J. Physiol. (I957) I35, (Received 20 July 1956) The interpretation ofthe experimental results ofthe preceding paper (Matthews

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1 263 J. Physiol. (I957) I35, THE RELATIVE SENSITIVITY OF MUSCLE NERVE FIBRES TO PROCAINE BY PETER B. C. MATTHEWS AND GEOFFREY RUSHWORTH From the Laboratory of Physiology, University of Oxford (Received 20 July 1956) The interpretation ofthe experimental results ofthe preceding paper (Matthews & Rushworth, 1957) depends partly upon a knowledge of the order in which procaine paralyses the different nerve fibres from muscle. Leksell (1945) recorded the compound action potential produced in a ventral root by large and small motor fibres, and found on applying 1/500 ethocaine to the nerve central to the stimulating electrodes that 'the effect upon the y wave was greater than the effect on the a wave'. But there has been no direct comparison of the relative sensitivity of muscle afferent and muscle efferent fibres. Gasser & Erlanger (1929) first showed by direct recording of action potentials that 'in general small fibres are blocked before large ones' by cocaine but 'the blocking is not effected with any precision'. The same was found for the action of procaine on several mammalian nerves (Heinbecker, Bishop & O'Leary, 1934). However, Toman (1952) in reviewing the action of procaine stated that 'although a preferential attack on small fibres is generally assumed, the rule is not universal'. It was therefore essential to determine the relative sensitivity of large motor, large afferent and small motor nerve fibres to the particular method of applying procaine used in the preceding paper to produce a selective paralysis of the stretch reflex of soleus muscle. This paper shows that the large fibres, whether afferent or efferent, are about equally sensitive, while the y motor fibres are much more rapidly paralysed. METHODS The experiments were performed on cats anaesthetized with pentobarbitone sodium (Nembutal, Abbott Laboratories) 40 mg/kg intraperitoneally, supplemented by further injections as necessary. The nerve to the right soleus muscle was cut peripherally and dissected out of the lateral head of gastrocnemius through which it runs; the common nerve to soleus and lateral head of gastrocnemius was then separated from the sciatic for about 5 cm. In other experiments the nerve to the medial head of gastrocnemius was similarly separated. The rest of the sciatic, the femoral, superficial obturator and hamstring nerves were cut in the right leg. The lower lumbar nerve roots were then exposed and those on the right side cut close to the spinal cord. Liquid paraffin,

2 264 P. B. C. MATTHEWS AND G. RUSHWORTH enclosed in two separate baths made by reflecting skin and muscle, covered the spinal roots and peripheral nerves. Compound action potentials set up on stimulating the central end of a muscle nerve with brief shocks were recorded from the peripheral end of an appropriate dorsal and ventral root through a pair of bipolar silver electrodes, and were amplified and displayed on a double-beam cathode-ray tube. The amplification of the recording system began to decrease for frequencies over 1 kc/s and had fallen to half for frequencies of 5 kc/s. The form of the action potentials must have been distorted, but this does not affect our conclusions. The amplifier time constant was set between 50 and 200 msec. The method of applying procaine was precisely the same as that described in the preceding paper (Matthews & Rushworth, 1957) for producing a selective paralysis of soleus reflexes. It consists of soaking a 1-5 x 1.5 cm square of filter-paper in 0-2 or 0.5% procaine solution, folding the filter paper almost double and placing it astride the nerve under paraffin. RESULTS The compound action potential set up by stimulating the nerve to the soleus muscle was recorded from the L 7 dorsal and ventral roots. The fast-conducting fibres produce an initial wave of ,uV in both roots, as shown in Fig. 1a. The fastest afferent fibres conducted at about 110 m/sec, while the fastest motor fibres conducted at about 90 m/sec; the threshold of the motor fibres was slightly higher than that of the afferent fibres. These differences were not found in the nerve to the gastrocnemius and are in accordance with the histological evidence that the soleus motor fibres are smaller than soleus large afferent fibres (Eccles & Sherrington, 1930; Lloyd & Chang, 1948). The initial wave in the dorsal root is produced by group I afferents, which come from both muscle spindles and Golgi tendon organs (Hunt, 1954). The initial wave in the ventral root is produced by the large oc motor fibres which supply ordinary striated muscle fibres, and not the intrafusal fibres (Leksell, 1945; Kuffler, Hunt & Quilliam, 1951). The potential produced by the y motor fibres, which supply the intrafusal fibres, was regularly seen after the oc motor wave, but was less than 20,uV in amplitude. In Fig. 3a the y wave is seen as an irregularity in the base line after the oa wave. The y fibres had a higher threshold than the a fibres (Leksell, 1954) and conduction velocities in the range m/sec. In one experiment the conduction velocity, calculated from the over-all conduction distance, was confirmed by moving the stimulating electrodes 2 cm along the nerve to the soleus after it had been directed away from the nerve to the lateral head of gastrocnemius. Despite the smallness of the y wave its presence or absence during nerve block with procaine could be fairly certainly decided by varying the strength of the stimulus while watching a repetitive trace (less than 10/sec) on the cathode-ray tube. The potentials produced by small afferent fibres (groups II and III) were also frequently observed, but were often largely obscured by spontaneous afferent discharges in large fibres from non-denervated muscles. Fig. la shows a particularly large group II wave immediately after the initial group

