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Glutamate receptors mediate the majority of rapid excitatory synaptic transmission in the central nervous system (CNS) and play important roles in synaptic plasticity and neuronal development. Recently, protein-protein interactions with the C-terminal domain of glutamate receptor subunits have been shown to be involved in the modulation of receptor function and clustering at excitatory synapses. In this paper, we have found that the N-ethylmaleimide-sensitive factor (NSF), a protein involved in membrane fusion events, specifically interacts with the C terminus of the GluR2 and GluR4c subunits of AMPA receptors in vitro and in vivo. Moreover, intracellular perfusion of neurons with a synthetic peptide that competes with the interaction of NSF and AMPA receptor subunits rapidly decreases the amplitude of miniature excitatory postsynaptic currents (mEPSCs), suggesting that NSF regulates AMPA receptor function.

The molecular mechanisms underlying the targeting and localization of glutamate receptors at postsynaptic sites is poorly understood. Recently, we have identified a PDZ domain-containing protein, glutamate receptor-interacting protein 1 (GRIP1), which specifically binds to the C termini of AMPA receptor subunits and may be involved in the synaptic targeting of these receptors. Here, we report the cloning of GRIP2, a homolog of GRIP1, and the characterization of the GRIP1 and GRIP2 proteins in the rat CNS. GRIP1 and GRIP2 are approximately 130 kDa proteins that are highly enriched in brain. GRIP1 and GRIP2 are widely expressed in brain, with the highest levels found in the cerebral cortex, hippocampus, and olfactory bulb. Biochemical studies show that GRIP1 and GRIP2 are enriched in synaptic plasma membrane and postsynaptic density fractions. GRIP1 is expressed early in embryonic development before the expression of AMPA receptors and peaks in expression at postnatal day 8-10. In contrast, GRIP2 is expressed relatively late in development and parallels the expression of AMPA receptors. Immunohistochemistry using the GRIP1 antibodies demonstrated that GRIP1 is expressed in neurons in a somatodendritic staining pattern. At the ultrastructural level, DAB and immunogold electromicroscopy studies showed that GRIP1 was enriched in dendritic spines near the postsynaptic density and was expressed in dendritic shafts and in peri-Golgi regions in the neuronal soma. GRIP1 appeared to be clustered at both glutamatergic and GABAergic synapses. These results suggest that GRIP1 and GRIP2 are AMPA receptor binding proteins potentially involved in the targeting of AMPA receptors to synapses. GRIP1 also may play functional roles at both excitatory and inhibitory synapses, as well as in early neuronal development.

Semaphorins provide signals that guide growing axons to their appropriate destinations. The secreted semaphorin, Sema3A, mediates repulsive effects on axons from various neuronal populations in embryonic rats. The authors localized Sema3A mRNA expression in the primary olfactory pathway during development, in adult rats, and in adult rats that were subjected to a unilateral olfactory bulbectomy. Developing rats at ages from embryonic day 14 (E14) to E19 expressed Sema3A in the olfactory receptor neurons (ORNs) of the olfactory epithelium and in chondrogenic structures surrounding the nasal cavity. In vitro, ORN axons at E14 avoided substrate-bound Sema3A. Low levels of Sema3A expression persisted in the normal adult epithelium both in ORNs scattered throughout the epithelium and in small clusters. Three days after a unilateral olfactory bulbectomy, Sema3A transcript levels increased in regenerating neurons. High levels of Sema3A transcript were found at 1 week postbulbectomy, persisted for 2 weeks, and diminished by 3 weeks. Several other murine semaphorins (Sema4A, Sema4B, and Sema4C) were expressed differentially in the primary olfactory pathway both during development and regeneration. These findings suggest that Sema3A and perhaps other semaphorins play a role in directing ORNs out of the epithelium and to the olfactory bulb, their target structure, during both development and regeneration.

Semaphorins provide signals that guide growing axons to their appropriate destinations. The secreted semaphorin, Sema3A, mediates repulsive effects on axons from various neuronal populations in embryonic rats. The authors localized Sema3A mRNA expression in the primary olfactory pathway during development, in adult rats, and in adult rats that were subjected to a unilateral olfactory bulbectomy. Developing rats at ages from embryonic day 14 (E14) to E19 expressed Sema3A in the olfactory receptor neurons (ORNs) of the olfactory epithelium and in chondrogenic structures surrounding the nasal cavity. In vitro, ORN axons at E14 avoided substrate-bound Sema3A. Low levels of Sema3A expression persisted in the normal adult epithelium both in ORNs scattered throughout the epithelium and in small clusters. Three days after a unilateral olfactory bulbectomy, Sema3A transcript levels increased in regenerating neurons. High levels of Sema3A transcript were found at 1 week postbulbectomy, persisted for 2 weeks, and diminished by 3 weeks. Several other murine semaphorins (Sema4A, Sema4B, and Sema4C) were expressed differentially in the primary olfactory pathway both during development and regeneration. These findings suggest that Sema3A and perhaps other semaphorins play a role in directing ORNs out of the epithelium and to the olfactory bulb, their target structure, during both development and regeneration.

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