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SUMMARY1. Resting membrane potentials of rat diaphragm muscles were measured in vitro after previous denervation for 0-10 days. In some experiments denervated muscles were incubated in vitro for 3 hr while in others they were cultured for 15-24 hr to allow adequate exposure to drugs before recording.2. It was found that resting membrane potentials within 2-5 mm of the site of nerve section were significantly lower, within 3 hr, than resting membrane potentials measured more than 9 mm away from site of nerve section. This difference could be reduced or abolished by bathing preparations in solutions containing adrenaline (10 Sam), noradrenaline (10 /SM) or isoprenaline (10 /LM) or dibutyryl cyclic AMP (10 /uM-0-25 mm in the presence of 2 mm theophylline). Cyclic AMP (0 5 mM) was ineffective. 3. Application of solutions containing dibutyryl cyclic AMP for 3 hr also raised the resting membrane potential of muscles denervated 4-5 days previously. Culture studies showed that this effect was sustained when the time of incubation was 24 hr.4. Incubating freshly denervated preparations with cycloheximide (22 jug/ml.) puromycin (10 or 50 jug/ml.) or actinomycin D (1 jug/ml.) did not prevent the development of the early (3 hr) fall in resting membrane potential despite a concomitant inhibition of RNA or protein synthesis. Culturing freshly denervated muscles in solutions containing cycloheximide (10 or 25 jug/ml.) which blocked 93 % of protein synthesis, did not prevent the expected drop in resting membrane potential after 15 or 24 hr.5. It was found that exposure to ouabain (1 or 5 mM) produced a rapid (15 min) fall in resting membrane potential in innervated and denervated preparations treated with dibutyryl cyclic AMP but not denervated J. J. BRAY AND OTHERS preparations. After 5 days denervation cyclic AMP levels in muscle were increased by about 40 %.6. It is suggested that upon denervation an electrogenic action of a Na+-pump is blocked and that dibutyryl cyclic AMP and catecholamines are capable of stimulating this pump.

SUMMARY1. Nerve impulses in the rat sciatic nerve were blocked for long periods by tetrodotoxin (TTX) released from capillary implants. The TTX capillaries did not block axonal transport, nor did they cause any sign of nerve degeneration.2. A comparison of the effects of TTX paralysis and denervation was made on both extensor digitorum longus (e.d.l.) and soleus muscles over 21 days, a time when the products of nerve degeneration were unlikely to contribute to the changes associated with denervation. The resting membrane potential of TTX-paralysed muscles was significantly different (P < 0005) from that of the denervated muscles at all periods and at 21 days the decrease that can be attributed to inactivity was 61 % (e.d.l.) and 49 % (soleus) of that which follows denervation. This disparity was even more pronounced for the ACh receptor density where the increase in receptors due to inactivity was only 34 % (e.d.l.) and 21 % (soleus) of that due to denervation.3. A similar comparison was made on muscles which had been reinnervated by TTX-inactive nerves. These muscles were found to have a significantly higher resting membrane potential and lower ACh receptor density than the denervated muscles (P < 005).4. The experiments on reinnervated muscles preclude the possibility that nerve degeneration products are solely responsible for the difference between the TTXparalysed and denervated muscles and suggest that the difference can be attributed to the trophic influence of the nerve.5. An observed increase in the m.e.p.p. frequency of the TTX-paralysed muscles indicated that nerve action potentials play a role in regulating the spontaneous release from nerve terminals.

