On the mechanism of acetylcholine receptor accumulation at newly formed synapses on chick myotubes

Role, L. W.; Matossian, VR; O'Brien, RJ; Fischbach, G D

doi:10.1523/jneurosci.05-08-02197.1985

Cited by 109 publications

(96 citation statements)

References 18 publications

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“…There are two possible explanations for the decrease in surface nAChRs caused by α7/α3 overexpression: competition for local insertion into the postsynaptic membrane, and disruption of targeting to the synapse, which may cause endogenous nAChRs to be localized extrasynaptically where they are less metabolically stable. There is support for both possibilities from studies of nAChRs in skeletal muscle 19,20 . In contrast to α7/α3, overexpression of α7/α5 chimeric subunits results in a perisynaptic localization and no alteration in endogenous nAChR surface levels.…”

Section: Discussionmentioning

confidence: 96%

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

et al. 1998

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ResultsUsing PCR, we constructed chimeric subunits in which the α7 cytoplasmic loop immediately after TM3 and before TM4 was replaced with the homologous region of the α3 or α5 sequence (Fig. 1a). The N-terminus up to TM2 regulates intersubunit assembly into receptor complexes, and α7 subunits do not coassemble with nAChR subunits, as demonstrated for endogenous subunits in CG neurons and for chimeric α7 subunits expressed in Xenopus laevis oocytes 4,5,12,13 . In particular, we have observed that coexpression of chimeric α7 subunits containing the α3 cytoplasmic loop together with wild-type α3 and β4 subunits in Xenopus oocytes results in the formation of two distinct receptor types that have the pharmacological properties of Bgt-nAChRs and nAChRs, respectively (data not shown). Thus, a difference in the distribution of chimeric α7 as compared to wild-type α7 on the infected CG neuron surface would result from the added α3 or α5 cytoplasmic loop. It cannot be explained by assembly with an endogenous nAChR subunit that can target to the synapse. Different types of neurotransmitter receptors coexist within single neurons and must be targeted to discrete synaptic regions for proper function. In chick ciliary ganglion neurons, nicotinic acetylcholine receptors (nAChRs) containing α3 and α5 subunits are concentrated in the postsynaptic membrane, whereas α-bungarotoxin receptors composed of α7 subunits are localized perisynaptically and excluded from the synapse. Using retroviral vector-mediated gene transfer in vivo, we show that the long cytoplasmic loop of α3 targets chimeric α7 subunits to the synapse and reduces endogenous nAChR surface levels, whereas the α5 loop does neither. These results show that a particular domain of one subunit targets specific receptor subtypes to the interneuronal synapse in vivo. Moreover, our findings suggest a difference in the mechanisms that govern assembly of interneuronal synapses as compared to the neuromuscular junction in vertebrates.1998 Nature America Inc.• http://neurosci.nature.com 1998 Nature America Inc.• http://neurosci.nature.com

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Section: Discussionmentioning

confidence: 96%

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

et al. 1998

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“…Over the next several days all but one of the processes retract, and the neurons remain unipolar for at least 3 wk, the longest time examined. We previously reported that more than one primary process was associated with receptor clusters (Role et al, 1985). In this paper we describe the distribution of NARIs at early times in more detail, the distribution during the period of process pruning, and the distribution at later times.…”

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confidence: 89%

“…(NARPs), appear at ciliary ganglion-myotube contacts with about the same incidence as they do along processes of spinal cord motoneurons (Role et al, 1985).…”

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confidence: 94%

“…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.…”

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confidence: 99%

“…Several lines of evidence suggest that NARPs mark sites of synapse formation in chick and Xenopus cultures. They are present on innervated muscle cells Fischbach and Cohen, 1973;Frank and Fischbach, 1979), and they are apparently uniquely associated with neurites of cholinergic neurons (Cohen and Weldon, 1980;Role et al, 1985). In Xenopus cultures, the amplitude and frequency of mepps are correlated with the extent of receptor accumulation (Anderson et al, 1979).…”

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

Role¹,

Roufa²,

Fischbach³

1987

The Journal of Cell Biology

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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.

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Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: Refinements and applications

Daniels

Lowe

Shah

et al. 2000

Microsc. Res. Tech.

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Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve‐muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better‐defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon‐induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon‐induced AChR aggregation on muscle cell species, by the use of chick‐rat chimeric co‐cultures. (5) We provide evidence for the role of alternatively‐spliced agrin isoforms in synapse formation by using single cell RT‐PCR with neurons collected from co‐cultures after observation of axon‐induced AChR aggregation. Microsc. Res. Tech. 49:26–37, 2000. Published 2000 Wiley‐Liss, Inc.

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On the mechanism of acetylcholine receptor accumulation at newly formed synapses on chick myotubes

Cited by 109 publications

References 18 publications

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

The long internal loop of the α3 subunit targets nAChRs to subdomains within individual synapses on neurons in vivo

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

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: Refinements and applications

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