Downing and I(1) found that the duration of the absolute refractory

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1 6I2.8I6:6I2.59 THE EFFECT OF TEMPERATURE UPON THE ABSOLUTE REFRACTORY PERIOD IN NERVE. BY WILLIAM R. AMBERSON'. (From the Department of Physiology and Biochemistry, University College, London, and the Department of Physiology, School of Medicine, University of Pennsylvania, Philadelphia.) IN the course of a study of the electric response of nerve to two stimuli, Downing and I(1) found that the duration of the absolute refractory period in nerve can be determined with good accuracy by the observation of the stimulus interval at which an increase occurs in the maximal deflection of a sensitive Downing moving-magnet galvanometer, over and above that previously given when the two stimuli were sent in at shorter intervals. The appearance of a second action potential wave is indicated by this increase in electric response, which comes in suddenly as the stimulus interval is gradually increased. The technique used for this determination is fully described in our paper. As an extension of this study I became interested in the effect of temperature upon the duration of the absolute refractory period, concerning which relationship the literature yielded rather meagre and incomplete information. I therefore carried out a group of determinations on the sciatic nerves of large R. esculenta, between 90 and 270 C., with results which were so regular and consistent that it seems wise to put them on record. The data here submitted are in good agreement with values published by a number of other workers, where comparison is possible; their chief advantage lies in the fact that they cover a wider temperature range than has previously been studied by any one investigator, and are more consistent than any to be found in the older literature. Previous measurements of the duration of the absolute refractory period in nerve have followed either the method of Helmholtz(2), in which determination is made of the least interval between two stimuli applied to the nerve which just produces a summated contraction in muscle, or the method of Got c h and B u r c h (3) which measures the least interval required for the appearance of a second electrical response in 1 Guggenheim Memorial Fellow.

$The values secured represent, at all temperatures, the duration of the absolute refractory period in the fibres which recover most quickly.$

2 TEMPERATURE AND REFRACTORY PERIOD OF NERVE. 61 nerve. The technique used in the present work is an application of this second method. The values secured represent, at all temperatures, the duration of the absolute refractory period in the fibres which recover most quickly. The weight of recent evidence appears to indicate that all medullated fibres have the same absolute refractory period (4,5); if this is true the values here given must hold for all such fibres. Although many determinations of the duration of the absolute and relative refractory period in nerve have been made either by the muscular summation method(6,7,s,9,l0,11,12,13,14,15), or by the method of second electrical response (11,16,17,18,19,20,21,4,5), there have been few systematic studies of the effect of temperature upon duration. Using the muscular summation method, B a z ett(7) applied the stimuli directly to the muscle, but believed that, in non-curarized preparations, the refractory period measured was that of the intramuscular nerves; he secured readings at temperatures ranging from 10 to 150 C. Adrian(9,ii), by the same method, but stimulating the nerve directly, concerned himself mainly with the effect of temperature upon the form of the whole recovery curve and in most cases did not directly determine the duration of the absolute refractory period. By the method of second electrical response no systematic study of this relationship has been made since the work of Gotchl6) who reported values between 30 and 120 C. only. Other investigators have worked at higher temperatures but have not been directly interested in the effect of temperature itself, and have often failed to specify at what temperature their work was done. Working at low temperatures Lucas (18) reported that even in fresh preparations, the least interval between stimuli which just gives a summated muscular contraction is definitely longer (20-25 p.c.) than the least interval required for the appearance of a second electrical response in nerve, indicating that a second action potential wave, subnormal in size, may be present in the nerve before it is able to affect the muscle. Adrian(ll) finds that this difference is not always present. Kato, Hayashi and Takeuchi(15) submit evidence that this disparity in time does not exist above 150 C., but their argument is somewhat indirect. On the whole it appears that direct measurements of the electric response in the nerve must give the more trustworthy values for refractory period. It must, however, be recognized that even this method fails to give absolute values for several reasons. Forbes, RRay and Griffith(19) have pointed out that, if the second stimulus be made sufficiently strong, the absolute refractory period, in the classical sense, may be reduced to zero. Such stimuli, while entering the nerve in the period which Lucas(22) once

$In such a case the stimulus interval is not a true measure of the "irresponsive" period, or response interval, which they suggest as a new definition of absolute refractory phase.$

