The long term goal of this proposal is to use a molecular genetic approach to investigate the function of nicotinic acetylcholine receptors (nAChR) in the vertebrate central nervous system. Although considerable molecular data regarding the structure and function of nAChR subunits is available, far less is known about neural circuitry or roles subserved by the individual receptors. Moreover, little is known about the physiological consequences of mutations within these genes, the precise roles of receptor subtypes in the normal ontogeny and modulation of central nervous system synapses, or in the establishment of nicotine-induced dependence, tolerance and withdrawal among habitual tobacco users. As a first step towards addressing these issues, this proposal outlines experiments which use homologous recombination to introduce null or altered-function mutations of selected nAChR subunit genes into mice. The rationale for these exploratory studies rests on the assumption that deficits or alterations in targeted genes can, upon analysis of subject animals, reveal or clarify the function of the mutated gene. Specifically, the extant application proposes to utilize recombinant DNA methodologies to construct two strains of mice, the first of which will harbor a deletion of the alpha6 nAChR subunit gene. Since the alpha6 subunit gene is actively transcribed in catecholaminergic nuclei (ventral segmental area, substantia nigra, and locus coeruleus), it is anticipated that null mutants will help to define central nicotinic cholinergic circuits which modulate locomotion. Likewise, an alpha6 null mutation is anticipated to have profound effects on behaviors which originate in the mesolimbic dopamine system and are relevant to the reinforcing properties of nicotine, or to basic motivational processes which underlie learning and cognitive behavior. The second genetically engineered mouse will harbor a specific mutation (Ser248Phe) in the alpha4 nAChR subunit gene. In vitro this mutation potentiates onset and slows recovery from receptor desensitization, while in vivo this mutation is responsible for a human, brain-specific phenotype known as autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The purpose of this part of the proposal is to provide an animal model to analyze the molecular pathology of partial epilepsy, and to offer a paradigm for the development and evaluation of cholinergic therapeutic strategies.
