Comparative Electro-Physiology: A Physico-Physiological Study

Specimen I.—Centripetal T; ransmisston The distance traversed by stimulus was 27 mm. Spectmen 11.—Centrifugal Transmission The distance traversed by stimulus was 38 mm. With regard to the effect of temperature, I found that cold reduced the velocity of transmission. Thus, in one experiment, slight cooling reduced it to one-third, and when carried still further, it abolished the conductivity altogether. A rise of temperature, on the other hand, had the effect of enhancing velocity of transmission. The following table shows that a rise of temperature from 30° C. to 35° C. doubled the velocity, and that at 37° C. the rate was. almost three times that at the first temperature. The velocity was in this case determined in the centrifugal direction.

TABLE SHOWING THE EFFECT OF RISE OF TEMPERATURE ON VELOCITY. Transmission of excitation, as I have shown elsewhere, and shall show again, is depressed or abolished by the action _of anesthetics. We shall also see, further, that the polar effects of currents on the velocity of transmission are the same in the plant as in the animal, being opposite, accord- ing as it is the anode or kathode. In the case of a so-called ‘sensitive’ plant, by taking advantage of the motile indica- tions afforded by the leaf or leaflet, it is possible to determine the velocity of transmission of excitation and its modifica- tions. With ordinary plants, however, no such indications being available, it is obvious that we must find some other means of detecting and observing the excitatory wave during transit. Onesuch I have described elsewhere as the Electro- tactile Method. It is found that the passage of the excitatory wave, even through an ordinary tissue, brings about minute form-changes. These give rise to pressure-variations as between two enclosing contacts. And this variation of pressure, in turn, can be recorded by means of a sensitive electrical device.

There is, however, a more direct way of detecting the excitatory wave during its passage through a vegetable tissue. In this—the Electro-motive Method—the galvano- meter takes the place of the motile leaflet. It has been shown that. when the plant tissue is directly excited, the state of excitation is invariably accompanied by an electro- motive variation, the excited point becoming galvano- metrically negative. Hence any excitatory wave which is transmitted through the tissue will always have an electro- motive wave as its strict concomitant. The moment, there- fore, at which excitation reaches any given point, may always be determined by observing the arrival at that point of the excitatory electrical disturbance of galvanometric negativity. In order to prove that the arrival of excitation at the given point is attended by this specific electrical response, we may perform an experiment on a plant such as Biophytum, which is provided with motile leaflets. One of the indicating leaflets is attached to the optic lever, its base being connected with one of the electrodes of the galvanometer, while the second is attached to a distant point on the leaf. The two spots of light, one from the optic lever indicating the mechanical response, and the other from the galvanometer, indicating the electrical, are so adjusted as to lie one above the other, on the same revolving-drum. On now applying a stimulus, say thermal, at.a distant point, it will be found, after the lapse of a definite interval; that both spots of light are deflected at the same time, showing that both alike give an outward indication of that state of molecular disturbance which is synonymous with excitation. These manifestations, of both kirids, would therefore take place at an identical moment, if only the inertia of the two indicators were absolutely the same.

But, just as the same impulse would be indicated at slightly different times, if one indicating-lever were light, and the other heavy, so here also there may be a slight difference as regards time between the appearance of the mechanical and electrical responses, according as the virtual inertia of the one indicator exceeds that of the other. In determining velocity of transmission by the Electro- motive Method, a previous experiment gives us the loss of time due to the inertia of the galvanometer. This, deducted from the observed interval between the application of stimulus and response, gives the time required for trans- mission through the given distance. In this manner I have been able to determine the rate of transmission of excitation in ordinary plants. I give below a table which shows these velocities as determined by me in the case of sensitive plants, and of ordinary plants, and for the purpose of comparison, those obtained by other observers, in the nerves of some of the lower animals, from which it will be seen that all these are more or less of the same order.

