The effects of intracellular pH changes on the acetylcholine (ACh) release and cytoplasmic Ca2+ concentration at developing neuromuscular synapses were studied in Xenopus nerve‐muscle co‐cultures. Spontaneous and evoked ACh release of motoneurons was monitored by using whole‐cell voltage‐clamped myocytes. Intracellular alkalinization with 15 mm NH4Cl slightly reduced the frequency of spontaneous synaptic currents (SSCs). However, cytosolic acidification following withdrawal of extracellular NH4Cl caused a marked and transient increase in spontaneous ACh release. Another method of cytosolic acidification was used in which NaCl in Ringer solution was replaced with weak organic acids. The increase in spontaneous ACh release paralleled the level of intracellular acidification resulting from addition of these organic acids. Acetate and propionate but not isethionate, methylsulphate and glucuronate, caused an increase in intracellular pH and a marked increase in spontaneous ACh release. Impulse‐evoked ACh release was slightly augmented by intracellular alkalinization and inhibited by cytosolic acidification. Cytosolic acidification was accompanied by an elevation in the cytoplasmic Ca2+ concentration ([Ca2+]i), resulting from both external Ca2+ influx and intracellular Ca2+ mobilization. In contrast, the increase in [Ca2+]i induced by high K+ was inhibited by cytosolic acidification. We conclude that cytosolic acidification regulates spontaneous and evoked ACh release differentially in Xenopus motoneurons, increasing spontaneous ACh release but inhibiting evoked ACh release.

NMDA receptors play important roles in synaptic plasticity and neuronal development. The functions of NMDA receptors are modulated by many endogenous substances, such as external pH (pH e ), as well as second messenger systems. In the present study, the nerve-muscle cocultures of Xenopus embryos were used to investigate the effects of both external and intracellular pH (pH i ) changes on the functional responses of presynaptic NMDA receptors. Spontaneous synaptic currents (SSCs) were recorded from innervated myocyte using whole-cell recordings. Local perfusion of NMDA at synaptic regions increased the SSC frequency via the activation of presynaptic NMDA receptors. A decrease in pH e from 7.6 to 6.6 reduced NMDA responses to 23% of the control, and an increase in pH e from 7.6 to 8.6 potentiated the NMDA responses in increasing SSC frequency.

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.

Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission. Here we report the non‐quantal release of ACh from embryonic myocytes. Co‐cultures of spinal neurons and myotomal muscle cells were prepared from 1‐day‐old Xenopus embryos. Single channel currents were recorded in the non‐innervated myocytes. When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d‐tubocurarine (d‐Tc). The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic‐type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution. When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration‐dependent manner increased channel open probability; it was reduced by d‐Tc or α‐bungarotoxin. Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine. Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release. The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts. These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non‐quantal manner. Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.

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