Comparative Electro-Physiology: A Physico-Physiological Study

intervening period of tetanisation will markedly enhance the negative response. We have now seen that, by the direct mode of investiga- tion afforded in mechanical response, we are able to trace out the causes which determine the three types of response found in nerves. It has been shown that all ‘these are brought about by the varying tonic condition of the tissue. From this it is easy to understand that the three types of | electromotive responses in nerve are also due to the same cause. In the experimental method there employed, the variations of con- ductivity appropriate to the tonic condition are superposed on parallel modifications of the excitability. Thus not only is the responsive change of a sub-tonic responding point positive, but the effect which is transmitted to it through sub-tonic conducting tissues is also positive; after tetanisation, how- ever, the tonic condition of the tissue is raised. The power of transmitting true excitation, previously in abeyance, is now not only restored, but gradually

case Effect in Me- The excitability also undergoes a similar chanical Response of : positive to normal negative, which latter again becomes enhanced to a degree that depends, within limits, on previous excitation. These effects, seen in electrical response to transmitted stimulation applied at a distance, I find repeated also in the mechanical response of nerve, under similar circumstances. That is to say, an isolated nerve, by the very fact of its being cut off from its normal sources of energy in the body of the intact animal, is apt to be rendered sub-tonic, and under these conditions no true excitation is transmitted, and it is only when the tonic

condition of the tissue has been raised, by the application of fresh energising stimulus, that the conducting power can be gradually restored. 2 This leads me to what is theoretically a very interesting mode of determining the velocity of transmission in nerve, by the mechanical response of the nerve itself, which will be understood from the diagram already given (fig. 315). In that figure, A B C is the nerve, so clamped at B as to prevent any mechanical slip, but not tightly enough to obstruct the transmission of excitation. The nerve, when brought to a normal excitatory condition, is first excited at A a by a pair of electrodes in connection with an induction coil. The transmitted excitation, reaching B C, induces a contractile mechanical response there, observed by the highly. magnify- ing optic lever. Records of the transmitted effect of stimulus obtained in this manner. will be given later in the chapter. The interval of time, 7, between the application of stimulus and the initiation of response is accurately determined by the usual methods. Stimulus is next applied at B 4, and the interval of time 7’ between stimulus and response again determined. The difference (¢—7’) is the time required for the stimulus to travel the intervening distance a 4. By this means, I found the velocity of transmission in a certain specimen of nerve of fern to be 50 mm. per second.

It is thus easy, by means of two successive experiments, to eliminate from the observation the element of the latent period. It is to be understood that the molecular change, ultimately to be expressed as contraction, begins to be initiated as soon as excitation reaches the responding area. As the contractile effect exhibited by the nerve is relatively small, we can see that a certain time will elapse before it becomes sufficient to be perceptible, unless the magnification employed is very high. With a magnification of the order of 100,000 times, however—which, as I shall show, is quite practicable—this loss of time is much lessened.

It was while working out this investigation that I realised how indefinite must be any determination of the velocity of transmission in an isolated nerve. The conductivity, even in the intact organism, we have seen to be liable to modifica- tion from various factors such as fatigue, and it is easy to understand that it will become still more fluctuating when the conducting tissue is isolated. The inevitable changes consequent on separation from the natural sources: of energy at once begin totake place. As the result of this sub-tonicity, even a typically conducting tissue, like nerve, will cease to. be the conductor of true excitation, and there will then be, properly speaking, no physiological distinction between such a structure and a non-conducting tissue. By the absorption of stimulus, however, a transformation sets in, and the non- conducting becomes gradually reconverted, first, into a feebly, and then into a very highly conducting structure. The possible variations in conductivity, therefore, are not a matter of some few units per cent. quantitatively, but even considered qualitatively range from non-conductivity to the highest conductivity. And even, further, when the nerve has been once more rendered conducting, its velocity of transmission will vary greatly with the tonic condition conferred by previous stimulation. Over-stimulation, again, by inducing fatigue, diminishes the power of conduction of true excitation. This fact I shall be able to demonstrate by special experiments.

