BY W. W. DOUGLAS AND J. L. MALCOLM From the National Institute for Medical Research, Mill Hill, London, N.W. 7

Transcription

1 53 J. Physiol. (1955) I30, 53-7I THE EFFECT OF LOCALIZED COOLING ON CONDUCTION IN CAT NERVES BY W. W. DOUGLAS AND J. L. MALCOLM From the National Institute for Medical Research, Mill Hill, London, N.W. 7 (Received 9 February 1955) It is an old observation that cold blocks nervous conduction and that nerve fibres subserving different modalities differ in their susceptibilities. Commonly it is believed that susceptibility to cold block is related to the diameter of the individual nerve fibre. The evidence for this belief is, however, fragmentary and conflicting. Comparing C fibres with A fibres, Lundberg (1952) found that the former were most resistant to cold block, but in Sinclair & Hinshaw's (1951) experiments the fibres most resistant to cold were not C fibres but certain fibres of the A group. A similar divergence of results is also apparent in experiments to assess the differential susceptibilities of different sized fibres within the large (myelinated) fibre group itself. In some experiments the largest of these fibres have proved the most sensitive to cold block (Torrance & Whitteridge, 1947; Whitteridge, 1948), while in others (Lundberg, 1952; Dodt, 1953) they have proved least sensitive. The work reported here describes the effects of localized cooling on several nerves in the cat. METHODS Most experiments were made on cats under chloralose anaesthesia ( mg/kg intravenously after ethyl chloride and ether). Some cats were anaesthetized with Nembutal (40 mg/kg intraperitoneally, supplemented as necessary). Nerves and spinal roots prepared for study in situ were immersed in liquid paraffin held in a bath formed by suitable arrangement of the skin flaps. In some experiments this paraffin bath was warmed by an immersion heater. Sometimes it was saturated with 5% CO2 in 02. In other experiments the nerve studied was dissected free of the animal and lightly stretched across silver electrodes in a paraffin bath saturated with 5% CO2 in 02. The nerves were stimulated at one or more points by square pulses delivered through silver electrodes. Action potential records were taken through silver electrodes and displayed on a double-beam cathode-ray oscillograph. Cold was applied to a length of the nerve between the point of recording and the point of stimulation by the thermode mllustrated in Fig. 1. Several interchangeable heads were used with this thermode to allow lengths from 2 to 20 mm to be cooled, and to accommodate nerves of different diameters.

2 54 W. W. DOUGLAS AND J. L. MALCOLM of coolant Temperature recording Heating coil 12 V X r FC ~~with ^ <t ~~nerve Silver 5mm head Fig. 1. Diagram of the thermode. Refrigerated ethylene glycol was used as coolant. Temperature of the head was recorded by thermocouple. The heating coil was incorporated to permit rapid rewarming. RESULTS Effect of cold on afferent fibres The saphenous nerve was selected first for examination for the following reasons: it is an almost pure sensory nerve and any results are not confused by possible differences between sensory and motor fibres; it contains a wide range of fibres of different sizes; it is long and the action potential complexes are well separated; and it splits readily into small bundles well suited to cooling. The propagated response that followed a maximal stimulus to the saphenous nerve consisted of a rapidly conducted complex, with conduction velocities of m/sec, and a much more slowly conducted response with conduction velocities of 1-2 m/sec. The former was identifiable as the A (myelinated fibre) complex and the latter the C (unmyelinated fibre) complex, as described by Gasser (1941). On cooling a 6 mm length of the saphenous nerve, failure of conduction was first apparent in the A complex which became progressively smaller and finally disappeared. At the temperature causing the A complex to disappear, the C complex remained. It was somewhat slowed and dispersed