3 62 W. R. AMBERSON. proposed to designate as "irresponsive" may endure for such a time, or may produce local effects of such a magnitude that the nerve is stimulated after an interval. In such a case the stimulus interval is not a true measure of the "irresponsive" period, or response interval, which they suggest as a new definition of absolute refractory phase. Even this definition must be qualified, however, when the response interval is determined from string galvanometer or oscillograph records, and measured at some distance from the site of stimulation, since it is known that the second action potential wave suffers a retardation in speed of conduction when travelling in the relative refractory phase of the first (16,19,23). The response interval therefore appears to be longer than when measured directly under the stimulating electrodes. Measurements of the latter sort have so far been made only through the use of the cathode ray oscillograph(21); it would appear that great accuracy in such readings is not possible (4). The values here reported approximate the absolute refractory period, in the classical sense, but must depart from the true value of the "irresponsive" period, as discussed above, by an error determined by the strength and duration of the second stimulus, and by these alone. The retardation in conduction time between the stimulating and lead-off electrodes does not introduce any error in the method here used, since, for the short intervals involved, the galvanometer deflection approximates to a ballistic summation of both action potential waves. The response interval is not, and cannot be, measured. Concerning the magnitude of the error introduced by the second stimulus it is possible to make an approximate estimate. Through the courtesy of Mr B. H. C. Mathews I have obtained oscillograph records of the form of the break shocks delivered by the coreless induction coils used in the present study. He obtained these records under conditions approximately similar to those of these experiments. The two coils give similar results. For the second stimulus the time to maximum is about 012a, with a total duration of about 028ar. It is not possible to know at what time, within this period, stimulation occurs, but the difference between the true value of the response interval and the known stimulus interval can hardly be more than the total duration of the second stimulus, and is probably considerably less. Since the magnitude of the error within these limits cannot be determined, and since the error in the determination of the stimulus interval itself may be as large as 0 la, I have made no attempt to apply a correction factor to the data. At least it can be stated that the values were secured by a uniform procedure at

4 TEMPERATURE AND REFRACTORY PERIOD OF NERVE. 63 all temperatures, and the presence of a systematic error should operate to shift the whole curve slightly along the time axis, without materially altering its form. Throughout these determinations the first stimulus was set to be somewhat supermaximal; the second stimulus was then made ten times as strong. Under those conditions a least interval is obtained which is not further shortened by a considerable increase in the magnitude of the second stimulus (15); this constant value may be considered as the closest approximation to the true value of the absolute refractory period which can be obtained by this method. The reduction of that period to zero, as described by Forbes, Ray and Griffith(19), is observed only when the second stimulus is made very much stronger, and local effects become very highly developed. In Fig. 1 are given the results of 34 determinations of the least interval, E a 3 4a Duration of absolute refractory period Fig. 1. Influence of temperature upon the duration of the absolute refractory period in the sciatic nerve of the frog. 34 determinations made on 13 nerves from eight animals. The values secured from each nerve are numbered, in theorderinwhich theexperiments were made.

5 64 W. R. AMBERSON, carried out on 13 nerves from eight animals (large R. esculenta). The values secured from each of the 13 nerves are indicated by the numerals enclosed within each circle; the nerves are numbered in the order in which the experiments were made. Of the eight animals six (nerves 1-10) had previously been kept in the ice-box for several months, at a temperature slightly above 00 0.; the other two (nerves 11-13) had previously been kept in an open tank on the roof, at summer temperatures. Since all of the values group together along one curve it appears that this difference in previous history has no effect upon the value of the absolute refractory period. Although the values are quite consistent there is sufficient variation to make any mathematical formulation of the results open to some question. As a first approximation a simple exponential curve seems to give a reasonably good fit. The curve drawn through the points in Fig. 1 is of this type1. If we accept it as giving a correct picture of the relationship the Q10 is 3-06 over the whole temperature range. This value is not far from 3-6, that found by Bazett(7), and lies very close to Adrian's figures of 2-9 (9), and 3.27 (11) for the time to 50 p.c. recovery. The exponential form of this relationship was recognized by Adrian. With this value for the Q10 a fall of temperature of 10 C. will lengthen the refractory period by 12 p.c. In any one nerve this change can easily be detected by the method here used. Gotch(16), using R. temporaria, worked at low temperatures, and gives no determination of refractory period above 120 C. The values here given are in good agreement with his over the range where comparison is possible. If we assume that the Q10 is constant down to 30 C., we may calculate values for the lower temperatures by extrapolation from the curve of Fig. 1. Such calculations give results not far different from what Gotch observed; considering the difference in material and method the coincidence is very good. The degree of coincidence can be seen in the following comparative values: The equation for this curve is: Calculated from Gotch Fig C. 7-5a 6-8a x=9 4(1.118)-', where x =refractory period in a, y =temperature in degrees Centigrade.