3 PROCAINE ON MUSCLE NERVES 265 I wave. However, this record was produced by superimposing forty traces, and as this has not been our usual practice we have not studied the paralysis of small afferents by procaine. Both large and small nerve fibres could be reversibly paralysed by applying procaine solution to the nerve to soleus and lateral head of gastrocnemius. Fig. 1 shows the progressive decrease in the dorsal and ventral root large fibre action potentials of soleus after applying procaine, and their recovery after covering the nerve in Ringer's solution. Before complete paralysis a b c d e 400 V VṘ.L _\ 2 msec Fig. 1. Simultaneous paralysis by procaine; of large afferent and large efferent fibres in the nerve to soleus; compound action potentials recorded in L 7 dorsal and ventral roots on stimulating nerve to soleus; 40 traces superimposed. Records taken before, during and after applying 0-2% procaine to the nerve for 20 min; a taken 5 min before applying procaine; b, c and d taken 5, 12 and 18 min after applying procaine; e taken 20 min after removing procaine. Fig. 2. V R-procaine VR0-2%. I100,80 ~f E60 ~40 -cr20 -x wave present Ringer r 0777, I p Time (min) Procaine block of conduction in motor and sensory fibres of the soleus motor nerve (same experiment as Figs. 1 and 3). The amplitude of the action potentials of the large fibres is plotted as a percentage of their amplitude 1 min before applying 0.2% procaine; the presence of the y wave in the ventral root record is shown at the bottom. the conduction time increased and also the duration of the compound action potential, both resulting from a slowing of conduction in the partially anaesthetized region of nerve. Fig. 2 is a graph prepared from the results of this experiment, and shows that the potentials produced by the large motor fibres and the large afferent fibres disappear and reappear at about the same rate. Fourteen other applications of procaine in this and three other cats have also paralysed the large motor and large afferent fibres closely together, though the time required for complete paralysis has varied from 5 min to

