Abstract

Factors that regulate the formation, spatial patterning, and maturation of CNS synapses are poorly understood. We used organotypic hippocampal slice cultures derived from developing (P5-P7) rat to test whether synaptic activity regulates the development and organization of postsynaptic structures at mossy fiber (MF) giant synapses. Antibodies to a prominent postsynaptic density (PSD) scaffold protein, PSD95, identified large (>1 microm) and irregularly shaped PSD assemblies that codistributed with synapsin-I or metabotropic glutamate receptor 7b (mGluR7b) -immunolabeled MF terminals in area CA3. To investigate the spatial organization of synaptic PSDs on individual pyramidal cells, neurons in slice cultures were transfected with a vector encoding a GFP-PSD95 fusion protein. Confocal three-dimensional reconstructions revealed clusters of PSDs along proximal dendrites of transfected pyramidal neurons in area CA3, but not in CA1. Clusters averaged 7.6 microm in length (range, 2.2-29 microm) and contained up to 35 individual PSDs (mean, 8.3). PSD clusters failed to form when slices were cultured without MFs, indicating that MFs induce cluster assembly. Chronic blockade of N-methyl-D-apartate- and AMPA/kainate-type glutamate receptors did not disrupt MF targeting or de novo formation of PSD clusters with a normal distribution on target cells. Additionally, glutamate receptor blockers did not alter the ultrastructural development of MF giant synapses containing multiple puncta adherens-like junctions and asymmetric synaptic junctions at dendritic shaft and spine domains, respectively. The results indicate that MF axons can induce the assembly and clustering of PSD95-containing postsynaptic complexes, displaying a normal subcellular and tissue distribution, by mechanisms that are independent of ionotropic glutamate receptor activation.

Copyright 2001 Wiley-Liss, Inc.