3 DIFFERENTIAL COLD BLOCK OF NERVES 55 but apparently little reduced. A considerable further lowering of temperature was required to block it (Fig. 2). This difference in susceptibility of the two groups was regularly observed in each of a number of preparations. The absolute values of temperature required to block the two groups varied somewhat from one preparation to another. In all the branches of the saphenous nerve that were examined, the A complex of the conducted action potential was characterized by two prominent and well-separated elevations. The more rapidly conducted of these was taken as representing the a component and the slower the 8 component. Between these, two other minor elevations were usually discernible and were considered 10 msec 100 msec Fig. 2. The differential blocking effect of cold on A and C complexes in the saphenous nerve: a, c and e, A complex; b, d andf, C complex. a and b at 370 C, c and d at 120 C, e andf at 105' C. b, d and f recorded at an amplification seven times that used for a, c and e. to represent fi and y elevations (Gasser, 1941). Within this A complex the blocking effect of cold was observed to be differential: but, in contrast to the differential effect observed on A and C complexes, the effect of cold was manifest first on the fibres of slower conduction rate. Thus the 8 component was appreciably diminished in several instances when the thermode temperature was 26 C, and was usually abolished at about 220 C, while at the time of its disappearance the a component was unaffected or very nearly so (Fig. 3). It did not prove possible in these experiments to discern with certainty whether this order of sensitivity to cold block regularly obtained for the other components of the A complex, since the 8 and y components were relatively small and not readily distinguished from the oa component, but in those records where fi and y components were particularly well represented, the y component was seen to be more susceptible to cold block than the,b, and the,b more susceptible than the a.

4 56 W. W. DOUGLAS AND J. L. MALCOLM In some experiments observations were made on the isolated saphenous nerve. The results obtained on cooling a segment of this preparation did not differ from those obtained on the nerve in situ. Since A fibres are peculiarly prone to repetitive firing (Skoglund, 1943), experiments were next conducted to demonstrate that the elevation taken to represent the 8 fibres was not an artifact. The experimental results which follow show that the 'A' elevation, and the other components 'f' and 'y', were independently conducted action potentials and not due to repetitive firing of faster conducting fibres D-5 Fig. 3. The differential blocking effect of cold on the components of the A complex: each of the six traces shows the A complex elicited with supramaximal stimulation. The figure beside each trace indicates the temperature (0 C) of the thermode at which the record was obtained. Time scale, 09 msec intervals. (a) Stimulation of the nerve at two different points showed that the separation of the several elevations varied directly with the distance between the stimulating points (Fig. 4). Calculation then showed that the '8' elevation was conducted at about 25 m/sec; that is to say, at a rate approximately four times slower than the a. (b) The a elevation of a second volley, initiated at suitable interval, summed completely with the 8 elevation of a preceding volley (Fig. 5). (c) When a stimulus strong enough to initiate a maximal a elevation alone was applied before a maximal stimulus at such a time that it caused the a elevation of the maximal stimulus to fall within its absolute refractory period, then it was found that the a elevation of the second stimulus was abolished, but that the 8 elevation remained unchanged (Fig. 6).

5 DIFFERENTIAL COLD BLOCK OF NERVES 57 The length of nerve cooled as afactor influencing nerve block. It is known that propagation of nerve impulses in myelinated fibres is not a continuous process but is effected at each node by the summing of electrical currents induced by the depolarization of the several antecedent nodes. Furthermore, it has been Fig. 4. Fig. 5. Fig. 6. Fig. 4. The saphenous nerve A complex recorded 42 mm (upper record) and 92 mm (lower record) from the point of stimulation. Fig. 5. Summation of the 8 component of one A complex with the ac component of a subsequent complex. (a) Two A complexes set up by stimulation of the saphenous nerve at two points; (b) the effect of shortening the interval between the two stimuli; (c) the effect of further shortening of the stimulus interval, showing summation of first S with second ac. Time, msec. Fig. 6. The effect of evoking a maximal at response (a) at diminishing intervals (records 1-4) before a maximal A complex (b). The a component of the latter is abolished (record 4) without the S component being affected. Time, msec. established that even in the absence of normal electrical responsiveness in one or more nodes, the extrinsic current flow may suffice to activate the nodes beyond the unresponsive region, and hence re-establish the propagated activity (Tasaki, 1939). In designing the thermode, this ability of the nerve