That such changes are not peculiar to the isolated nerve, where the manifestation can be traced unmistakably to its true cause, is seen in those cases of living animals where, owing to mal-nutrition, or for other reasons, the tonic condition of the nerve falls below par, with growing non- conductivity and paralysis as the effect. And here it may be said that the transformation again from non-conducting or feebly-conducting to the normal state of conductivity may in general be brought about by the same means as are employed with the isolated nerve, namely, by the frequent repetition of tetanising electric shocks.

The photographic method of recording the response of nerve, employed in the Kunchangraph, has the advantage that, as the record is made by the moving spot of light, the recording-point, as it were, encounters no friction, and the characteristic form of the response curve is thus unmodified. But prolonged work in the photographic dark-room is very fatiguing to the observer. I was, therefore, desirous of so perfecting the ordinary mode of record by the movement of the tracing-point of a lever over a smoked surface, that it would be adequate for most purposes. The difficulties involved in carrying this out lie, first, in the obtaining of a sufficiently high magnification, and, second, in the overcoming of friction at the writing point. A long lever, such as is necessary for high magnification, entails a heavy weight. But this can be obviated by employing a light and thin aluminium wire, 50 cm. in length. The fulcrum-rod, to which the lever-index is attached, has a diameter of 2 mm. A thread attached to the contracting nerve is wound once round this fulcrum-rod. The radius of the latter being 1 mm., the magnification produced by this arrangement is 500 times. The magnification may in this manner be raised as high as 1,000, by taking a longer lever. For the tracing point the end of the lever is bent at right angles, and a fine bristle attached. Even this degree of magnification is not always necessary, as I have already said. The records which immediately follow have a magnification of only fifty times. The next difficulty, as already stated, lies in the friction to be overcome. The friction offered by a writing-surface of smoked paper is too great to be employed. A surface of plate-glass, coated with a thin and uniform layer of smoke, offers considerably less resistance. But even this retards the free movement of the tracing-point. I was therefore led to the construction of my Oscillating Recorder.

The glass plate, on which the record is made, is carried on a primary frame, which is moved at a uniform rate, regulated by clock- work, on wheels, over rails. The plate is mounted on this primary frame in a secondary frame, which is held away from the primary, at a certain fixed distance, by means of spiral springs. This secondary frame, by means of an electro- magnetic arrangement, can be maintained in a state of to- and-fro oscillation, always strictly parallel to the primary. The recording-index moves in a vertical plane, and the smoked plate backwards and forwards, at right angles to this, the extent of its oscillation being about 1 mm. The recording point is adjusted, barely to touch the smoked » surface. Thus the oscillation of the plate brings it periodi- cally in contact with the tracing-point, which is thus practi- cally free to execute its movements unimpeded. When the oscillation frequency of the plate is sufficiently high, and the speed of the recording-surface low, the curve of record

Fic. 323. Pho ographic Reproduction of Record of Mechanical Re- sponses of Frog’s Nerve (left-hand record) and Plant-nerve (right-hand record) obtained on Smoked Glass Surface of Oscillating Recorder appears as continuous. In other experiments, where the determination of time-relations is important, a high speed can be given to the plate by the regulation of the clockwork, and the record will then appear as a succession of dots. From these, and a knowledge of the oscillation-frequency of the plate, the time-relations of different parts of the curve can be determined with accuracy. I give here two different series of uniform mechanical responses recorded with this instrument, obtained from the nerves of frog and of fern respectively (fig. 323).

I have also been able, by means of this instrument, to demonstrate a very important fact, namely, that the responses of the afferent or sensory nerves are in every way the same as those of the efferent, or motor. The numerous records already given are of the latter. For the demonstration of the former I took the optical nerve of Ophzocephalus, and recorded its responses to uniform electrical stimuli, on a smoked surface. The following (fig. 324) is a photographic reproduction of the record. Owing to sub-tonicity, the first response is seen here to be abnormal positive. Successive stiniulation converts this, through diphasic, into normal negative, in a manner exactly the same as has already been observed in the sciatic nerve of frog. Another interesting record obtained with the optical 3 nerve is given later (fig. 404).

