, by the almost complete abolition of the excitatory factor, the response has undergone an apparent conversion to positive; this positive response is, however, subsequently abolished by the death of the plant.

Arrangement for interference of excitatory waves—Effect of increasing difference of phase—Interference effects causing change from positive to negative, through intermediate di-phasic—Diametric balance—Effect of unilateral application of KHO—Effect of unilateral cooling. I HAVE explained how the variations of excitability brought about by various agencies may be determined, by recording the corresponding amplitudes of response, I shall now pro- ceed to describe a new and interesting method of making such determinations, by means of which it will be found possible to elucidate certain questions which without it must remain obscure. This method is, moreover, of extreme delicacy, enabling the investigator to detect the slightest variation of excitability, induced by any agent.

Let two points in the experimental tissue, say A on the right, and B on the left, be suitably connected with the galva- nometer, and let the occurrence of excitation at A on the right be represented by an ‘up’ response record, the excita- tory effect at B, on the left, being represented as ‘down.’ If now the two points, A and B, be excited simultaneously, the resultant electrical response will be due to the algebraical summation of the two excitatory electro-motive effects E, and Ey, these standing for the individual electrical effects at the two points A and B, Now if the intensities of the two effects be the same, and if their time-relations be also the same, it is evident that these two excitatory electrical waves, being of equal amplitude and having the same ‘phase but of opposite signs, will, by their mutual interference, neutralise

each other. Under such balanced conditions, therefore, on simultaneous excitation of A and B, the resultant response will be zero. If now, under the modifying action of any external agency, the excitability of A be enhanced, it is clear that the resultant response will be ‘up, showing the greater excitability of the right-hand point. A similar effect will also be produced if the excitability of B be depressed. | Similarly the depression of the excitability of A, or enhance- ment of B, would cause a resultant response which would be ‘down.’ If, again, the two waves of excitation be not of the same phase, we shall obtain various di-phasic effects resulting from the algebraical summation of the constituent response- curves. The resultant zero-response may thus be converted into di-phasic, by the action of any agency which is capable of changing the time-relations of either of the constituent responses.

_ I shall now proceed to describe the experimental arrange- ments by which two points in connection with E and E’ may be excited, and the resulting electrical disturbances made to interfere with each other. For this purpose we may use the vibrational stimulation which has already been described, with certain necessary additions .(fig. 99). The angle of torsional vibration which regulates the intensity of excitation is determined by two stops, P and Q. An elastic piece of brass, B, projects from the torsion-head. When a single stroke is given to this, a quick to-and-fro vibration is induced, the backward pull being supplied by the attached spring, s. The amplitude of this vibration remains always the same, as determined beforehand by the setting of the stops P and Q. The stroke is given by the striking-rod R, set in motion by the turning of a handle. What has already been said about the excitation of the right-hand side of the specimen applies equally to the left-hand, arrangements for the purpose being a duplicate of those just described. After deciding on a suitable angle of torsional vibration for the right, and taking the response at that point, we proceed to adjust the torsional angle on the left, so that the response there may be exactly

the same as that on the right. If the excitability of the two points had been exactly the same, equal amplitudes of vibra- tion would have resulted in the equal stimulation of both. But in practice the excitabilities are found to be slightly different and the angle of vibration of the one must, therefore, be so adjusted as to induce an excitatory effect exactly equal to that of the other. The two striking-rods, one on the right, R, and the other on the left, R’, can be adjusted so that both are in the same

Nee” : Fic. 99. R, R’, striking-rods for stimulation of two ends of specimen ; B, elastic brass tongue projecting from torsion-head. For producing phase-difference R is adjustable in azimuth. . vertical plane, or so that one is in advance of the other. The left rod is permanently fixed to the rotating axis, but the right can be set at any angle that is desired, with the other. When the right striking-rod is set, pointing to zero of the scale, the two rods are in the same vertical plane, and the rotation of the handle causes equal vibrational stimulus by the two at the same moment. The excitatory reactions on right and left are now, therefore, of the same phase and of equal intensity, but opposed to each other. In fig. 100, a, are reproduced the two separate and equal constituent responses given by a specimen of stem of Amaranth. The

‘down’ curve was given by the individual excitation of the left, and the ‘up’ by the right. On the simultaneous excita- tion of the two points, the resultant response was zero (6). But if the excitation of one—say, the right—be increased by increasing the angle of vibration, the resultant differential response is found to be ‘up.’ It is obvious that’ a similar effect would have been observed had the stimulation of the right been kept the same, while its excitability was increased . by any external agent. In these cases we have two opposed excitatory waves of similar phase, and of the same or unequal intensities, interfering with each other.

