Development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos and rat pups at birth

We studied the development of excitatory synapses in cultured neurons dissociated from the cortices of rat embryos at the 18th day of gestation (E18) and rat pups at birth (PO). Between 7 and 14 days in vitro (DIV), large increases in the amplitudes and frequencies of the spontaneous excitatory postsynaptic currents (EPSCs) of both cultured E18 and PO neurons were observed. The EPSCs of E18 neurons were mediated primarily by alpha-amino-3-hydroxy-5-methyl-4-iso-xazole-propionic acid (AMPA) receptors at 7 DIV and by both N-methyl-D-aspartate (NMDA) and AMPA receptors at 14 DIV. Consistently, immunostaining indicated significant increases in the proportion of the clusters of NR1, an NMDA receptor subunit, which were associated with the accumulation of synaptophysin, a presynaptic marker, in cultured E18 neurons between 7 and 14 DIV. The proportion of NR1 clusters residing in synaptic regions and the proportion of synapses that colocalized with NR1 clusters in 7-day-old PO neurons were not different statistically from those found in 7-day-old E18 neurons. However, cultured PO neurons at 7 DIV displayed clear EPSCs mediated by NMDA receptors. Our results suggest that the targeting of NMDA receptors to synaptic regions lag behind the synaptic clustering of AMPA receptors during the in vitro development of cultured rat El 8 cortical neurons. The results further suggest that the cortical neurons at PO differ from those at El 9 in certain cellular properties; as a result, the currents mediated by the synaptic NMDA receptors in 7-day-old PO neurons are larger than those mediated by the synaptic NMDA receptors in 7-day-old E18 neurons. (C) 2002 Wiley-Liss, Inc.