I also give in: the next figure (fig. 325) a series of effects of transmitted. stimulation, which show.in avery interesting manner the effect of fatigue in the modi- fication of the conductivity of a nerve. Itis customary to suppose that the nerve is indefatigable. Responses to Electrical Stimu- But I shall be able to show that lus obtained on Smoked Glass, : gs ds and given by the Optic Nerve not only is the conductivity of a of Fish Ophiocephalus

nerve liable to fatigue, but its Note the abnormal positive re- _excitability also. The demon- natch a ree ed stration of the latter will be given in a succeeding chapter. For the demonstration of the effect of fatigue on conductivity I selected a length of 10 cm. from the sciatic nerve of gecko. This was attached for experiment to the Kunchangraph, in the manner diagrammatically represented in fig. 315. The length Bc, which showed contraction, in response to stimulus trans- mitted from A, measured 5 cm. The two exciting elec- trodes, A a, were 2 cm. apart. The intervening tract, through which excitation was transmitted, was, therefore, 3 cm. At the beginning of the experiment, owing to the depression of tone which the nerve had undergone, from

isolation, its conductivity was below par, and the responses obtained were positive. After a series of stimulations, however, the true excitatory wave was transmitted, with the concomitant negative or contractile responses. In order to demonstrate the effect of fatigue on conductivity, the recording of this series was commenced only after many normal responses had already been given. In the series recorded we can see that the responses at first exhibit periodic fatigue. The accentuation of fatigue is then mani- fested by a rapid decline in the amplitude of the responses. A remarkable change next begins to appear. It has been

Fic. 325. Record, obtained on Smoked Glass, ot Transmitted Effect of Stimulation on Nerve of Gecko Note here the progressive effect of fatigue, seen first as periodic fatigue ; second as diphasic effect ; and third as reversal into abnormal positive. shown that the true excitatory negative response contains a masked positive element. Owing now to growing fatigue, the exhibition of the negative is delayed, and the positive thus shows itself as a preliminary down-curve in a diphasic response. Afterwards, the excitatory negative is completely abolished, and the positive response by expansion alone remains, as seen in the last of the series. Ultimately, when the nerve is killed, by excessive stimulation, even the positive response disappears.

We may notice here the interesting fact that nerve, which is regarded as a conductor, par excellence, will sometimes become a non-conductor. Conduction, therefore, is not alone dependent on anatomical structure, but requires also a certain molecular condition. A nerve, whose continuity remains uninterrupted, may nevertheless undergo paralysis and cease to conduct. Recovery may then, in many in- stances, be brought about by tetanisation. Thus, by means of mechanical response, obtained with a magnification of only fifty times, we have been able to demonstrate not only those results which may be observed by the most sensitive galvanometer, but also others which were never so detected. The magnification thus employed in the Kunchangraph, however, is here, as already stated, only in its lowest terms. When this is further exalted, still further and important phenomena regarding the exci-

tatory changes in nerve are revealed, and some of these will be described in the next chapter. Great sensitiveness of the high magnification Kunchangraph—Individual con- tractile twitches shown in tetanisation of nerve—Sudden enhancement of mechanical response of nerve on cessation of tetanisation—Secondary excita- tion—Multiple mechanical excitation of nerve by single strong stimulation — Multiple mechanical excitation of nerve by drying. WE have already seen that, in order to detect the excitatory _ changes in nerve by the electrical method, the moderate sensitiveness of an ordinary galvanometer has to be exalted more than a million times. Galvanometric indications, more- over, are liable, as we have seen, to be complicated by the occurrence of differential effects at the two contacts. In - the Kunchangraphic method of record, however, there is no possibility of such complications, for the response curve here represents the direct effect of stimulus. We also saw that, according to this method, a very moderate magnification would give us all the variations that could be detected by the most sensitive galvanometer, and, besides this, owing to its simplicity, it makes it possible to observe other phenomena, whose occurrence the galvanometer could not satisfactorily have demonstrated.