SUMMARY1. Resting membrane potentials of rat diaphragm muscles cultured in Trowell T8 medium were measured in vitro. After 3 hr in culture the resting membrane potential of muscle fibres within 2-5 mm of nerve section ('near') was -683 + 0 4 mV (nineteen preparations). This was significantly lower (PI< 0001) than the resting potential (-74 0 + 0 4 mV) measured in muscle fibres 8-10 mm from the site of nerve section ('far') in the same preparations. A difference between the 'near' and the 'far' fibres was maintained in muscles cultured for 6 and 12 hr. Miniature end-plate potentials were present in both 'near' and 'far' fibres cultured for 3 and 6 hr and ceased after 12-15 hr.2. Thepresenceofearbamylcholine (10-orr 10-8 M) maintainedtherestingmembrane potential of 'near' fibres close to that of 'far' fibres at 3, 6 and 12 hr. For example, at 3 hr in the presence of 10-8 M-carbamylcholine the mean resting potential was 75'6 + 0'5 mV in 'near' fibres and 76-1 + 04 mV in 'far' fibres (four preparations). A similar effect was produced in preparations exposed to anticholinesterases: diisopropylphosphorofluoridate (DFP) (10-7 M), neostigmine (10-7 M) or physostigmine (10-5 M).3. Agents that blocked acetylcholine receptors had the reverse effect. In the presence of oc-bungarotoxin (1 ,sg/ml.) or d-tubocurarine (10-5 M) the resting membrane potential of 'far' fibres was reduced to the level of 'near' fibres over the 24 hr period of observation. For example, at 3 hr in the presence of a-bungarotoxin the mean resting potential was 67-2 + 0-5 mV in 'near' fibres and 68-5 + 0-6 mV in 'far' fibres (six preparations). The effect of d-tubocurarine was reversible.4. When muscles were cultured in Ca2+-free medium containing 1 mM-EGTA and 10 mM-Mg2+, there was no difference in membrane potential between 'near' and 'far' fibres and physostigmine (10-5 M) was ineffective in raising the membrane potential of 'near' fibres.5. It is suggested that non-quantal acetylcholine released from nerve terminals maintains the membrane potential of muscle fibres through a Ca2+-dependent mechanism.* Authors' names are in alphabetical order.

Radioactively labelled leucine and orotic acid were injected into the ventral horn of the lumbar region of the spinal cord. The outflow of labelled products into the sciatic nerve was studied. Leucine is rapidly incorporated into protein and to a lesser extent into lipid. The labelled protein is transported down the nerve, apparently by axoplasmic flow. This labelled protein was present in all sub-cellular fractions of nerve although the soluble fraction had the highest specific activity. Orotic acid is converted to a number of nucleotide derivatives and RNA. Both the nucleotides and RNA move down the sciatic nerve, but the pattern of movement is diffuse whereas protein appears to move as a discrete band. The results suggest that two systems may be operating, one in which part of the RNA is transported by axoplasmic flow along the nerve and the other involving a synthesis of RNA in the nerve from precursors which flow down the nerve. This labelled RNA was membrane bound and on sedimentation analysis, proved to be predominantly of ribosomal type.

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Small doses of botulinum toxin can produce partial blockage of transmitter release at the nerve‐‐muscle junction. 2. Subthreshold e.p.p.s, 3‐‐10 days after poisoning, show a distribution of amplitudes that is fitted by Poisson statistics. Successive e.p.p.s. in a short train show a marked facilitation. 3. Two weeks or more after poisoning with a dose of toxin that paralyses the whole muscle, when nerve‐‐muscle transmission is in course of recovery, subthreshold e.p.p.s have an amplitude distribution that is fitted by binomial statistics. This property of transmission is similar to those described in newly formed nerve‐‐muscle junctions, during embryogenesis or regeneration. 4. Muscle fibres with subthreshold transmission in the 5‐‐10 day group of muscles were all supersensitive to ACh, as were a number of fibres in which nerve stimulation still produced an action potential. 5. Two weeks or more after poisoning, muscle fibres with subthreshold transmission had lost their extrajunctional ACh‐sensitivity, as had many fibres with m.e.p.p.s of roughly normal frequency but no response to nerve stimulation. 6. In diaphragm muscles poisoned with botulinum toxin between 1 and 4 days previously, the rate of fast axonal transport of radioactively labelled proteins down the phrenic nerve is not greatly affected, but the amount of materials carried is reduced to about one quarter of normal. These labelled proteins accumulate in the intramuscular portion of the phrenic nerve, in or near the nerve terminals, to a much greater extent than in controls, showing that the normal release of some of these materials has been prevented by the toxin. 7. It is concluded that the blockage of the trophic effects of nerves by botulinum toxin is due to a blockage of release of trophic factors other than ACh. 8. The muscle nerve cannot maintain a muscle in its normal state simply by activation of contraction, and a regenerating nerve terminal can restore a muscle towards its normal state before it can release enough ACh to produce muscle contraction.