Fic. 100. (a) Isolated response of left side (down) and right side (up) ; (4) null-effect when excitations are simultaneous ; (c), (d@), (e) di-phasic responses obtained with increasing difference of phase. We shall next take some simple instances in which, while the stimulation is maintained constant, there is an increasing difference of phase. If the right-hand striking-rod R, instead of being set at zero, be set to the right, or at a p/us angle, the rotation of the handle will cause a slightly earlier excita- tion of the right than of the Jeft. If, on the other hand, the rod be set at a mznus angle, the excitation of the right will be later than that of the left. Under these circumstances, instead of the null-effect due to continuous balance, we shall have a di-phasic response. It is also clear that as the phase difference is increased, the neutralisation of -effects will become. less and less perfect, the separate constituent respon- ses being thus rendered increasingly apparent, In fig. 100, ¢,

is seen the di-phasic effect which was induced when the excitation of the right was made to lag slightly behind that of the left, by the adjustment of the striking-rod at a small mznus angle. The first of the two twitches, which is downwards, indicates the relatively earlier excitation of the left-hand contact. As the phase-difference was increased progressively as in (d) and (e), it is seen that the constituent elements of the di-phasic response are increased corre- spondingly. It is also clear from this that, having obtained the null-effect, if any agents were afterwards applied locally which would make the excitation of the one point earlier than that of the other, we must then expect the null-effect to be modified to di-phasic. An earlier ‘up’ twitch would now indicate that the right-hand contact, having had its re- action quickened, was the first to respond ; an earlier ‘down’? twitch the opposite. 3

We thus see how the conversion of the null-effect into a resultant ‘down’ negative or ‘up’ positive, could .be utilised as a test of the excitatory or depressing nature of a given reagent. We alsosee how the conversion of this null into a di-phasic effect would give us indications as to the change of time-relations induced by the reagent. I shall here, before going on to describe the results obtained with plants, give a photographic record (fig. 101) of certain positive, nega- tive, and di-phasic effects obtained in the electrical response of the inorganic substance, tin, under appropriate modification of the excitability of its two contacts by various chemical reagents.’

Turning now to the question of the determination of the effects of the various reagents by the Method of Interference, we may, as we have seen, cause simultaneous excitation of right and left, by means of the apparatus which has just been described, and which I shall distinguish as the Longitudinal Balance. There is, again, another and simple method of accomplishing the same object, by means, namely, of the Diametric Balance, the diagram of which has already

been given in fig. 80. Using this arrangement, the specimen is clamped at one end, the vibration-head being at the other. Electrical connections are now made with the two dia- metrically opposite points, A and B, of which one, say A, is the upper, and B the lower. Ina tissue -which is isotropic, vibrational stimulus will induce equal and simultaneous excitation at the two points A and B. The effect of any given agency is tested by applying it locally, say at A, and observing the resultant variation of the response. I shall

(a) (4) (c) Fic. 101. Photographic Record showing Negative, Di-phasic, and Positive Resultant Responses in Tin under appropriate modifications of excitation of the two contacts here give examples of results obtained by both these methods, thus affording an indication of the extent of their applicability in various investigations. , We have seen in the previous chapter that the application of strong solution of potash will abolish the excitability of a tissue. Using the Longitudinal Balance, I took a petiole of Bryophyllum and first made such adjustments that the right ‘up’ and left ‘down’ responses were almost equal. On now producing simultaneous excitation of the two ends, a di-phasic response was obtained, due to the fact that the left-hand point was the quicker to respond. Strong solution

of potash was next applied on the right-hand point, and from the record it is seen that the ‘up’ part of the di-phasic response, due to the excitation of the right-hand side, was thus completely abolished, the ‘down’ response being at the | same time increased. by the suppression of this opposing response (fig. 102). In order to demonstrate the use of the Diametric Balance Method, I undertook to investi- gate by its means the influence of the lowering of tempera- ture on excitability. For this