We propose to continue our studies on brain mRNAs so as to better our understanding of patterns of brain gene expression, to identify and solve the structures of novel brain proteins with spatially restricted expression within the brain, and to gain insights into their functional nature and their contribution to the behavioral and physiological operation of the organism. We will use the newly developed and automated TOGA technology to identify cDNA clones of mRNAs highly enriched in their expression in the hypothalamus, so as to identify those whose expression is restricted to discrete hypothalamic nuclei, or the striatum, so as to compile a complete dossier on the molecular nature of medium spiny neurons. Full-length clones of novel ensemble members will be isolated and their sequences determined so as to team the identity of their encoded proteins. The sites of expression will be determined by in situ hybridization and unmunohistochemical methods using antisera to synthetic peptides corresponding to domains of the deduced protein sequences. We will use the acquired information to form hypotheses about function and test these by biochemical measurements on the recombinant protein expressed by bacteria or transfected manimalian cells and by gene inactivation studies. For hypocretin, an excitatory neoromodulatory peptide that is produced by a few thousand hypothalamic neurons, that has been implicated in multiple homeostatic systems, including sleep and feeding, and that is implicated in all human narcolepsies, and for cortistatin, a somatostatin-like sleep-promoting peptide of cortical inhibitory intemeurons, we will generate lines of transgenic mice that express the intracellular portions of either diphtheria toxin or cholera toxin and thus either ablate or hyperactivate the neurons that normally produce these peptides. The hypocretin-DT mice will serve as a model for HLA-linked narcolepsy, which is thought to be caused by autoimmune destruction of hypocretin neurons. We will isolate RNA from these several transgenic mouse lines and use TOGA to identify the entire ensemble of mRNAs that are selectively expressed in hypocretin and cortistatin neurons. We will characterize knockout mice with null alleles in the genes encoding: a) 5-HT7, a serotonin receptor expressed prominently in ventral hypothalamus, which is coupled to stimulation of adenylyl cyclase, and whose pharmacology we have shown to be uniquely consistent with that of the 5-HT receptor that mediates circadian phase shifts in hypothalamic slice preparations, and which is a target for the sleep-promoting effects of oleamide, b) RC3/neurogranin, a forebrain-specific calmodulin-binding phosphoprotein which has been implicated in determining the availability of calinodulin and the calcium set point in dendritic spines and possibly in the development of dendritic spines, and c) cortistatin, a neuropeptide of cortical intemeurons that antagonizes the desynchronizing effects of acetylcholine on paired pulse and EEG measurements and enhances slow wave sleep.
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