4 266 P. B. C. MATTHEWS AND G. RUSHWORTH over 30 min. On average the large motor fibres of soleus have been slightly more sensitive than the large afferent fibres, for the oc motor wave has sometimes disappeared at a time when the group I afferent wave was still 30% of its original amplitude. a Before b During c After 400 i-45 msec Fig. 3, Selective paralysis of y efferents in the soleus nerve by procaine. Same experiment as Fig. 1, but on slower time base to show the y wave (irregularity in base line after a wave); 20 traces superimposed; records 5 min before applying 0-2% procaine, 5 min after applying procaine, 20 min after removing procaine. V.R. Before During After I mv I &.R.Gnl 4K9nL100yV.-5 msec l l l ~~~~1 mv D.R.J Fig. 4. Selective paralysis of y efferents in the gastrocnemius nerve by procaine: compound action potentials recorded from S, dorsal and ventral roots on stimulating nerve to medial head of gastrocnemius; single traces. The ventral root potential was recorded at two amplifications, taken a few seconds apart, to show both a and y waves clearly; records 3 min before applying 0-2 % procaine, 5 min after applying procaine, and 30 min after removing procaine. In contrast to the similar sensitivity of the large fibres to procaine has been the much greater susceptibility of the small motor fibres. They were regularly and completely paralysed at a time when the large fibre potentials were only beginning to decrease in amplitude, and only recovered after the large fibre potentials had returned to near their original size. Thus in Fig. 3b, taken 5 min after applying procaine, the irregular wave produced by the y fibres of soleus has disappeared though the a motor wave is little changed. The disappearance of the y wave was more clearly shown by examining the cathoderay tube during the experiment. Fig. 3 is from the same experiment as Fig. 1, and the presence of the y wave is indicated at the bottom of the graph of Fig. 2. Other experiments on the soleus nerve have given similar results. I As the y wave of soleus is so small the experiments were repeated using the nerve to the medial head of gastrocnemius which produces a y wave of uV instead of 20EV. These experiments have entirely confirmed the greater sensitivity of the y

5 PROCAINE ON MUSCLE NERVES 267 motor fibres to procaine; in nine cats the y wave of gastrocnemius disappeared at a time when the large fibre action potentials, both motor and afferent, were always over 80% of their original amplitude, and were frequently unchanged. Fig. 4 shows a record from such an experiment where the ventral root compound action potential was recorded at two different amplifications to show both oc and y motor waves clearly. Five minutes after applying procaine the y wave disappeared though the large motor and large afferent waves were unchanged. Procaine was left on the nerve for 25 min without completely paralysing the large fibres. Half an hour after removing procaine the y wave had returned, though the large afferent potential had become spontaneously smaller. The y wave produced by stimulating the common nerve to the lateral head of gastrocnemius and soleus was also selectively paralysed. When the nerve to the medial head of gastrocnemius was stimulated, the large motor and afferent fibres, like those of soleus, were paralysed at about the same rate. However, in this case the large afferents were slightly more sensitive than the large motor fibres. In two extreme cases the cx motor wave persisted at half its original amplitude when the group I afferent wave had almost disappeared, but the y motor wave had disappeared long before. Before anaesthetized nerve fails to transmit a single action potential it fails to transmit a train of action potentials (Wedensky inhibition). We have stimulated the nerves at frequencies of /sec at various times after applying procaine, and have seen Wedensky inhibition interfering with the transmission of progressively lower frequencies as narcosis progressed. At the time when the y wave disappeared the large fibres were always able to transmit 100 impulses/ sec, usually 300 impulses/sec and sometimes 500 impulses/sec. The more slowly the nerve was being paralysed the greater was the ability of the large fibres to conduct at the time when the y wave vanished. There has been no great difference between the Wedensky inhibition of large motor and large afferent fibres, and the slight differences found were those expected from the relative rate of decline of their action potentials. Thus, for soleus, Wedensky inhibition in large motor fibres was more marked, while for gastrocnemius it was more marked in the large afferent fibres. There has never been any sign of facilitation of successive action potentials at the anaesthetized region of the nerve. The second of two action potentials separated by 1-10 msec became progressively smaller than the first as the nerve was paralysed. It never became larger and there was no summation when the nerve was completely paralysed. DISCUSSION The result of these experiments is that our particular method of applying procaine paralyses the large motor and large muscle afferent (group I) fibres at about the same time. The small motor fibres, however, are paralysed much

