SPECIFIC PROTEOME OF MAMMALIAN CORTEX INHIBITORY & EXCITATORY SYNAPSES

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Four years ago, in collaboration with Nat Heintz (Rockefeller University), we initiated the study of the protein complement present at excitatory synapses in Purkinje cells. We used the Bacterial Artificial Chromosome (BAC) modification strategy to target the specific in vivo expression of GFP-fused GRID 2 to Purkinje cell's excitatory synapses. We performed dissections of mouse cerebella, and purified synapses bearing GFP-GRID2. Although challenging, our approach proved successful, as we isolated synapses and analyzed low-femtomol levels of proteins. During this last year, we continued our mass spectrometric analyses and identified ~70 synaptic proteins, confirming known excitatory proteins, the absence of inhibitory proteins, and identifying novel signatures of excitatory synapses. We have published a manuscript describing this work (F. Selimi, I. Cristea, E. Heller, B.T. Chait, N. Heintz "Proteomic studies of a single CNS synapse type: the parallel fiber/Purkinje cell synapse" PLoS Biology, 2009 Apr 14;7(4):e83). Using a similar approach to the one described above, we hare currently studying the protein composition of inhibitory synapses by isolating GABA receptors from specific cell populations in the Cortex. A paper describing this work has been prepared for submission. The following is the abstract from this manuscript: Electron microscopic studies of the mammalian brain revealed that there are two major classes of synapses (1). Type 1, excitatory synapses were defined as "asymmetric" based on the electron dense material directly apposed to the post- but not the presynaptic membrane. Biochemical studies of this postsynaptic density (PSD) have established it as a complex signal-processing machine that controls synaptic plasticity (2-5). Type 2, inhibitory synapses were defined as "symmetric" because the PSD is greatly reduced or absent. We report here that symmetric synapses contain a variety of neurotransmitter receptors, neural cell-scaffolding and adhesion molecules, but that they are entirely lacking in cell signaling proteins. This fundamental distinction between the functions of excitatory and inhibitory synapses in the nervous system has far reaching implications for models of synaptic plasticity, rapid adaptations in neural circuits, and longer term homeostatic mechanisms controlling the balance of excitation and inhibition in the mature brain.