TEMPERATURE AND REFRACTORY PERIOD OF NERVE. 65 By the method of second electrical response Adrian (11) located the value between 2 and 2-5a at 130 C. Fig. 1 gives 2-2 for this temperature.

6 TEMPERATURE AND REFRACTORY PERIOD OF NERVE. 65 By the method of second electrical response Adrian (11) located the value between 2 and 2-5a at 130 C. Fig. 1 gives 2-2 for this temperature. With the method used in the present work it is difficult to secure satisfactory values above 250 C. and below 100 C. As the temperature rises the maximal deflection of the galvanometer is progressively reduced, until the error in reading renders accurate determinations impossible. On the other hand, the increase in maximal deflection which is taken to indicate the presence of a second action potential wave becomes less and. less abrupt as the temperature falls, until finally the least interval cannot be located with any certainty. Field and BrUcke(20) have recently reported that between 150 and 190 C. the absolute refractory period for the same material (sciatic nerve of R. esculenta), measured by another method, varies from 0-8 to 2-25a. Erlanger, Gasser and Bishop(4) have published values for R. catesbiana which vary from 0 73 to 1-77a. The temperatures are not given. In both cases the impression given by the values is one of considerable variability in the material. I am inclined to believe that this is not correct. In my experience the behaviour of the nerves is quite uniform, when the temperature is carefully controlled. In addition to the data here given, in which a special attempt was made to determine the value of the least interval accurately, I have secured many other determinations, on other nerves, which fall into good agreement with these. I therefore incline to the opinion that the variation observed by these authors probably arose mainly from temperature differences rather than from variations in the tissues. It might be stated, from an inspection of Fig. 1, that between 150 and 190 C. the value of the least interval varies between 1 and 2a. Such a description, while, strictly correct, gives a false impression of the data, since the experimental points fall closely along the mean curve over the whole range. Of the total variation approximately two-thirds is due to the changes in temperature themselves, and only one-third arises from variation in the material, and other experimental errors. Field and Briucke also indicate that the refractory period lengthens rather quickly after the preparation has been made and find that when, after an interval, the second nerve of the animal is prepared for study, the value obtained is longer. I have not observed this effect, but have, on the contrary, been impressed by the constancy of the value of the least interval over a considerable time, when temperature remains constant and the stimulation is relatively infrequent, and by single and double shocks only, as in all of the determinations here reported. I have PH. LXIX. 5)

7 66 W. R..AMBERSON. also found it possible to carry out a series of determinations at various temperatures, and then secure (as did Adrian(9) with the muscular summation method) a final reading at the initial temperature which is almost identical with that previoutsly obtained. SUMMARY. The influence of temperature upon the absolute refractory period of the sciatic nerve of R. esculenta has been studied. Over the temperature range examined (9-27 C.) the experimental values outline a curve which is approximately exponential in form. The Qlo is 3x06. REFERENCES. 1. Amberson and Downing. This Journ. 68. P Helmholtz. Monatsb. d. Berliner Akad. p Gotch and Burch. This Journ. 24. p Erlanger, Gasser and Bishop. Amer. J. Physiol. 81. p Heinbecker. Ibid. 89. p Boycott. This Journ. 24. p Bazett. Ibid. 36. p Adrian and Lucas. Ibid. 44. p Adrian. Ibid. 48. p Adrian. Ibid. 54. p Adrian. Ibid. 55. p Adrian and Olmsted. Ibid. 56. p Cooper. Ibid. 59. p Woronzow. Pfiuiger's Arch p Kato, Hayashi and Takeuchi. Amer. J. Physiol. 83. p Gotch. This Journ. 40. p Bramwell and Lucas. Ibid. 42. p Lucas. Ibid. 43. p Forbes, Ray and Griffith. Amer. J. Physiol. 66. p Field and Brucke. Pfluiger's Arch p Erlanger, Bishop and Gasser. Amer. J. Physiol. 78. p Lucas. This Journ. 39. p Gasser and Erlanger. Amer. J. Physiol. 73. p

University College, London.)

THE EFFECT OF FREQUENCY OF STIMULATION ON THE HEAT PRODUCTION OF NERVE. BY R..W. GERARD', A. V. HILL AND Y. ZOTTERMAN2. (From the Department of Physiology and Biochemistry, University College, London.)