? This revised application is being submitted to provide Dr. Michael Glass, with four years of mentored training in molecular neuropharmacological and stereological methods to study drug abuse under the primary sponsorship of Dr Charles Inturissi and co-sponsorship of Dr. Virginia Pickel. This project will allow the candidate to engage in full-time research which will be facilitated by his appointment to tenure-track assistant professor at the Weill Medical College of Cornell University. The projects in this proposal will address the role of amygdala gene expression of the essential NMDA-NR1 (NR1) receptor subunit in the development and expression of opiate dependence by creating spatial-temporal knockouts using a specific and versatile form of Cre-loxP technology. This Cre-loxP system has been successfully used to achieve conditional NR1 gene knockout in spinal cord (i.e. spatial knockout) in adult animals (i.e. temporal knockout) with changes in injury-induced pain. The viability of this conditional gene knockout approach to achieve significant reductions of NR1 gene expression in a specific brain region of adult animals, and its consequences for behavior, neural activation, and protein targeting in dendritic spines has never been determined. This proposal will integrate innovative methods for conditional gene knockout and high resolution quantitative electron microscopic methods along with established behavioral paradigms to achieve the following aims: 1). produce NR1 knockout limited to the amygdala of adult mice, 2). examine the effects of conditional NR1 knockout in the amygdala on the development and expression of morphine dependence and tolerance, 3). determine the effects of conditional NR1 knockout in the amygdala on neural activation induced by opiate withdrawal, 4). examine the effects of amygdala NR1 knockout on the cellular targeting of proteins that are structurally and functionally linked to NR1, including the NMDA-R2 (NR2) subunit and neuronal nitric oxide synthase (nNOS) in dendritic spines. These studies will provide important new information on the role of amygdala NR1 subunit gene expression in the development of opiate dependence, and provide novel insights into the mechanisms mediating neural plasticity induced by chronic opiate use with implications for maintenance of drug use and relapse. ? ? ? ?
