GABA is the major transmitter at inhibitory chemical synapses in the CNS and alterations in type A GABA receptor (GABAR) function have been hypothesized to be a critical component of epileptogenesis. Alterations in GABAR subunit-specific mRNA levels in vivo underlie changes in functional properties of dentate granule cells following pilocarpine-induced status epilepticus (SE). Increased expression of the alpha-4 subunit and decreased expression of alpha-1 after pilocarpine treatment produces benzodiazepine insensitivity and increased zinc blockade of GABA currents. It has long been suspected that a change in the number or composition of GABARs can produce profound changes in inhibitory synaptic transmission, however, it is not known whether the changes in GABAR properties seen in animal models of temporal lobe epilepsy (TIE) are either necessary or sufficient for epileptogenesis. We have shown that the minimal promoter of the alpha-4 subunit gene (GABRA4) when contained within AAV vectors controls region-specific transcription and condition-specific SE up-regulation. In addition, we have shown that viral-mediated delivery of alpha-1 subunits using the GABRA4 promoter inhibits the development of spontaneous seizures, establishing for the first time a connection between the expression of GABAR subunits and epileptogenesis in animals. Results of functional promoter assays and in vivo chromatin immunoprecipitation (ChIP) show that the condition-specific response of the GABRA4 gene most likely reflects the binding of the early growth response factor 3 (EGR3). The experiments of this proposal are aimed at determining the role of prolonged synaptic activity and release of growth factors to Egr mediated changes in GABRA4 transcription that occur in cultured primary embronic and postnatal dentate granule cells. Using viral-mediated delivery of RNAi to target both endogenous Egr3 and GABRA4 gene expression, we will test whether increased alpha-4 subunits are required for development of spontaneous seizures in TLE rats.
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