Sequential formation of GABAergic and glutamatergic synapses is thought to be crucial for constructing the stereotypic neural networks during brain development. However, why GABAergic synapses are formed earlier than glutamatergic synapses in the developing brain is not well understood. We used electrophysiology and fluorescence imaging to study GABAergic and glutamatergic synaptogenesis in embryonic hypothalamic cultures, which contain ∼40% GABAergic and ∼60% glutamatergic neurons. The newly dissociated embryonic hypothalamic neurons contained a significant pool of functional GABAA receptors but a very low level of glutamate receptors. Within the first week of culture, the time course of GABAergic synaptogenesis in embryonic neurons coincided with that of presynaptic vesicle cycling, but both measurements lagged behind the detection of functional GABAA receptors. Remarkably, the GABAA receptors of newly dissociated embryonic neurons can be rapidly clustered into postsynaptic apparatus and generate functional synaptic currents within 4-6 h when cocultured with mature neurons. Consistent with earlier expression of GABAA receptors in immature neurons, synaptic GABAergic events were always detected before the onset of glutamatergic events in both purely embryonic and heterochronic cultures. Interestingly, overexpression of glutamate receptors in embryonic neurons not only increased whole-cell glutamate currents but also significantly increased the frequency of excitatory synaptic events. We conclude that the sequential formation of GABAergic and glutamatergic synapses in immature neurons is likely governed by a sequential expression of GABAA and glutamate receptors during neuronal development.
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