Abstracts

Gene therapy offers an alternative strategy for treating some CNS disorders, but has been relatively unexplored as a therapy for epilepsy. As background for a gene therapy approach, it is important to gain some understanding about the details of expression for those genes exogenously introduced into the brain. Toward this end, we have begun to characterize the efficiency, cell type specificity, and subcellular localization of Kv potassium channel subunits introduced into neurons [italic]in vitro[/italic]. These studies will provide essential background for testing gene therapy approaches in intact preparations., Organotypic hippocampal slice cultures (OHSC) and dissociated primary neuronal cell cultures were prepared from wildtype mice and Kv1.1 knock-out mice (lacking the Kv1.1[alpha] subunit). Cultures were maintained for seven days [italic]in vitro[/italic], and then infected via an adenoviral vector or an HSV1 vector containing voltage-gated potassium (Kv) channel subunit genes (e.g., Kv1.1, Kv[beta]2, Kv1.4, Kv2.1). Cultures were fixed at 1-3 weeks post-infection, and subsequently processed for immunofluorescence and electron microscopic observation. Immuno-reacted cultures were analyzed for cell-type specific expression, subcellular distribution, and co-localization of Kv channel subunit expression., In both primary dissociated cultures and OHSCs, adenoviral vectors successfully infected a variety of neuron types with Kv channel subunits. Exogenously-expressed subunits were observed [italic]in vitro[/italic] at several days post-injection. Adenoviral gene transfer appeared to target larger populations of neurons than the HSV1 vector. Subcellular localization of exogenous subunits did not always reproduce the patterns seen for endogenous subunits. For example, endogenous Kv1.1 and Kv1.4 subunits are expressed only in the axons and terminals of hippocampal pyramidal and granule cells; however, these subunits appeared primarily in cell somata when expressed via viral infection. In the relatively complex and well-organized organotypic cultures, injections of adenoviral vectors resulted in infection of neurons primarily around the site of the injection. In these OHSCs, pyramidal cells, granule cells, mossy cells, and interneurons expressed the exogenous gene product., Viral vector-mediated infection with genes for Kv channel subunits resulted in successful [italic]in vitro[/italic] infection of limited neuronal populations. Expression patterns of exogenous Kv subunits did not always mimick the subcellular localization patterns seen [italic]in vivo[/italic]. Further studies are needed to optimize infection efficiency and to reproduce appropriate localization of expression for specific channel subunits., (Supported by American Epilepsy Society Research Initiative Award.)