6 268 P. B. C. MATTHEWS AND G. RUSHWORTH earlier, as has previously been shown by Leksell (1945). It is probable but not certain that the same results would have been obtained if procaine had been applied in a different manner, but with local anaesthetics as with cold (Douglas & Malcolm 1955) the length of nerve paralysed is probably rather important for determining the relative susceptibilities of different sized fibres. However, the results support the principle that 'in general small nerve fibres are blocked before large ones' (Gasser & Erlanger, 1929). The slightly greater sensitivity of soleus large motor fibres compared with soleus large afferent fibres is explained in terms of size, for the measurements of conduction velocity and threshold show that the motor fibres are smaller than the large afferent fibres. There is no such explanation for the occasional greater sensitivity of the large afferent fibres of gastrocnemius medialis than of the large motor fibres; but under our experimental conditions the difference in sensitivity to procaine between these large fibres was much less than the difference in sensitivity between either of these groups of large fibres and the y motor fibres. The results of this paper are applied in the preceding paper (Matthews & Rushworth, 1957) to support the contention that the selective paralysis of the stretch reflex of the soleus, which is produced by procaine, is caused by the selective paralysis of small motor fibres. SUMMARY 1. The compound action potentials set up on stimulating the nerve to soleus or the nerve to gastrocnemius have been recorded from an appropriate dorsal and ventral root in the anaesthetized cat. Procaine was applied to the nerve, between stimulating and recording electrodes, and the disappearance of the potentials observed. 2. The large cx motor fibres and the large group I afferent fibres are paralysed at about the same rate. 3. The small y motor fibres are paralysed at a time when the large fibre potentials are only beginning to decrease. 4. The method of applying procaine was the same as that previously used to paralyse the stretch reflex selectively. We wish to thank Dr C. G. Phillips for constant encouragement and criticism. REFERENCES DOUGLAS, W. W. & MALCOLM, J. L. (1955). The effect of localized cooling on conduction in cat nerves. J. Physiol. 130, ECCLES, J. C. & SHERRLNGTON, C. C. (1930). Numbers and contraction-values of individual motorunits examined in some muscles of the limb. Proc. Roy. Soc. B, 106, GASSER, H. S. & ERLANGER, J. (1929). The role of fiber size in the establishment of a nerve block by pressure or cocaine. Amer. J. Physiol. 88, HEINBECKER, P., BISHOP, G. H. & O'LEARY, J. (1934). Analysis of sensation in terms of the nerve impulse. Arch. Neurol. P8ychiat., Chicago, 31,

7 PROCAINE ON MUSCLE NERVES 269 HUNT, C. C. (1954). Relation of function to diameter in afferent fibres of muscle nerves. J. gen. Phy8iol. 38, KuImEm., S. W., HUNT, C. C. & QULLiAM, J. P. (1951). Function of medullated small-nerve fibres in mammalian ventral roots: efferent muscle spindle innervation. J. Neurophy8iol. 14, LERSELL, L. (1945). The action potential and excitatory effects of the small ventral root fibres to skeletal muscle. Acta phy8iol. 8cand. 10, Suppl. 31. LLOYD, D. P. C. & CIANG, H. T. (1948). Afferent fibres in muscle nerves. J. Neurophy8iol. 11, MATTHEWS, P. B.C. & RUSHWORTH, G. (1957). The selective effect of procaine on the stretch reflex and tendon jerk of soleus muscle when applied to its nerve. J. Phy8iol. 135, TOMAN, J. E. P. (1952). Neuropharmacology of peripheral nerve. Pharmacol. Rev. 4, Note added in proof. Our attention has been drawn to a paper describing the effect of intramuscular injection of procaine into the gastrocnemius soleus muscle of decerebrate cats. The tendon jerk and the after-discharge of the crossed extension reflex were selectively paralysed. To explain this it was considered that the afferent fibres were being paralysed before the motor fibres; selective paralysis of the y motor fibres now appears a preferable explanation. REFERENCE BREMER, F. & TITrECA, J. (1930). Du m6chanisme de l'action de la novocamne sur le tonus musculaire. C.R. Soc. Biol., Pari8, 105,