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Although the long-term effects of all-trans retinoic acid (RA) on neuronal growth and differentiation have been intensively studied, nothing is known about its effect on synaptic transmission. Here we show that RA rapidly and specifically enhances the spontaneous acetylcholine release at developing neuromuscular synapses in Xenopus cell culture using whole-cell patch-clamp recording. Acute addition of RA dose-dependently and reversibly enhances the frequency of spontaneous synaptic currents (SSCs). Application of the lipophilic RA analogue all-trans retinol or RA metabolites produced by light-induced decomposition failed to provoke similar changes in SSC frequency, indicating the specificity of RA-induced facilitation of spontaneous transmitter release. Protein synthesis inhibitors anisomycin or cycloheximide had no effect on RA-induced SSC frequency facilitation. Treating cells with pan RA receptor (RAR) selective agonist or RARbeta-selective agonist, but not RARalpha-, RARgamma- or retinoid X receptor (RXR)-selective agonists, mimicked the action of RA. These results suggest that RA acts through the activation of RARbeta, to induce a rapid, non-genomic increase in the frequency of spontaneous transmitter release at developing neuromuscular synapses.

1. Myocyte-dependent regulation of acetylcholine (ACh) quantal secretion from developing motoneurons was studied in day-3 Xenopus nerve-muscle co-cultures. Spontaneous synaptic currents (SSCs) were measured in manipulated synapses by using whole-cell voltage-clamped myocytes. Changes in SSC amplitude were assumed to reflect changes in the ACh content of secreted quantal packets. Compared with natural synapses, motoneurons without any contact with a myocyte (naive neurons) released ACh in smaller quantal packets. 2. Bipolar cultured motoneurons, which were in contact with a myocyte with one axon branch (contact-end) but remained free at another axon branch (free-end), were further used to examine quantal ACh secretion. The ACh quantal size recorded at free-end terminals was similar to that of naive neurons and was smaller than that at the contact-end, indicating that myocyte contact exerts differential regulation on quantal secretion in the same neuron. 3. Some of the neurons that formed a natural synapse with a myocyte continued to grow forward and ACh quantal secretion from the free growth cone was examined. The ACh quantal size recorded at free growth cones was inversely proportional to the distance to the natural synapse, implying localized regulation of quantal secretion by the myocyte. 4. Chronic treatment of day-1 cultures with veratridine and d-tubocurarine, respectively, increased and decreased the neurotrophic action of myocytes when assayed on day 3. 5. Taken together, these findings suggest that the myocyte is an important postsynaptic target in the regulation of quantal secretion and that the trophic action is spatially restricted to the neighbourhood of the neuromuscular junction.

1. The ability of different neurotrophic factors to maintain and regulate synaptic function at the developing motoneuron was studied in Xenopus nerve-muscle co-cultures. Spontaneous synaptic currents (SSCs) were measured by using whole-cell voltage-clamped myocytes. 2. Compared with natural synapses, motoneurons without contact on a myocyte (naive neurons) released ACh in smaller quantal packets, the amplitude being inversely proportional to the days in culture. The mean SSC amplitudes of naive neurons, which were measured by manipulating a myoball into contact with the myocyte-free nerve terminals to form a manipulated synapse, were 99.5 +/- 6.7 and 48.2 +/- 1.9 pA for day-1 and day-3 cultures, respectively. 3. Chronic treatment of day-1 cultures with brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), ciliary neurotrophic factor (CNTF) or glial cell line-derived neurotrophic factor (GDNF) for 2 days, increased the ACh quantal size of naive motoneurons in a concentration-dependent manner, whereas insulin-like growth factor-1 (IGF-1) and basic fibroblast growth factor (bFGF) had no effect, even at high concentrations. 4. The interaction of various neurotrophic factors was examined, using concentrations that gave maximal effects. Combination of CNTF plus BDNF or CNTF plus NT-3 had synergistic effects in potentiating SSC amplitude of the manipulated synapse of naive neurons, whereas NT-3 plus BDNF, NT-3 plus GDNF, BDNF plus GDNF or CNTF plus GDNF had no synergistic action. 5. Chronic treatment with d-tubocurarine for 2 days resulted in a reduction of the quantal size of natural synapses. Concomitant treatment with BDNF, NT-3, GDNF, CNTF but not bFGF or IGF-1, reconstituted the SSC amplitude. 6. Taken together, these findings suggest that BDNF, NT-3, NT-4, CNTF and GDNF may regulate and maintain the synaptic function of developing motoneurons, and different neurotrophic factors utilizing distinct signalling mechanisms may have synergistic actions.

Neurotrophic factors derived from postsynaptic muscle cells may play important roles in the development of presynaptic neuronal functions. In 3-d-old Xenopus nerve-muscle cultures, embryonic spinal neurons that had made natural contact with co-cultured myocytes exhibited spontaneous release of larger packets of acetylcholine (ACh) quanta than those released by the isolated neurons having no contact with any myocyte. Treatment of isolated neurons with neurotrophin-3 (NT-3) for 2 d increased the average sizes of quantal ACh packets at newly formed nerve-muscle synapses, whereas treatment with antibody against NT-3 or with K252a, a specific inhibitor of tyrosine kinase receptors, decreased the quantal size at existing synapses, which suggests that NT-3 supplied by the postsynaptic muscle cell may be responsible for the development and maintenance of the quantal packets. The muscle effect seems to depend on synaptic activities mediated by postsynaptic ACh receptor channels, because chronic treatment of the culture with D-tubocurarine (D-Tc) for 2 d resulted in a marked reduction of the quantal sizes, when assayed after extensive washing of the culture with Ringer's solution. The curare treatment did not affect the postsynaptic ACh receptor sensitivity, because iontophoretically applied ACh induced current responses similar to those of control. Finally, co-treatment of the culture with NT-3 and D-Tc reversed the effect of D-Tc on the quantal size, and this reversal effect was abolished when K252a was also applied concomitantly. Our results suggest that muscle-derived NT-3 participates in the maturation of normal transmitter packets in developing neurons, and the secretion of NT-3 depends on spontaneous synaptic activity.