6 58 W. W. DOUGLAS AND J. L. MALCOLM impulse to 'hurdle' a certain length of inactive nerve was borne in mind, but at the same time, the dimensions of the cooling head were kept small to minimize the length of nerve which had to be freed for cooling. Consideration of these requirements led to a 6 mm channel being used. Assuming correct the data by Tasaki (1939) and Hodler, Stimpfli & Tasaki (1952), this length would seem adequate to prevent abnormal hurdling, for they found that block of 2-3 nodes, that is to say 2-3 mm in the largest fibres, was sufficient to block transmission. Nevertheless, since the larger fibres, in virtue of their greater internodal distances, are those best suited to allow hurdling of a length of block, and since the most persistent component of the A complex on cooling was found to be that conducted by the largest fibres, the question arose whether the persistence of the large fibre component was due to saltation, and, indeed, whether the differential effect of cooling within the A group was not so much the result of differences in fibre response to cold, but rather an expression of the different hurdling abilities in the different fibres. This question was settled by cooling a 20 mm length of the saphenous nerve, where any possibility of 'hurdling' of the conducted response could be excluded. When this longer length of nerve was cooled, the results obtained were qualitatively similar to those obtained when a 6 mm length was cooled. Progressive cooling caused first the A complex to disappear and then, at a substantially lower temperature, the C complex. Within the A complex the blocking action was again differential, and again the 8 fibres were most susceptible and the a fibres least susceptible. The dissociation, however, appeared to be less clear-cut than that obtained when 6 mm of the nerve was cooled. The abolition of the 8 component was only obtained after the spike height of the cx group had begun to diminish. But cooling the longer length of nerve obviously caused a greater dispersal of the cx component from slowing of conduction in the cooled region, which must necessarily have reduced its height, even in the absence of block in any of its contributing fibres. When a comparison was made of the areas of the conducted a and 8 elevations obtained during cooling, the poorer discrimination which resulted from cooling 20 mm of nerve was found to be more apparent than real, for the oc area was scarcely affected at the temperature at which no 8 activity was detectable (Table 1). TABLE 1. Comparison of the effects on action potential (AP) height and area on cooling 20 mm of saphenous nerve AP height, % AP area, % Temperature,, a ( C) a a

7 DIFFERENTIAL COLD BLOCK OF NERVES 59 It was clear from these experiments that the differential blocking effect of cold observed when 6 mm or more of the saphenous nerve was cooled was not attributable to the differing saltatory abilities of the various fibres, but that it must reflect some other difference in fibre behaviour. Nevertheless, since the order of sensitivity to cold paralleled the saltatory abilities of the fibres, and since the differential effect of cold tended to become obscured when longer lengths of nerves were cooled, experiments were done to see whether the differential effect could be improved by cooling a length sufficiently short to permit saltation. It was found that cooling a 2 mm length of the saphenous i -1%W1MW -o: Fig. 7. The effect of cooling a 2 mm length of the saphenous nerve. Records from a small twig. a and c: records of the A complex (the 8 component is unusually large). b and d: records of the C complex. Records a and b were obtained with the thermode at 340 C: records c and cd with the thermode at 50 C. Time marks: a and c, 1 msec intervals; b and di, 20 msec intervals. nerve did lead to a more discriminant block of the A fibres than did cooling a 6 mm length. The temperature required to block 8 fibres was about the same (c. 230 C) as when the longer lengths of nerve were cooled, but the temperature necessary to block all cx fibres (c. 50 C) was about 50 C lower. Moreover, the cx component was comparatively little dispersed at the temperature blocking 8 fibres. On the other hand, cooling 2 mm of nerve differentiated A fibres and C fibres poorly. For example, although the 8 response was abolished by a degree of cold insufficient to influence the C response, further cooling to block oc fibres always diminished the C component. Indeed, in several of these experiments the component most resistant to cold was not the C elevation but the A. Such an experiment is illustrated in Fig. 7.

8 60 W. W. DOUGLAS AND J. L. MALCOLM Experiments on small nerve bundles. A few experiments were made which permitted observation of the effect of cold on individual fibres of the A group. In these, records were obtained from small bundles of about 10 active fibres dissected from a twig of the saphenous nerve. The first effect of cooling a 6 mm length of the parent fascicle of such a twig was a slowing in the conduction rate of the cx and 8 elevations. Then, as the temperature was further lowered, the individual fibre action potentials contributing to these elevations could be discerned as some became more slowed than others. Finally, after further slowing and separation of the spikes, a temperature would be reached which was critical for conduction in one or another fibre. At such a temperature its action potential would suddenly disappear from the oscilloscope record. Upon varying the thermode temperature by a fraction of a degree centigrade about this critical level, the particular action potential could be made to reappear or disappear in all-or-none fashion. At this critical level it was observed that the conduction time from the stimulating to the recording point was approximately doubled. In each of these experiments the slower A group fibres, those of 8 velocity, were blocked by cold before the faster conducting fibres of the ac group. The effect of cooling on the recovery cycles of oc and 8 fibres. In several experiments it was observed that when the saphenous nerve was cooled to an extent insufficient to diminish the single maximal response, the highest frequency at which responses could be conducted through a cooled region of nerve was decreased. This suggested to us that measurement of the duration of recovery cycles of the a and 8 components during cooling might give a more sensitive indication of the action of cold than measurement of the heights or areas of the single conducted potentials. Experiments were therefore carried out in which the recovery cycles of a and 8 components were measured at different temperatures by exciting the saphenous nerve maximally with two stimuli delivered at varying intervals of time, and measuring the height of the action potential elicited by the second stimulus. The results showed a distinct difference in the behaviour of the oc and 8 components. The recovery cycle of the 8 component was much more prolonged than that of the; oc component (Fig. 8). Effect of cold on efferent fibres Experiments were made to determine whether a differential sensitivity to cold could similarly be demonstrated among efferent fibres. Branches of the medial and lateral popliteal nerves were stimulated at one or two points in the region of the knee, the thermode was applied to the corresponding fascicle in the thigh, and the action potentials recorded in the corresponding ventral root. With an adequate stimulus to most branches, action potentials produced by