Such a magnification, however, as I have already said, is in its first stage only. With due precautions it is possible to obtain a Kunchangraphic magnification of a hundred thousand times. It will easily be seen that this places at our disposal an instrument of incomparable sensibility, by whose aid many of the phenomena of the nervous change, hitherto beyond our power of observation, may be brought within the sphere of investigation. This magnification, of the order of a hundred thousand times, may be accomplished in either of two different ways. A magnified image of the end of the long lever may, in the first place, be thrown on a distant screen. By this compound magnification the sensitiveness of the record may be raised to the extent desired. Or, in the second place, we may employ a battery of two levers in series. The first of these gives a magnification, say, of five hundred times, and is connected with a second optical lever, by which a multiplying magnification of two hundred times is easily obtained. It is unnecessary to point out that special care should in this second case be taken to ensure the steadiness of the support. With due precautions it is, however, not difficult to secure the entire elimination of all disturbing elements.

When the spot of light from the second lever is thus thrown on a distant screen, it is very interesting to watch the various changes induced in the nerve by the environmental conditions. An isolated nerve in a moist chamber, cut off from its natural sources of energy, becomes increasingly sub-tonic. This process is attended by an abnormal relaxa- tion, which causes a steady movement of the spot of light in one direction. When the nerve has become very sub- tonic, the effect of stimulus, as that of electric shocks, is to enhance the tonic condition, and by this the downward drift of the spot of light is retarded or arrested. In cases of extreme sub-tonicity there is no further response, beyond this arrest. But where the sub-tonicity is less pronounced, stimulus will induce the abnormal positive response by a sudden positive variation of the drift, which is followed by recovery in the opposite direction. The after-effect of absorption of stimulus is further effective in causing the gradual retardation and final arrest of the downward drift. By such absorption of stimulus the tonic

' The abnormal positive response is also obtained from ‘nerve in ordinary tonic condition, it should be remembered, by the application of excessively feeble stimulus. condition is raised, and the normal excitability consequently enhanced. From this point onwards the responses are con- tractile or normal negative. At this stage the response of the nerve exhibits the staircase increase, the nerve itself showing a certain amount of tonic contraction. The excitability of the nerve then attains a maximum, and the successive responses become uniform. Long and intense stimulation will, after this, bring on fatigue. This stage is characterised, again, by a growing relaxation of the nerve as a whole, and its responses may become, first, diminished in amplitude, second, of a diphasic character, and, thirdly, reversed to the abnormal positive, according to the amount of fatigue which supervenes. We may thus, for the sake of convenience, distinguish four stages in the response of nerve. The first of these is the initial phase, SUB-TONIC RELAXATION, and the characteristic response to individual stimuli is here abnormal positive. The second phase is that of the TRANSITION to normal response. The characteristic responses to individual stimuli here show a staircase increase, with more or less permanent contraction as its after-effect. If at this stage the nerve is allowed to remain long without stimulation, it slowly reverts to the first stage of sub-tonic relaxation, with its growing relaxa- tion and abnormal positive response. Stimulation, how- ever, brings it back once more to the second or transition stage. In the third stage of UNIFORM responsiveness, the responses are normal and take place by equal contraction. In the fourth, or FATIGUE stage, there is a tendency, as already said, to relaxation on the part of the nerve as a_ whole, and it thus outwardly mimics the stage of sub-tonicity. The responses now, therefore, diminish in amplitude, and show the diphasic or the abnormal positive character.

Further characteristics of these four stages, and_ their relations to each other, will be treated in detail in Chapter XLI. A few words may be said about the mechanical response of the nerve, when it is in a favourable condition of excitability. We have seen that in order to obtain a galvanometric record of the electrical response of nerve, one or even a few shocks will not be sufficient to induce the necessary electromotive change. For this, tetanic shocks of a certain duration are necessary, and the responsive electromotive change is not immediately perceptible. In consequence of this intensity of stimulation, moreover, complete electrical recovery can only take place after a perceptible interval. With the low magnifi- cation Kunchangraph, too, tetanic shocks of something like a second in duration are necessary, and complete recovery here also requires a period of about one minute. But with the greater sensitiveness available in the highly magni- fying apparatus, response with a highly excitable specimen of nerve is obtained with even so short-lived a stimu- lation as that of two or three vibrations of the vibrating interrupter of the secondary coil, lasting less than one-tenth of asecond. The responsive contraction of this short-lived stimulus, and its recovery, are also quick. It is in conse- quence of the rapidity of this response and recovery that the responsive contractions due to the rapidly intermittent tetanising shocks do not become fused, but show themselves in the response-curve as consisting of successive twitches, corresponding to the component shocks. Owing to the high amplitude of these responses, and the trend of the base-line either up or down, it is difficult in practice to obtain photo- graphic records of these effects. But it is easy enough to obtain definite visual demonstration of various characteristic effects in the response by the employment of the following device. The spot of light from the optic lever is made incident on a revolving mirror, and reflected from it to a large white screen at some distance.

