Abstract
BackgroundActivity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons.Methodology/Principal FindingsGluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures.Conclusions/SignificanceThe enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde ‘reward’ signal generated by WT neurons, although in this paradigm there was no ‘punishment’ signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous system.
Highlights
Synaptic activity mediated by NMDA type glutamate receptors sculpts the wiring of the nervous system, regulating functional and structural connectivity [1,2]
We find that GluN1 -/- and wild type hippocampal neurons develop activity-dependent differences in synapse density only when forced to compete in co-cultures of defined ratio
Wild type hippocampal neurons when cultured together with predominantly GluN1 -/- neighbours developed increased synapse density compared with their GluN1 -/- neighbours and compared with sister wild type neurons cultured alone
Summary
Synaptic activity mediated by NMDA type glutamate receptors sculpts the wiring of the nervous system, regulating functional and structural connectivity [1,2]. NMDA receptors sculpt much of the developing circuitry in the nervous system [1,2,4], the precise rules of competition and underlying molecular mechanisms are not well understood. Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. We develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons
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