9 DIFFERENTIAL COLD BLOCK OF NERVES 61 the cx and y fibres could be readily identified. The separation between them, however, was frequently poor, and observation of differential effects consequently unreliable. This difficulty was overcome by stimulating at two points on the nerve, after adjusting stimulus strengths so that the proximal stimulus fired oc fibres only, and the distal stimulus ac and y fibres. The interval between these two stimuli was adjusted so that the stimulus to the distal point was applied during the refractoriness produced by the volley set up at the proximal point in the oc fibres only, and conducted away from the recording electrodes. There then appeared at the recording electrodes, first the action potential of 1 00.,c,, o. I ~... P o _oo 80 o.o* a 0 (a) c (b) * ~ D cm 20~~~~~~~~~~~~~~~~~ log10 0 cooled ,0 >-.o 15 Time (msec) Fig. 8. Comparison of recovery cycles of y fibres (0) and t fibres (0) in saphenous nerve. Abscissa, interval in msec between maximal conditioning stimuls and a test stimulus of the same size. Ordinate, height of action potential evoked by test stimulus, expressed as percentage of control response. Cooling applied to 6 mm saphenous nerve between stimulating and recording electrodes: (a) nerve cooled to 240 C; (b) nerve cooled to C; (c) nerve cooled to 180 C. the cc fibres initiated at the proximal point, and later the action potential of the y fibres initiated at the distal point. This procedure not only increased the separation between theoc and y complexes by the conduction time for cc fibres between the distal and proximal stimulating points, but also provided a means of checking that the two complexes whose sensitivities to cold were being compared were related to fibres of different thresholds and conduction velocities. As the difference in size of the cc and y action potentials was considerable, it was found convenient to amplify each separately and to record them simultaneously on the two beams of the oscilloscope. Using a 12 mm thermode, the y complex was always the first to be affected, slowing of conduction preceding its disappearance. The temperature at which the y complex was indiscernible, however, always produced some slowing and C (c)

10 62 W. W. DOUGLAS AND J. L. MALCOLM spreading of the ot component (Fig. 9), and sometimes also a significant degree of block. As in the case of afferent fibres, cooling prolonged the recovery cycle of the smaller (y) fibres more than that of the larger (oc) fibres. Irecc Fig. 9. Effect of cold on the fast and slow efferent fibres of the peroneal nerve. Action potentials were recorded in the motor root of L 7. The lower record in each instance shows the cx and y components; the upper, taken at higher gain, shows better the -y response. Length of nerve cooled, 12 mm. Thermode temperature (a) = 330O' C, (b) = C, (c) C, (d) = C. Comparison of the effects of cold on efferent and afferent fibres This comparison was made by recording simultaneously from dorsal and ventral roots, after stimulating a peripheral mixed nerve. Cold was applied over a 12 mm length of nerve. In five out of ten cats in which the lateral popliteal nerve, nerve to gastrocnemius, and the nerve to the hamstring muscles were investigated, the dorsal and ventral root action potentials diminished equally when cold was applied to the nerve trunk. In the other five cats the action potentials recorded in the ventral root were more sensitive to cold. In two of these cats the temperature ranges over which dorsal and ventral responses diminished overlapped to a considerable extent. In the