Nerve of Eledone (observed by Uexkiill) ‘5 to 1 mm. Ms (6) Sensitive Plants. Subject | Velocity | eens Mimosa pudica: petiole. : : ; | 14 mm. per second Neptunia oleracea: petiole . : ; . | I°I mm. ie Biophytum sensitivum : | Petiole of, direction centripetal . 7 2°I mm. Petiole of, direction centrifugal . 3°38 mm. os Peduncle of . F : : ; | 3°7 mm. ze (c) Ordinary Plants. Subject | Velocity = = - po LiLE Fern: isolated nerve of . , . 2 50 mm. per second Ficus religtosa: stem. : ; ; a 9°4 mm. ‘3 Cucurbita : tendril ; ; | 5 mm. ¥3 Jute: stem P e ‘ : ‘ - | 3°5 mm. Pe Artocarpus: petiole . ; ; ; Fae *54 mm. ae

Since the conduction of excitation takes place by the transmission of protoplasmic changes, it is evident that it must occur most easily along those paths in which there is greatest protoplasmic continuity. It is clear, then, that certain elements in the fibro-vascular bundles will furnish the best conducting medium. Cells of indifferent tissue, on the other hand, like the parenchyma of the leaf, are divided from each other by more or less complete septa, the fine filaments, by which neighbouring cells may be protoplasmically connected, being so minute that the conduction of stimulus through such imperfect channels must be comparatively feeble. Such tissues are, therefore, indifferent conductors of excitation, the stimulus remaining more or less localised in them. Plant-organs, then, which contain fibro-vascular elements, such as the stem, peduncle, and petiole, are for that reason relatively good conductors. Conductivity in such an organ, again, is,as we should expect, much greater along the length than across.

I shall now describe an important method by which the relative conductivity of a tissue in different directions may be experimentally determined, verifying by its means the difference in the power of a tissue to transmit stimulus longitudinally and transversely. For this purpose I took a thick peduncle of J/usa, and made two electrical con- nections, of which one was at a fixed point B, transversely situated as regards C, the point of application of stimulus. The second point, A, was longitudinally above Cc, and its distance from it could be varied in successive experiments (fig. 273).

If we now take a point, A, in such a position that CA is equal to CB, then, on account of the better conductivity along CA, the excitation will reach the A contact earlier than that at B, making that point galvanometrically negative. The direction of the first responsive current, therefore, will be from A->B in the tissue. If, next, the longitudinal contact be moved to A”, that is to say, so far that the excitation reaches the B contact first, then the responsive current. will

be reversed, flowing now from B>A”. A point of transition, or of balance, A’, may now be found by searching, at which the movement of the exploring contact, nearer or further, will give rise to opposite responsive currents. The con- ductivity along the longitudinal direction will then be, to that in the transverse direction, as the balancing-distance CA’ is to CB. With a given specimen of the peduncle of Musa the transverse distance CB was 3°7 cm., and the longitudinal balancing-distance CA” was determined at 10°4 cm. Hence the longitudinal velocity was 2°8 times that in the transverse direction.

Fic. 273. Experimental Arrangement for Comparing the Relative Conductivities in Transverse and Longitudinal Directions C, point of application of stimulus ; B, permanent transverse contact ; A, A’, A’, exploring points of longitudinal contact for obtaining balance, It has been shown that different tissues in the plant may possess extremely different powers of conducting stimulus. In animals there are specialised channels of conduction known as nerves, and in plants also I have been able to discover similar conducting tissues, which can be isolated for the study of their responsive peculiarities. Experiments on this subject will be related in detail in Chapter XXXII. It may be said here, however, in anticipation, that the velocity of transmission of true excitation through these nervous channels is, generally speaking, fairly high, being at the rate of about 50 mm. per second in the case of isolated

nerve of fern. This, for the relatively sluggish vegetable tissue, is undoubtedly very high. In connection with this question of velocity of trans- mission, a fact not hitherto taken into account is, that there are two distinct kinds of nervous impulses, travelling with different velocities—namely, the hydro-positive and the true excitatory negative. Of these the velocity of the former is greater. In the nerves of higher animals, where the velocity of transmission of true excitation is also great, it is not generally easy to distinguish one from the other, so rapid is their succession. But their occurrence as distinct waves, even in animal tissues, I shall be able to demonstrate in a subsequent chapter. In plants, however, where the velocity of transmission of true excitation is not very high, it generally lags perceptibly behind the positive wave (p. 59). Burdon Sanderson, in his determination of the velocity of trans- mission of excitation in Dzonea, arrived at the exceptionally high result of 200 mm. per second. I have shown, however, that the wave whose velocity he measured was not of true excitation, but of hydro-positive disturbance (p. 231).

