The distribution of acetylcholine receptor clusters and sites of transmitter release along chick ciliary ganglion neurite-myotube contacts in culture.

Abstract

Abstract. Acetylcholine receptors accumulate along the length of cholinergic neuron-skeletal muscle contacts in vitro. The main purpose of this study was to describe, in a quantitative way, the distribution of acetylcholine receptor clusters induced by ciliary ganglion neurons over a period of time extending from hours to weeks after contacts are established. Neurites were filled with Lucifer Yellow and receptor clusters were identified with rhodamine-bungarotoxin. A cluster located within 5 gm of a nerve process or I0 l~m of the base of a growth cone was considered to be a neuriteassociated receptor patch (NARP). The first synaptic potentials were evoked 20 min after growth cone-myotube contact, and, after 24 h of co-culture, >60% of the nerve-muscle pairs tested were functionally connected. NARPs appear rapidly; the first clusters were detected "o6 h after the neurons were plated. They were composed of several small subclusters or speckles of rhodamine-bungarotoxin fluorescence. The initial accumulation of receptors may occur at the advancing tips of nerve processes because NARPs were found at >80 % of the growth cone-muscle contacts examined between 12 and 24 h of co-culture. Over the 3-wk period examined, the mean incidence of NARPs ranged between 1.0 and 2.6 per 100 gm of neurite-myotube contact, with the peak observed on the second day of co-culture. During the first 3 d in culture, when the neurons were multipolar, nearly all of the primary processes induced one or more clusters. With time, as the neurons become unipolar (Role and Fischbach, 1987) NARPs persisted along the remaining dominant process. Measurements made during the third day of co-culture suggest that NARPs disappear along shorter neurites before they retract. Synaptic currents were detected by focal extracellular recording at 55 % of the NARPs. The fact that spontaneous or evoked responses were not recorded at 45 % suggests that contacts with clusters exhibit two functional states. Two types of presynaptic specialization at identified NARPs observed by scanning electron microscopy appear to be correlated with the functional state.MBRYONIC motoneurons promote the accumulation of acetylcholine (ACh) I receptors (AChRs) at newly formed nerve-muscle junctions in vivo and in vitro Cohen, 1980;Steinbach and Bloch, 1986;Schuetze and Role, 1987). In chick cultures, it is clear that receptors accumulate soon after nerve-muscle contact is established, and that each neuron is capable of inducing more than one receptor cluster (Frank and Fischbach, 1979;Role et al., 1985). However, little information is available 1. Abbreviations used in this paper: ACh, acetylcholine; AChR, acetylcholine receptor; NARP(s), neurite-associated receptor patch(es); R-BTX, rhodamine-conjugated alpha-bungarotoxin.Portions of this work have appeared in abstract form (1982. Soc. Neurosci.

Changes in the number of chick ciliary ganglion neuron processes with time in cell culture.

“…All of the processes were thin at their point of exit from the cell body and they did not taper rapidly with distance from the soma. Moreover, all of the processes tested conducted impulses and they formed synapses on contacted myotubes (see Role et al, 1987). The number of processes per neuron and the time course of their elimination in vitro is consistent with their identity as axons.…”

“…This was demonstrated by recording spikes with focal extracellular electrodes positioned along individual processes at different distances from the cell bodies (Fig. 9), and also by the fact that soma stimulation reliably evoked synaptic potentials in innervated myotubes (Role et al, 1985(Role et al, , 1987. From the delay in stimulus-spike or stimulus-epp intervals, the conduction velocity of ciliary neurites was *0.1 m/s.…”

“…3). This technique, which has proved extremely useful (Role et al, 1987) rents of <1 nA. Slightly larger currents were required when the stimulating electrodes were filled with a solution containing 140 mM NaC1 instead of 140 mM KC1, presumably because the conductance of the sealed-off membrane patch was lower in this solution.…”

Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor

“…These neurons have previously been demonstrated to form functional synaptic connections with myotubes in vitro and to induce the AChR to cluster beneath the nerve terminal (Cohen and Fischbath, 1977;Role et al, 1985Role et al, , 1987. In Figure lA, a nerveinduced AChR cluster is shown at the site of a nerve-muscle contact.…”

Local accumulation of acetylcholine receptors is neither necessary nor sufficient to induce cluster formation

“…Prior to innervation, the AChRs are distributed diffusely; clustering begins shortly after neuronal contact both in vivo (Blackshaw and Warner, 1976;Creazzo and Sohal, 1983) and in vitro (Frank and Fischbach, 1979;Kidokoro et al, 1980;Role et al, 1987). The development and stabilization of receptor clusters continues over a period of days to weeks and is likely to involve multiple mechanisms acting in concert as the cluster matures.…”