11 I DIFFERENTIAL COLD BLOCK OF NERVES 63 remaining three the ventral root response was so much more sensitive to cold that it was almost completely blocked by a fall in thermode temperature insufficient to affect significantly the dorsal root response (Fig. 10). I8_ J 5 c Fig. 10. Comparison of the effect of cold on efferent and afferent fibres in the peroneal nerve. The upper record shows the action potential recorded in the ventral root of L 7; the lower record, that recorded in the dorsal root of L 7 at the same time. The responses to cooling a 12 mm length of the peroneal nerve to different temperatures are shown. The figure against each record gives the temperature of the thermode in 'C. In four of these experiments a more sensitive measure of the effect of cold on efferent and afferent axons was obtained by examining their recovery cycles during cooling with the 2-stimulus technique already described. In each instance cooling prolonged the recovery cycles of efferent axons more than those of afferent axons (Fig. 11). In two of the experiments cold had been seen to reduce the ventral root response to a single stimulus more than the dorsal root response, but in the other two no differential effect of cold on the single action potentials had been apparent.

12 64 W. W. DOUGLAS AND J. L. MALCOLM 100 * 100 0; 0o0 0 C 0 C. 00 (a) _ (b) (c) 0~~~~~~~~~~ o o Q!D ol~~--ai4,1-o Time (msec) Fig. 11. Comparison of recovery cycle of large x efferent fibres (0) and ca afferent fibres (0). Abscissa: interval in msec between maximal conditioning stimulus and a maximal test stimulus. Ordinate: height of action potential evoked by test stimulus expressed as percentage of control response. Cooling applied to posterior tibial nerve in mid-thigh region; stimulating electrodes on the posterior tibial nerve in the popliteal space; recording electrodes on the 7th lumbar dorsal and ventral roots. Dorsal and ventral root action potentials recorded simultaneously. (a) Nerve cooled to 250 C; (b) nerve cooled to 130 C; (c) nerve cooled to 9.50 C. Effect of cold on the vagus nerve The results described above are at variance with those obtained by Torrance & Whitteridge (1947) and Whitteridge (1948) on the vagus, which showed the larger of the medullated fibres to be more easily blocked by cold than the smaller. We therefore carried out several experiments on vagi, either in situ or isolated, to ascertain whether the discrepancy was to be explained by a difference in the nerves studied, or by a difference in technique. In the experiments on the vagus in situ, the animal was maintained under artificial respiration. The stimulating electrodes were applied to intrathoracic branches of the vagus and the recording leads were placed just below the nodose ganglion. The results were similar in both types of preparation. The vagal action potential elicited by maximal stimulation showed in each instance two major elevations, the first corresponding to the medullated fibres of A and B groups and the second, more slowly conducted, corresponding to C fibres. When cold was applied to a 12 mm length of the vagus, the A-B component was first abolished and then, at a considerably lower temperature, the C component (Fig. 12). Within the medullated group, however, no clear-cut differential sensitivity to cold was observed. In contradistinction to the saphenous nerve records, where the separation between the component due to oc fibres and that due to the 8 fibres was so well defined that the differential effect of cold was readily discerned by inspection, the vagal medullated complex appeared to be due to a substantially continuous spectrum of fibres and any differential effect would evidently be less easily detected. Nevertheless, it was

13 DIFFERENTIAL COLD BLOCK OF NERVES 65 to be expected that if the faster and slower conducting fibres differed considerably in their susceptibilities to cold, the front or rear of the action potential complex would be seen to subside during cooling. This did not occur: on cooling the complex subsided along its whole length. In one experiment, for example, when block was about 60% complete (the area of the complex elevation being reduced to 40 % of its original value), measurement showed that within the remaining elevation there were both fast and slowly conducted components. Further lowering of the temperature caused such dispersion and U. 10 mnsec 100 msec Fig. 12. The differential effect of cold on the vagus. Records (a) and (c): response of the A-B group. Records (b) and (d): response of the C group. a and b at 36 C.; c and d at 9.50 C. reduction of the height of the complex that satisfactory measurements could not be made. Attempts were therefore made to clarify the results by two modifications of the technique. First, the conducted A-B complex was split artificially into two components by a two stimulus technique similar to that described for the experiments on the oc-y complex of motor nerves. To do this the vagus was stimulated maximally at one end and submaximally at a point nearer the recording leads. The time interval between the two stimuli was then adjusted so that the potential recorded showed two clear elevations, a faster conducted component (elicited by the submaximal stimulus) and a slower conducted component (elicited by the maximal stimulus), the two being separated by a distinct 'notch' due to refractoriness of the fast component of the response to the second stimulus. After cooling sufficient to reduce the total area of the two elevations to about 5 PHYSIO. Cxxx