During a period of repose the quiescent spot traces a more or less horizontal line of light. This may trend either in a downward or an upward direction continuously, according as there is induced a continuous sub-tonic relaxation or a growing contraction, due to the after-effect of stimulus. Somewhere between these two extremes may be obtained a condition of more or less stability, where the record made by the spot of light appears as a horizontal line. Under normal conditions, then, the response to excitation is a sudden movement, due to con- traction, say upwards, followed by recovery down. In the response-curve projected on the screen, the vertical movement or ordinate represents the amplitude of the re- sponse, and the horizontal abscissa the time. Under tetani- sation a series of curves corresponding in frequency to the | frequency of the shocks is observed as serrations.

Another very interesting observation often made in the mechanical response of nerve is that of the after-effect on the cessation of continuous stimulation ‘by tetanic shocks. It has been found, it will be remembered (p. 428), that the response of the retina to the action of continuous light often shows on its cessation a sudden transient increase. This phenomenon has been regarded as peculiar tothe retina. But I have found exactly parallel effects to occurin the mechanical response of nerve. Under continuous stimulation there is a tendency to the attainment of a maximum contraction, which suddenly, on the cessation of stimulation, overshoots, to be followed by the usual recovery. I have already referred to the two different effects of an opposite character caused by incident stimulus, namely, the effect of negativity, and its converse positivity. In the case of mechanical response, it is the former which is effective in inducing contraction, while the latter is associated with expansion, and is a factor in re- covery. It will also be seen, in a general way, that by the - antagonistic action of these two elements, and by the differ- ing relative intensities of their after-effects, many diverse results may be exhibited. In the case of the after-effect in question, which occurs by a sudden positive variation of the contraction, the excitatory effect would appear to be pre- dominant. Even when, after this brief positive variation, recovery is taking place, the excitatory element, with its contractile tendency, appears to persist; for if a second stimulus be applied, some time before the recovery is com- plete, the consequent contractile response takes place almost

instantaneously. But when the recovery is once complete, a similar stimulation will not induce a similar immediate response. Instead of this there will be a brief period of hesitation or latency before its initiation. Another interesting phenomenon, which I was first able to observe by the help of the highly magnifying Kunchan- graph, was the occurrence of multiple response in nerve under intense stimulation. I was led to this discovery by an investigation which I had undertaken for the demon- stration of the identity of response in animal and vegetable nerves. After showing the extended parallelism which exists between the two, under similar conditions and varia- tions of conditions, as already described, I was desirous of seeing whether a plant nerve could be substituted in certain experiments for the animal nerve. In accordance with this I used the vegetal nerve in the experiment known as secondary contraction. Here a nerve-and-muscle preparation of frog is taken, and a second piece of frog’s nerve is suitably laid, with one end lying upon the end of the other nerve. On excitation of this second detached nerve, say by electric shocks, excitatory electrical variation is found to cause responsive contraction in the muscle of the first preparation. In my own rendering of this experiment I employed, instead of the second piece of: frog’s nerve, a length of nerve of fern. In order that the experiment should not be open to any objection arising from the escape of stimulating current, I employed a non-electrical form of stimulus. This was done by touching the plant nerve with a strongly heated wire. The terminal muscle would then, under favourable conditions, begin to respond by strong spasmodic contraction. When this had subsided, a new series of tetanic contractions began ; and this was repeated at short intervals, for nearly half an hour. When this series of spasms had come to a stop, I have often succeeded by a fresh application of the hot wire to the vegetal nerve in obtaining a second series of such repeated responses.

