siRNA Knockdown of PSD-95 in the Developing Rat

Massachusetts Institute Of Technology, Cambridge MA

Investigators

Abstract

[unreadable] DESCRIPTION (provided by applicant): Our goal is to test the postulate that N-methyl-D-aspartate (NMDA) receptor (NR) scaffolding proteins are crucial for activity-dependent glutamate-mediated developmental plasticity. These studies will focus on the molecular suppression of the PSD-95 synaptic scaffolding molecule. Lentivirus vectors will introduce an eGFP reporter and DMA hairloop coding for siRNA to specifically suppress PSD-95 message. In vitro studies using cultured superior colliculus (SC) and visual cortex neurons will be used to characterize the protein and message knockdown of the PSD-95 genes. Another set of in vitro studies will also be performed to determine whether PSD-95 is critical in synaptic scaffolding and glutamate currents. We will ultimately study changes in glutamate currents and synaptic refinement in the visual pathway of intact rats. Small populations of neurons in the SC will be transfected with one of the viral constructs to study a few affected cells in an otherwise normally developing SC. Controlled eye opening will be used to coordinate known events and assess the effects of PSD-95 knockdown. These experiments will be studied using quantitative RT-PCR, confocal/deconvolution microscopy, immunohistochemistry and whole-cell patch-clamp recording of synaptic currents. [unreadable] [unreadable]