Initial synaptic transmission at the growth cone in Xenopus nerve-muscle cultures.

Abstract

The excellent visibility ofcultured cells allows the early events during formation of the neuromuscular junction to be suitably studied. It has been shown in various culture systems that synaptic transmission occurs early after nerve-muscle contact. Early synaptic potentials are small in amplitude and slow in time course reflecting a low acetylcholine receptor density at the site of nerve contact. Acetylcholine receptors accumulate later at the contact region. We have examined initial synaptic transmission at the growth cone-muscle contact in Xenopua nerve-muscle cultures. The approaching growth cone was observed under a phasecontrast microscope while the membrane potential of its target muscle cell was continuously monitored by using an intracellular microelectrode. The innervating neuron was stimulated extracellularly at the cell body. No synaptic potential was evoked when the growth cone was contacting the muscle only at the tip of filopodia. However, as soon as the main portion ofthe growth cone contacted the muscle membrane, nerve-evoked synaptic potentials were detected after stimulation of the nerve. This immediate appearance ofsynaptic potentials raises the possibility that acetylcholine could be released at the growth cone even prior to contact with muscle cells. As the area ofcontact enlarged during the observation period the amplitude of end-plate potentials also increased. Spontaneous synaptic potentials (miniature end-plate potentials) were rarely observed in these early growth cone-muscle contacts. Although there were several inherent difficulties, quantal analysis of the end-plate potentials was attempted by using binomial statistics. This analysis suggests that nerve-evoked transmitter release at the growth cone occurs in a quantal fashion.Functional synaptic transmission occurs early during formation ofthe neuromuscular junction, shortly after nerve-muscle contact (1-5). For example, in chicken nerve-muscle cultures the growth cones form functional synaptic contacts but the amplitudes of the nerve-evoked synaptic potentials are vanishingly small (50 uV on average) and only detectable after summation of the signal (3). The small amplitudes of these early synaptic potentials could be accounted for by relatively low acetylcholine (AcCho) sensitivities of chicken myotubes at regions of low AcCho receptor density, although one cannot exclude the possibility that the amount of AcCho released at the growth cone is smaller. Studies on Xenopus nerve-muscle cultures have shown that early spontaneous synaptic potentials (miniature end-plate potentials, MEPPs) are small in amplitude and slow in time course, probably reflecting a low AcCho receptor density at the contact area. Indeed, staining AcCho receptors with tetramethylrhodamine-conjugated a-bungarotoxin revealed that at this stage the AcCho receptor density is no higher at the site of nerve contact than in the surrounding region (6, 7). Because this indicates that spontaneous release ofAcCho can be detected at any part of the muscle membrane ...

Optical Measurements of Presynaptic Release in Mutant Zebrafish Lacking Postsynaptic Receptors

“…Our observation that presynaptic exocytotic machinery can develop in the absence of postsynaptic receptors agrees with previously published studies showing that nerve-muscle synapses form in the presence of antagonists that block receptor function (Cohen, 1972;Ding et al, 1983;Landmesser and Szente, 1986). Our results are also consistent with the findings that dissociated spinal neuron somas and associated growth cones are capable of both spontaneous (Kidokoro and Yeh, 1982;Hume et al, 1983;Young and Poo, 1983;Chow and Poo, 1985) and evoked (Sun and Poo, 1987) transmitter release. However, studies using Xenopus have further indicated that responses to both spontaneous and stimulusevoked release of ACh from spinal neurons are potentiated on contact with muscle.…”

“…Postsynaptic modulation of transmitter release machinery has been provided from studies that use Xenopus nerve and muscle wherein the formation of synaptic contacts can be strictly controlled. Measurement of synaptic responses from myocytes shows that both motor neuron somas and growth cones can spontaneously release ACh before contact with muscle (Kidokoro and Yeh, 1982;Hume et al, 1983;Young and Poo, 1983;Chow and Poo, 1985). However, contact with muscle potentiates transmitter release, as reflected in the increased size (Evers et al, 1989;Liou et al, , 1999 and frequency of spontaneous events (Xie and Poo, 1986;Evers et al, 1989).…”

Direct Measurements of Presynaptic Calcium and Calcium-Activated Potassium Currents Regulating Neurotransmitter Release at CulturedXenopusNerve–Muscle Synapses

“…Disaggregated cells were then plated onto glass coverslips or plastic tissue-culture dishes and incubated at room temperature (22-24°C) for 24 -48 hr in a medium composed of 49% NFR and 51% L-15 (Life Technologies, Gaithersburg, MD) supplemented with 3 mg/ml glutamine, 0.1 mg/ml insulin, 0.7 mg/ml sodium selenite, 0.6 mg/ml transferrin, 1 mg/ml sodium pyruvate, and 35 ng/ml brain-derived neurotrophic factor [kindly provided by Amgen (Thousand Oaks, CA) and Regeneron (Tarrytown, NY)]. In as little as 12 hr, spinal neurons extended neurites, occasionally elaborating varicose regions on the substrate and in contact with spindle-shaped muscle cells (Kidokoro and Yeh, 1982;Takahashi et al, 1987;Evers et al, 1989;Tabti and Poo, 1994). In 1 and 2 d cultures, the presynaptic contact often takes the form of a varicosity sufficiently large to be accessed with patch electrodes, allowing one to correlate presynaptic electrophysiological events with transmitter release.…”

Physiological properties of newly formed synapses between sympathetic preganglionic neurons and sympathetic ganglion neurons

“…The best information available on the probability of forming a synapse comes from elegant studies of neuromuscular junctions in culture. These studies indicate that functional synapses form with high probability within a few seconds after motor neurons contact muscle (Kidokoro and Yeh, 1982;Sun and Poo. 1987;Evers, Laser, Sun, Xie.…”

“…Vogel, and Nirenberg, 1973;Bevan and Steinbach, 1977), and presynaptic neurons can package and release transmitter before they encounter a muscle cell (Hume, Role, and Fischbach, 1983;Young and Poo, 1983). A consequence of this precocious development is that functional interactions can be seen as soon as the growth cone arrives at the muscle surface (Frank and Fischbach, 1979;Kidokoro and Yeh, 1982;Sun and Poo, 1987). However, even neuromuscular synapses undergo an extended penod of maturation during which their functional properties are extensively modified (see Purves and Lichtman, 1985 for a review).…”