The overall goal of this project is to develop tools for cell-type-specific regulation of gene expression in the brain. The basolateral region of the amygdala (ABL) of the rat brain will be used as a model system. The project will develop lentiviral vectors to target transgene over-expression in identified subpopulations of ABL neurons and to selectively knock-down endogenous gene expression in these same cell groups.
Aim 1 - Tarqeted Over-expression: The proposed studies will test the hypothesis that lentiviral vectors can be constructed to restrict gene over-expression to defined subpopulations of ABL neurons by incorporating cell- type-specific promoters. Lentiviruses designed to target a marker gene or c-myc-tagged GABAA receptor a1 subunit to pyramidal cells (CAM kinase II promoter) or discreet populations of GABAergic interneurons expressing VIP and calretinin (calretinin promoter) or parvalbumin (pan/albumin promoter) will be constructed, tested and optimized for specificity of cell-type targeting.
Aim 2 -Targeted Knock-down of Expression: The proposed studies will test the hypothesis that lentiviruses incorporating cell-type-specific promoters can be used to selectively reduce gene expression in defined subpopulations of ABL neurons by expression of antisense RNA. After identifying antisense constructs that effectively reduce mRNA for the NMDA receptor subunit 1 and for GABAA receptor a1 subunit in cultured cells, the antisense constructs will be inserted into lentiviral vectors under control of the CaM kinase II, calretinin or parvalbumin promoters. These lentiviral vectors will be tested for cell-type specificity in knock-down of target gene expression following stereotaxic delivery to the ABL. Development of tools for cell-type selective manipulation of gene expression will greatly facilitate determining the roles of specific gene products in subpopulations of neurons participating in defined neuronal circuits of intact animals. This knowledge will, in turn, permit development of new drugs and genetic therapies for neurological and behavioral disorders. In the ABL, the development of these tools is particularly relevant to treatments for stress and anxiety disorders and for epilepsy. It is also expected that the vectors developed in this project will be useful to study synaptic transmission and the regulation of neuronal networks in other brain areas including the cortex and the hippocampus. These tools will have wide applicability in elucidating the functional anatomy of the central nervous system, particularly in determining the roles of individual proteins found in specific groups of brain cells in the behavior of the whole organism. Because of the brain region being studied, this work is of particular relevance to the understanding and treatment of stress, anxiety and epilepsy. It is expected that this work will ultimately lead to new drugs or genetic therapies for these and other disorders of the nervous system. ? ?