14 66 W. W. DOUGLAS AND J. L. MALCOLM 50 % of its original value, measurement showed that the two elevations were diminished proportionately. The second device employed was again based on the use of two stimuli, one maximal and the other just above threshold. But in this instance both stimuli were applied at the same point alternately and some seconds apart. Cold was applied to the nerve until the area of the conducted complex elicited by the maximal stimulus was reduced by about half. Application of the near-threshold stimulus was then found still to cause the appearance of a small elevation. DISCUSSION Our experiments have shown that in sensory and motor nerves of the cat hindlimb, the block exerted by localized cooling is to a greater or lesser degree selective on fibres of differing conduction velocities, and that the order of susceptibility is the following: first, small medullated fibres, then large medullated fibres, and finally unmedullated fibres. Within the medullated group, the greatest degree of differentiation achieved was in the saphenous nerve, where complete suppression of the 8 component was in almost all instances readily achieved by cooling without there being any failure of conduction in the ac complex, and where the y and P components, when readily visualized, fell out in that order as the temperature was lowered and before there was significant block of the oa component. These findings are in accord with Dodt's observation (1953) that, in the cat lingual nerve, 8 fibres were blocked by cold more readily than,b fibres, and with Lundberg's finding (1952) that the electroneurogram of the whole cat saphenous nerve cooled to 100 C showed an ac but not a 8 elevation. Our experiments on the large and small motor fibres of the sciatic nerve, although less striking than those on the saphenous nerve, showed in each instance that the smaller medullated fibres were more sensitive to cold. In comparing the results obtained on the sciatic with those obtained on the saphenous nerve, consideration must be given to the fact that the disparity between the conduction velocities of the large (cx) and small (8) sensory fibres of the saphenous nerve is much greater than that between the large (a) and small (y) motor fibres in the sciatic nerve. If size is a factor which determines a nerve fibre's sensitivity to cold, then less discrimination is to be expected in the latter nerve. It is clear, however, that size alone cannot determine a fibre's sensitivity to cold block. Although the first fibres blocked on cooling are the smallest of the myelinated group, the last fibres blocked are smaller than these and belong to the unmyelinated group. Moreover, fibres of very similar size may behave quite differently, as is shown by the differential effect of cold on motor and sensory fibres of similar conduction velocities in the peroneal nerve. (The explanation of this latter finding may reside in chemical differences between sensory and motor fibres.)

15 DIFFERENTIAL COLD BLOCK OF NERVES 67 On occasion we have found that the differentiation between medullated and unmedullated fibres may not be clear-cut, although the length of nerve cooled is adequate, and that some block of the unmedullated fibres may occur before all medullated fibres fail to transmit. The reason for this, and indeed for the sometimes indifferent discriminant effects of cold within the medullated group itself, is not apparent. We have found no explanation in terms of the state of the animal, local circulation, or the use of in situ or isolated preparations of the nerve. Somewhat similar and unexplained variation in discrimination has been observed by Leksell (1945) when producing block by pressure. All published work on the effect of local cooling on vagal fibres suggests that their order of susceptibility to block is the reverse of that which holds for the other nerves so far examined. Most of the evidence is indirect and depends on correlating the effect of local cooling on vagal reflexes with the fibre population presumably responsible for these reflexes. For example, local cooling is known to suppress the stretch reflex at higher temperatures than it suppresses the reflex initiated by phenyl diguanide, yet the fibres mediating the former reflex are considerably larger (for references see Paintal, 1953). There is also some direct evidence. Torrance & Whitteridge (1947) and Whitteridge (1948), observing single fibre preparations of the vagus, noted that among the several fibre types examined the most sensitive to cold were stretch fibres, which conducted at the highest velocity. In our own experiments on the vagus, the largest fibres did not prove especially susceptible to cold, as witness the findings that both the early and late portion of the compound action potential subsided equally during the early stages of block, and that stimuli of strength sufficient only to excite the largest fibres of the nerve still produced a response after 50 % block. Our technique, however, did not allow us to discern differential effects below about 50 % block in the vagus, and our results must be regarded as equivocal. It is of importance that this apparent discrepancy between the behaviour of medullated vagal fibres and those of the limb nerves be confirmed or refuted by further work, for its existence would indicate a difference between somatic and visceral fibres. In presenting our results on somatic nerves, the inverse relationship between internodal distance and susceptibility to block was considered and dismissed as the factor leading to differential block when adequate lengths of nerves were cooled. Nevertheless, the differences in internodal length might be expected to give rise to differential blocking effects when the length of nerve cooled is sufficiently short. The experiments of Hodler et al. (1952) and Tasaki (1939) have shown that conduction in myelinated fibres fails when three or more nodes are blocked, but that when only two nodes are blocked the current flowing beyond the inactive nodes is still sufficient to excite the fibre. Since the internodal distance in the largest fibres is close to 1 mm, it follows that some 2-3 mm of nerve must be cooled to block conduction 5-2