In the present chapter it has been my object to demon- strate the reality of true excitatory propagation in plants similar to that in the animal. The examples given will be found more fully described on referring to my book on ‘ Plant Response.’ I shall, however, in the course of the present work describe new and extremely delicate means by which the modifications of conductivity may be studied in plants unde- varying physiological conditions, 7 Drawbacks to use of electrical stimulus in recording electrical response— Response to equi-alternating electrical shocks—Modification of response by decline of injury—Positive after-effect—Stimulation of nerve by thermal shocks — Enhancement of normal response after tetanisation—-Untenability of theory of evolution of carbonic acid—Abnormal positive response converted into normal negative after tetanisation—Gradual transition from positive to negative, through intermediate diphasic—Effect of depression of tonicity on excitability and conductivity—Conversion of abnormal into normal response by increase of stimulus-intensity—Cyclic variation of response under molecular modifica- tion.

IN the study of the electrical effects of excitation on the nerve, the chief experimental difficulty lies in the selection of a form of stimulus which can be made quantitative. In such investigations it is usual to employ the electrical form of stimulus, because of the great facilities which it offers. A marked drawback to its use, however, lies in the fact that unless extraordinary precautions are taken it is liable to lead to serious error. It must be remembered that for the detec- tion of responsive variations in the nerve an extremely sensitive galvanometer has to be employed. The excitatory effect which is to be detected being indicated by the relatively feeble electrical response, and the form of stimulus being also electrical and being of high intensity, the results are liable to be disturbed in an unknown manner by leakage of the stimu- lating current.

In some cases it is possible to take the bold step of including the experimental nerve itself in a circuit in which the exciting coil and the galvanometer are in series. Under these circumstances, and employing strictly equi-alternating shocks, we have seen that the resultant response is due to the differential excitabilities of the two nerve-contacts A and B. If, for instance, we wish to obtain the responsive reaction of one point only, say A, uncomplicated by that of 8, it is” only necessary to abolish the excitability of the latter. This can be done to a greater or less extent by injury, as, say, by making a transverse section, or by scalding. Response will then take place by the induction of relative galvanometric negativity at A. In fig. 274 is seen a series of records obtained in this manner. The responses here apparently indicate growing fatigue of the nerve. They also exhibit the positive after- effect.

With reference to the method of obtaining response by injuring one con- tact, commonly employed, it may be said that the assumption that the ex- citability of the injured point is totally abolished is not justified; for I have found that though recent injury causes a great depression of excitability, yet py. 274. Response of after a lapse of time the injured point — Frog’s Nerve under ‘ F ‘ar Simultaneous Excita- tends to recover its excitability to a tion of both Contacts,

greater or less extent. In such a case by Equi - alternating . é Electrical Shocks, one we may expect two different effects to Contact being Injured be exhibited in the responses. The re- Note the positive after- sultant response being due, as we have i 3 seen, to the differential excitability of A and B, the gradual restoration of the excitability of B will progressively diminish the amplitude of the resultant response, thus giving it the appearance of fatigue. Under these conditions, and after a sufficiently long interval, response may almost disappear. This appears to me to be the true explanation of the gradual fall in the amplitude of response, when the specimen is a nerve, having one contact at the transverse section. It also explains why, in such a nerve, a fresh section, causing

renewed depression of excitability, is necessary in order to obtain renewed amplitude of response. The second effect due to this depression, without abolition of the excitability of B, is seen in the diphasic character of the responses. The positive after-effect observed in the record shown in fig. 274 may thus be ascribed to the later induction of negativity at the depressed point B. The electrical re- sponse of the nerve is apparently liable in this way to great _ variations, when the method of record employed is differential. But it must be remembered that true characteristic variations of the response as determined by physiological modification can only be obtained by finding some means which shall be strictly independent of this differential factor. With this object, I have succeeded in devising a new mode of observing and recording the direct effect of stimulus on the nerve, uncomplicated by the differential factor. In a subsequent chapter we shall, using this method, be able to determine the conditions which induce the characteristic variations in the response of nerve, from the staircase increase to the fatigue- decline, or even reversal, through the intermediate phase of uniform reponses.

