Hippocampal mossy fibers induce assembly and clustering of PSD95-containing postsynaptic densities independent of glutamate receptor activation.

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.