16 68 W. W. DOUGLAS AND J. L. MALCOLM in these fibres, and a somewhat shorter length to block the smaller fibres with shorter internodal distances; and also that about this critical length saltation will influence greatly the results. The effects we observed when cold was applied to 2 mm of the saphenous nerve, i.e. the paradoxical persistence of large A fibres after block of the C fibres, and the heightened discriminant effect of cold within the A group itself, are doubtless to be accounted for by effects of this sort. Cooling of such a short length of nerve is obviously contraindicated when differentiation between A and C fibres is sought, but it would appear to be a useful procedure when it is required to block differentially within the medullated group. Complete block probably occurs in such experiments only after cold has spread beyond the edges of the 2 mm long thermode; certainly the temperature to which the small thermode needed to be lowered to block a given fibre group was lower than with larger thermodes. Slowing of conduction, affecting as it did some fibres more than others, led to temporal dispersion of the action potential. This introduced a difficulty in assessing the degree of block during cooling, for temporal dispersion in the absence of block reduced the height of the compound action potential. The difficulty was overcome by taking area measurement of the response rather than height as an index of block. In the experiment on the limb nerves where 12 mm or less of the total length of the nerve was cooled, the slowing in conduction time from stimulating to recording point was seldom more than doubled before block occurred. This was especially evident when single action potentials were observed in small branches of the nerve, as in these conditions an approximate doubling of this conduction time preceded the sudden disappearance of the individual fibre's action potential. In such circumstances the compound action potential retains sufficient synchronism to provide a trace whose area can be measured with reasonable accuracy. In the vagus, however, where the conduction velocities contributing to the action potential cover a very wide range, and where the compound action potential is normally dispersed, the additional temporal dispersion produced by cooling renders measurement of the area (and of block) extremely difficult. Prolongation of the rate of recovery after a conducted response has been an early feature of local cooling in all the nerves we have examined. Whitteridge's observation (1948) that Wedensky inhibition occurs in cardio-vascular afferent nerve fibres in the vagus on cooling to temperatures that just failed to block conduction can be accounted for in this way. In each nerve cooling has prolonged the recovery cycle of the slowest fibres most. At normal temperatures the recovery cycle of the smallest fibres is perceptibly longer than that of the larger, and it may be that cooling emphasizes this difference by a proportional increase in the refractory periods of both large and small. The effect, however, is such that fibres most susceptible to cold show the earliest and greatest prolongation of their recovery cycle. We have made use of this to reveal better

17 DIFFERENTIAL COLD BLOCK OF NERVES 69 the differential effect of cold. Moreover, it appears probable that the phenomenon may well be an important factor acting to enhance the differentiation by cold of different modalities activated by repetitive stimulation or firing in normal repetitive fashion to physiological stimulus. The explanation of the phenomenon may be that a relatively small amount of axoplasm has to repolarize a given number of nodes in the case of the smaller axon, and hence that there is a much greater chance of delay in completion of the recovery process. Another explanation must hold for the differential effect of cold on the recovery cycles of the fast sensory and motor fibres whose mean diameter and internodal distances are presumably similar. It is apparent from our experiments that caution must be used in employing the technique of localized cooling of nerves for analysis of the function of different kinds of nerves. Although in a given nerve the susceptibilities of the component fibres to cold block appear related in orderly fashion to their fibre sizes, it cannot be predicted with certainty whether the fibres of greater diameter will be most or least susceptible. Before full advantage may be taken from the technique, the differential effect on conduction in the nerve under study should be determined. Consideration should also be given to the effects of Wedensky inhibition. For example, in the somatic nerves we have examined, an effect mediated by fibres firing rapidly might be expected to be depressed by cold more readily than another mediated by fibres of the same size firing more slowly. Alternatively, where frequency of discharge is similar in two fibre groups of different sizes, the Wedensky phenomenon might be expected to suppress the small fibre effects even before there was any obvious change in the conducted response to a single isolated response in these fibres. Finally, it might even happen that in a nerve whose smaller nerve fibres prove most sensitive to cold block when tested by single stimuli, block of the physiological effect of larger fibres occurs first due to their firing at much higher rates than the smaller fibres. Our work gives no insight into the mechanisms underlying the differential effects of cold on nerve fibres. One possibility is that the effect is related to the availability of energy required for maintaining and restoring the resting state. Oxygen consumption of nerve, which is presumably devoted largely to maintaining the resting state, is lowered by cooling (Arvanitaki & Chalazonitis, 1954) and from this it would follow that cooling must prolong the rate of restoration of resting state following activity. At sufficiently low temperature, the rate of provision of energy may be so reduced that normal excitability is not maintained. This interpretation would account for the finding that the differential effect of cold on the saphenous nerve is similar to that of anoxia (Gasser, 1941). It also would explain the Wedensky inhibition which precedes cold block.