It thus appeared that the strongly excited plant nerve gave rise to a series of multiple excitations, the indications of which were afforded by the nerve-and-muscle preparation. The only perplexing feature of these responses was the abnormally long period of ten to fifteen seconds which was generally found to elapse between the application of the strong stimulus to the plant nerve, and the response subse- quently given by the terminal muscle. Here it must be remembered that the excitation applied at one end of the plant nerve has to travel the entire length before its excita- tory electrical variation can be communicated to the nerve of the frog-preparation. The transmitted excitatory varia- tion in the primary has, moreover, to reach a certain intensity before it can effectively excite the secondary preparation. We know, further, that an isolated piece of nerve is liable to fall into a sub-tonic or depressed condition, in which its conducting power is much lowered, to be gradually restored again under strong or long-continued stimulation. These considerations will probably be found to account for the delay in the occur- rence of the first of these responses. It would thus appear from the last experiment that a nerve, when subjected to a single strong stimulus, will give a multiple series of responses. In order to test this by direct experiment I employed the highly magnifying Kunchangraph, and subjected an experimental nerve of frog to a single strong thermal stimulation. This gave rise, at first, either to an abnormal positive response or to a moderate negative. But there followed, after a longer or shorter pause, a series of multiple contractile responses, which generally grew in intensity for a considerable time. There were in the series a number of short pauses, each followed by a veritable storm of excitation, in which individual responses were so rapid that the up or down movement of the spot of light appeared as brief flashes, in which all distinctness was obliterated.

This experiment conclusively shows that the nerve, like certain other tissues, is susceptible of multiple excitation. If the nerve in a nerve-and-muscle preparation be allowed to dry, the muscle is seen to be thrown into a series verify by experiment. As the individual responses in these multiple series were of fairly large amplitude, I ex- pected to be able to obtain a record of such a series by an ordinary magnification on smoked glass. In order to* obtain this record under normal conditions a stream’ of air, bubbling through water, was passed through the chamber at a uniform rate. Owing to the run- down of the latent energy in the nerve, we are able to observe a_ consequent growing relaxation. By the manipulation of a stop-cock the air is passed through a calcium chloride tube, in- stead of a vessel containing water. In this way the nerve is quickly subjected to dry air instead of moist vapour. This substitution is repre- sented in the record by an upward arrow 7, and it will be noticed how at this point

The nerve, owing to growing sub- tonicity, was showing a growing relaxation, as seen in the first part of the record. Air passed through CaCl, tube, and, thus dried, was now passed through nerve-chamber at point marked with upward arrow *. This gave rise to a large con- tractile response, followed by sub- sequent multiple responses. Original record on smoked glass here reduced photographically to 4. contractile movement is followed by a long-continued series of multiple responses, here seen to fall into a somewhat

Variation of resistance in Dionea, by ‘ modification’—Excitatory change, its various independent expressions—Characteristic difficulties of investigation— Morographic record by variation of resistivity—Inversion of curves at death- point—Similarities between mechanical, electro-motive and resistivity curves of death—The true excitatory effect attended by diminution of resistance — Response of plant nerve by resistivity variation—Independence of resistivity and mechanical variations—Responsive resistivity variation in frog’s nerve, and its modification under aneesthetics.

after ‘modification’ exhibited a diminished electrical resist- ance; and this ‘modification’ he found to be most easily induced after the passage of an electrical current through the tissue. Subsequent observers have also noticed a diminu- tion of resistance in many cases when a tissue has been subjected to electric shocks. These diminutions of resistance are observed as more or less permanent after-effects. The experiments in these cases depend on obtaining the galvano- meter deflections caused by a small E.M.F., before and after the modification. The larger deflection due to the same E.M.F. after modification shows that the resistance of the tissue has undergone a diminution.

This method, however, is open to several objections. The passage of constant or induction currents through the tissue would not only give rise to polarisation effects, but would also induce an unknown electromotive variation at the two contacts on the surfaces of the tissue, to an extent depending on their differential excitability. The observed deflection by a small testing E.M.F. is thus affected, not only by .the variation of resistance, but also by varying polarisation and. excitatory electromotive effects.

The question still remains, What is the nature and significance of this induced variation of resistance? As the effects which have been referred to are generally seen to be induced after excitation, and to constitute its after-effect, does it follow that the diminution of resistance takes place as a remote consequence of other excitatory changes? Or is it but a different manifestation of that fundamental molecular change, induced by excitation, of which the