The method which. has just been described, of exciting the nerve at both contacts by equi-alternating shocks, is not applicable, however, where the object of investigation is the conductivity of an intervening tract of nerve between the exciting and the led-off circuits. Here the employment of electrical shocks as exciting stimulus gives rise to disturbing unipolar effects, which persist even when the physiological conductivity of the intervening tract is destroyed as, say, by ligature or by crushing. Thus—

‘If the nerve of a frog’s leg is laid across two electrodes connected with the poles of a secondary coil, so as to close the induction circuit, a ligature being then applied to the myopolar tract, tetanus may still be observed in the isolated leg, on making the lead off from it at a certain distance of coil... These unipolar effects may obviously be very disturbing, and are indeed pro- ductive of fallacies in vivisection and also in experi- ments with the galvanometer, if not avoided by due precautions. Hering has pointed out that in experiments such as the investigation of the negative variation of nerve-currents, in which galvanometers and exciting circuits are separated by a long tract of nerve, the most complete insulation of the two circuits is no guarantee against the overflow of induced electricity through the interpolar part of the nerve into the galvanometer circuit... . This kind of unipolar stimulation is an obvious danger in all experiments on action-currents and negative variation in nerve, while it shows what narrow bounds restrict the intensities of current that may be safely used in these experiments.’ ?

From this it will be seen how important it is to have at our command some non-electrical form of stimulation, when the response to be recorded is electrical. Heidenhain. em- ployed a mechanical form of stimulation, by which the nerve was subjected to blows from an ivory hammer, which was kept vibrating by means of an electro-magnetic arrangement. The employment of this mode of stimulation would there- fore eliminate all that uncertainty—arising from the possible escape of current—which is inseparable from the use of electrical stimulus. Though this method must be regarded as one of great value, yet it is impossible to say how far the excitability of a given point in a structure so delicate as nerve will remain unmodified under the repeated action of such blows. In any case, it appeared desirable to inquire whether there was no other non-electrical form of stimulus that could be rendered practicable.

Besides the mechanical, the only remaining non-electrical forms of stimulus are the chemical and the thermal. Of these, the former is obviously incapable either of repetition or of being rendered quantitative. As regards the latter, I~have already shown its practicability for experiments on excitatory phenomena in vegetable tissues. Thus a single loop of platinum wire may be made closely to surround the experi- mental tissue. A definite current sent through the platinum loop for a given length of time will now subject the encircled area to a sudden thermal variation, which acts as a stimulus. Successive closures of the circuit for a definite length of time are ensured by means of a key actuated by a metro- nome. The intensity of stimulus may be graduated in a pre- determined manner by the adjustment of the heating-current. Excitation may then be caused either by one or by a summated series of thermal shocks.

I was now desirous of determining whether this form of stimulation would prove advantageous to experiments on the nerve, and in the course of the investigation I found it to be extremely convenient and appropriate. With good speci- mens of nerve I have been able, using thermal stimulus, to obtain long-sustained records of perfectly regular responses. As regards its pliability and facility of application this form of stimulus is quite unique. How many difficult problems -are made possible of attack by its means will be realised in the course of the two following chapters, where the responsive variations of different conducting tissues under changing conditions are subjected to investigation.

In order to obtain the electrical responses of animal nerve—that of frog, for example—the distal contact is killed and appropriate electrical connections made with the galvano- meter. The heating current is then adjusted for the desired amount of excitation.: The thermal variation, it must be remembered, should not be so great as to injure the tissue in any way. The platinum loop is not in this case in contact with the specimen, and this is the mode generally employed. Should a more intense stimulation be desired, however, the nerve may be allowed to rest on the platinum loop. In such a case care must be taken to see that the rise of temperature is not so great as in any way to injure the

tissue. The nerve, as usual, must be enclosed in a moist chamber, a convenient form of which, as employed i in 1 practice, will be seen in fig. 291. | I shall next give a few records in illustration of the ease and efficiency with which this mode of stimulus may be applied. These records will show the characteristic varia- tions of response given by the nerve under different con- ditions. When making records of electrical responses with frog’s nerve, under electrical stimulus, Dr. Waller obtained responses of three different types. The first of these was the normal, and consisted of negative responses ; the second was diphasic ; and the third was the abnormal positive. This last he regarded as characteristic of stale nerve.