18 70 W. W. DOUGLAS AND J. L. MALCOLM SUMMARY 1. Experiments have been carried out to determine the effect of localized cooling on impulse transmission along cat nerves, and especially to see if differential block according to nerve fibre size occurs. 2. Action potentials from single or multiple shocks have been recorded during cooling of 2-20 mm of nerve between the stimulating and recording points. 3. In the saphenous nerve a greater lowering of temperature was required to block the C fibres than the A fibres. 4. In the saphenous nerve, cold caused differential block within the A fibre group. The order of block during progressive cooling was first 8 then y, followed by,b and finally a fibres. 5. In motor branches of the sciatic nerve, progressive cooling blocked first the y, and then the a efferents. 6. In comparing the effect of cold on motor and sensory fibres of equal size (oc) in the sciatic nerve, a difference was observed in five out of ten cats: in these the motor fibres were blocked by a fall in temperature insufficient to block the sensory fibres. 7. In the vagus, a greater lowering of temperature was required to block C fibres than the fibres of the A-B group. No clear-cut differential effect of cold was seen in the A-B group. 8. The effect of varying the length of nerve cooled has been tried: when the length is short (2 mm) saltation may occur. Since this occurs most readily with large fibres, it enhances the differential effect of cold within the A group. It obscures, however, the differential effect of cold on A and C groups. 9. In all the nerve fibre groups examined, slowing of the conduction rate and prolongation of the recovery cycle occurred before block. The latter gives rise to Wedensky inhibition on repetitive stimulation. REFERENCES ARVANITAKI, A. & CmATAZoNrrIs, N. (1954). RWponses bioelectriques de l'axone geant a l'acc6leration thermique de sa respiration. C.R. Soc. Biol., Pari8, 148, DODT, E. (1953). Differential thermosensitivity of mammalian A fibres. Acta phy8iol. 8cand. 29, GASSER, H. S. (1941). The classification of nerve fibres. Ohio J. Sci. 41, HODLER, J., STXMPFLI, R. & TAsAK, I. (1952). Role of potential wave spreading along myelinated nerve fiber in excitation and conduction. Amer. J. Physiol. 170, LEKSELL, L. (1945). The action potential and excitatory effects of the small ventral root fibres to skeletal muscle. Acta phy8iol. 8cand. 10, suppl. 31, LUNDBERG, A. (1952). Potassium and the differential thermal sensitivity of the membrane potential, spike, and negative after potential in mammalian A & C fibres. Acta physiol. 8cand. 15, suppl. 50, PAiNTAL, A. S. (1953). The conduction velocities of respiratory and cardiovascular afferent fibres in the vagus nerve. J. Physiol. 121,

19 DIFFERENTIAL COLD BLOCK OF NERVES 71 SINCLAIR, D. C. & HiNsHAw, J. R. (1951). Sensory changes in nerve blocks induced by cooling. Brain, 74, SKOGLUND, C. R. (1943). The response to linearly increasing currents in mammalian motor and sensory nerves. Acta physiol. scand. 4, suppl. 12, TASAKI, I. (1939). The electro-saltatory transmission of the nerve impulse and the effect of narcosis upon the nerve fiber. Amer. J. Phy8iol. 127, TORRANCE, R. W. & WEITTERIDGE, D. (1947). Technical aids in the study of respiratory reflexes. J. Physiol. 107, 6-7P. WHITTERIDGE, D. (1948). Afferent nerve fibres from the heart and lungs in the cervical vagus. J. Physiol. 107,