Abstract
Numerous studies suggest that the extracellular matrix protein agrin directs the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). Strong support for this hypothesis comes from the observation that the high density of acetylcholine receptors (AChR) normally present at the neuromuscular junction fails to form in muscle of embryonic agrin mutant mice. Agrin is expressed by many populations of neurons in the central nervous system (CNS), suggesting that this molecule may also play a role in neuron-neuron synapse formation. To test this hypothesis, we examined synapse formation between cultured cortical neurons isolated from agrin-deficient mouse embryos. Our data show that glutamate receptors accumulate at synaptic sites on agrin-deficient neurons. Moreover, electrophysiological analysis demonstrates that functional glutamatergic and gamma-aminobutyric acid (GABA)ergic synapses form between mutant neurons. The frequency and amplitude of miniature postsynaptic glutamatergic and GABAergic currents are similar in mutant and age-matched wild-type neurons during the first 3 weeks in culture. These results demonstrate that neuron-specific agrin is not required for formation and early development of functional synaptic contacts between CNS neurons, and suggest that mechanisms of interneuronal synaptogenesis are distinct from those regulating synapse formation at the neuromuscular junction.
Highlights
Agrin is synthesized by motor neurons (Rupp et al, 1991; Tsim et al, 1992) and is present at high concentrations at the neuromuscular junction (NMJ), where its appearance coincides with the earliest forming acetylcholine receptors (AChR) clusters during development (Reist et al, 1987; Fallon and Gelfman, 1989)
Neurons appeared healthy for at least 3 weeks in culture with no obvious morphological differences at any stage of development between cultures prepared from cortices of homozygous wild-type control or agrin-deficient embryos
We analyzed morphological and functional correlates of synapse formation occurring between cortical neurons isolated from agrin-deficient mouse embryos developing in dissociated cell culture
Summary
Agrin is synthesized by motor neurons (Rupp et al, 1991; Tsim et al, 1992) and is present at high concentrations at the NMJ, where its appearance coincides with the earliest forming AChR clusters during development (Reist et al, 1987; Fallon and Gelfman, 1989). We hypothesized that if the role of agrin at interneuronal synapses is similar to its action at the NMJ, agrin-deficient mice should exhibit deficits in the clustering of neurotransmitter receptors in the postsynaptic membranes of neurons and related alterations in synaptic transmission in the CNS This prediction is difficult to test in vivo as mice homozygous for the mutant agrin gene exhibit a perinatal lethal phenotype (Gautam et al, 1996). The results of this study show clusters of glutamate receptors at synaptic sites and functionally active synapses between neurons lacking “active” agrin that were indistinguishable from those in wild-type neurons during the first 3 weeks in culture These data suggest that neuron–neuron synapse formation does not show the same agrin-dependence as the NMJ
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