These normal negative responses of the first of the three classes were found by him to undergo enhancement after a period of tetanisation ; while the third, that of the abnormal response of stale nerve, underwent a change into diphasic, or a reversal to normal, after tetanisation. From the fact that carbonic acid enhances the normal negative response of nerve, Dr. Walier has suggested that the enhancement of response in normal nerve after tetanisa- tion, and the tendency of the modified nerve to revert to the normal, are results of the hypothetical evolution ot carbonic acid in the nervous substance, due to metabolism accompany- ing excitatory reactions. It must be said, however, that no trace of the presence of carbonic acid has yet been detected in such cases. I shall be able to show, moreover, that these effects are in no way due to the evolutions of carbonic acid, but take place in consequence of molecular changes induced in the responding tissue, which find concomitant expression in changes of conductivity and excitability.

I shall now give records of responses of these various types obtained under the action of thermal stimulus. In order to exhibit the effect of tetanisation I give, in fig. 275, a series of normal responses by induced _galvanometric negativity, given by nerve of frog in its normal excitatory condition. ‘This nerve was then subjected to tetanic thermal shocks, after which its responses to individual stimuli of the former intensity were recorded once more. The subsequent responses show, as is seen in the record, an enhancement of amplitude. . The next series of responses, in fig. 276, exhibits abnormal galvanometric positivity. It may be mentioned here that ; these abnormal responses are not, as supposed by Dr. Waller, exclusively character- istic of the stale condition of the nerve. For employing other and more delicate methods of record I have found even fresh nerves, under certain conditions, to exhibit this effect. Neither is this positive response due in general to any chemical degradation. Instead of this, as we shall see in the present and succeeding chapters, it may be attributed to the run-down of the latent energy of the specimen, a process

of Response, as After-effect of which becomes accelerated in Thermal Tetanisation, in Frog’s . ; Notice , 5° isolation. When such a de- The first three responses are normal. pressed specimen is supplied Brief thermal tetanisation is here again with the requisite applied, and the responses subse- quently obtained under original energy, it becomes normally, stimulation are seen to be en- added: or even supernormally, ex- citable. The first part of the following record (fig. 276) gives a series of abnormal positive S §: #/9) § p responses obtained from a specimen of frog’s nerve, which was in a somewhat sub-tonic condition. After the appli- cation of tetanic thermal shocks it will be noticed that the responses in the second part of the figure have become

Between these two extremes of normal negative and abnormal positive responses there lies the intermediate diphasic. All these—positive, diphasic, and negative—may be exhibited in the same specimen, in the course of a sus- tained record of responses to single stimuli, without tetani- sation. This fact is illustrated in fig. 277, where the first series shows the unmixed abnormal positive. then passes by a gradual transition into diphasic— positive followed by negative —and this phase, lastly, is succeeded by a series of purely negative responses.

We come next to the ex- planation of these phenomena. We have seen that on account of isolation the tonic condition of a highly excitable tissue will undergo a graduai decline. On account of this its ex- citability and conductivity will fall below par. We have also seen that in this de- pressed condition the normal response by negativity tends to be reversed to positivity. With regard to the con- duction of excitation it may Positive into Normal Negative Re- sponse after Thermal Tetanisation

to left into normal’ negative on right, after intervening tetanisa- tion. be said that this condition of depression will lower the power of the tissue to conduct true excitation. Thus a stimulus of given intensity, capable under normal conditions of transmission to a certain distance, will, when the tissue is thus depressed, fail of conduction to the same distance. It will now, therefore, be the hydro-positive effect of stimulus which will make its appearance alone at the distant responding point. And the electrical expression of this will be galvanometric positivity.