Studies of nerve - muscle interactions in Xenopus cell culture: analysis of early synaptic currents (original) (raw)

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

Proceedings of the National Academy of Sciences, 1982

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.

Physiological regulation of synaptic effectiveness at frog neuromuscular junctions

The Journal of physiology, 1980

1. Nerve terminals in two different muscles of the frog, the sartorius and cutaneous pectoris (c.p.), have been found to differ sharply in safety factor. This difference is shown to be attributable to corresponding disparities in the amount of transmitter released, without evident correlated morphological differences. 2. In Ringer containing 0.3 mM-Ca2+ and 1 mM-Mg2+, quantal content of c.p. junctions exceeded that of sartorius junctions by 3-4 times. 3. When quantal content was corrected for nerve terminal size, c.p. terminals still released 2-4 times more transmitter per unit terminal length. 4. Light and electron microscopic examination of junctional morphology in the two muscles revealed no significant difference in the spacing of presynaptic active zones, the width of synaptic contact, or the density of presynaptic vesicles and mitochondria. It seems likely, therefore, that the greater release at c.p. junctions is due to a 'physiological' difference between the two popu...

Nerve, Muscle and Synaptogenesis

The vertebrate skeletal neuromuscular junction (NMJ) has long served as a model system for studying synapse structure, function and development. Over the last several decades a neuron-specific isoform of agrin, a heparan sulfate proteoglycan, has been identified as playing a central role in synapse formation at all vertebrate skeletal neuromuscular synapses. While agrin was initially postulated to be the inductive molecule that initiates synaptogenesis, this model has been modified in response to work showing that postsynaptic differentiation can develop in the absence of innervation, and that synapses can form in transgenic mice in which the agrin gene is ablated. In place of a unitary mechanism for neuromuscular synapse formation, studies in both mice and zebrafish have led to the proposal that two mechanisms mediate synaptogenesis, with some synapses being induced by nerve contact while others involve the incorporation of prepatterned postsynaptic structures. Moreover, the curren...

Simultaneous pre- and post-synaptic electrophysiological recording from Xenopus nerve-muscle co-cultures

Journal of visualized experiments : JoVE, 2013

Much information about the coupling of presynaptic ionic currents with the release of neurotransmitter has been obtained from invertebrate preparations, most notably the squid giant synapse. However, except for the preparation described here, few vertebrate preparations exist in which it is possible to make simultaneous measurements of neurotransmitter release and presynaptic ionic currents. Embryonic Xenopus motoneurons and muscle cells can be grown together in simple culture medium at room temperature; they will form functional synapses within twelve to twenty-four hours, and can be used to study nerve and muscle cell development and synaptic interactions for several days (until overgrowth occurs). Some advantages of these co-cultures over other vertebrate preparations include the simplicity of preparation, the ability to maintain the cultures and work at room temperature, and the ready accessibility of the synapses formed. The preparation has been used widely to study the biophys...