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The mechanism of the action of beta-bungarotoxin (beta-BuTx) in the facilitation of spontaneous transmitter release at neuromuscular synapse was investigated in Xenopus cell culture using whole-cell patch clamp recording. Exposure of the culture to beta-BuTx dose-dependently enhances the frequency of spontaneous synaptic currents (SSCs). Buffering the rise of intracellular Ca2+ with BAPTA-AM hampered the facilitation of SSC frequency induced by beta-BuTx. The beta-BuTx-enhanced SSC frequency was reduced when the pharmacological Ca2+ -ATPase inhibitor thapsigargin was used to deplete intracellular Ca2+ store. Application of membrane-permeable inhibitors of inositol 1,4,5-trisphosphate (IP3) but not ryanodine receptors effectively occluded the increase of SSC frequency elicited by beta-BuTx. Treating cells with either wortmannin or LY294002, two structurally different inhibitors of phosphatidylinositol 3-kinase (PI3K) and with phospholipase C (PLC) inhibitor U73122, abolished the beta-BuTx-induced facilitation of synaptic transmission. The beta-BuTx-induced synaptic facilitation was completely abolished while there was presynaptic loading of the motoneuron with GDPbetaS, a non-hydrolyzable GDP analogue and inhibitor of G protein. Taken collectively, these results suggest that beta-BuTx elicits Ca2+ release from the IP3 sensitive intracellular Ca2+ stores of the presynaptic nerve terminal. This is done via PI3K/PLC signaling cascades and G protein activation, leading to an enhancement of spontaneous transmitter release.

In the present study, Xenopus nerve-muscle cultures were used to explore the functional roles of A chain (a phospholipase A(2) subunit) and B chain (a non-phospholipase A(2) subunit) of Bungarus multicinctus beta-bungarotoxin. It was found that beta-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve-muscle cultures. Modification of beta-bungarotoxin with pyridoxal-5'-phosphate or substitution of Ca(2+) with Ba(2+) in buffer abolished the phospholipase A(2) activity of beta-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A(2) activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of beta-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve-muscle cultures.

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.

The effects of endogenously released ATP on the maturation of developing neuromuscular synapses were investigated in Xenopus nerve-muscle co-cultures. The potentiating action of ATP (1 mM) on spontaneous acetylcholine release was inhibited by P2-purinoceptor antagonists suramin (0.3 mM) and reactive blue 2 (RB-2, 3 microM) in day 1 cultures. Bath application of suramin (10 microM) or RB-2 in day 1 cultures and prolonged treatment for 2 days dramatically decreased the amplitude of both spontaneous synaptic currents (SSCs) and evoked synaptic currents (ESCs) in the same cultures on day 3. Chronic treatment with 8-cyclopentyltheophylline (4 microM) or 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX, 10 microM), P1-purinoceptor and glutamate receptor antagonists respectively, did not exert such an inhibitory effect. Chronic treatment with suramin or RB-2 for 2 days had no significant effect on the amplitude of either iontophoretic acetylcholine-induced whole-cell currents or single acetylcholine channel measurements in 3-day-old cultured myocytes. In addition, prolonged treatment for 2 days with various kinase inhibitors such as H-8 (10 microM), KN-62 (5 microM) and H-7 (10 microM) also decreased the amplitudes of both spontaneous and evoked synaptic currents in natural synapses, but not those of iontophoretic acetylcholine-induced currents. Furthermore, suramin and these protein kinase inhibitors also decreased the amplitude of spontaneous synaptic currents in manipulated synapses of 'vacated' nerve terminals. The results suggest that endogenously released ATP, acting in concert with various protein kinases, is involved in the maintenance and/or development of the quantum size of synaptic vesicles at embryonic neuromuscular synapses.

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.

1. Glutamate receptors play important roles in synaptic plasticity and neural development. Here we report that, at the developing neuromuscular synapses in Xenopus cultures, the activation of presynaptic glutamate receptors at motor nerve terminals potentiates spontaneous acetylcholine (ACh) release. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes using whole-cell recording. Bath application of glutamate (10 microM) markedly increased the frequency of SSCs, and the action of glutamate was reversible. 3. Pretreatment with 0.3 microM tetrodotoxin, which blocks Na+ channels and the conduction of action potentials, only slightly inhibited the potentiating action of glutamate on SSCs. Furthermore, the enhancement of ACh secretion was much more prominent when glutamate was applied locally to the synaptic region. 4. Three types of glutamate receptor agonists, kainate, quisqualate, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and NMDA (N-methyl-D-aspartate), were effective in inducing the potentiating effect. The ranking order was: glutamate > kainate > NMDA > AMPA > quisqualate. Glycine potentiated the effects induced by NMDA. Metabotropic receptors were not involved in the potentiating action of glutamate. 5. The potentiating effect of glutamate depended on the influx of Ca2+ through both L-type Ca2+ channels and NMDA-gated channels. 6. Since glutamate is known to be co-released with ACh at some cholinergic nerve terminals, the released glutamate may serve as a positive feedback regulation of ACh secretion at developing neuromuscular junctions via its action on presynaptic glutamate receptors.