We applied the directional tag PCR subtractive hybridization method to construct a rat hypothalamic cDNA library from which cerebellar and hippocampal sequences had been depleted, enriching 20-30-fold for sequences expressed selectively in the hypothalamus. We studied a sample of 94 clones selected for enrichment in the subtracted library. These clones corresponded to 43 distinct mRNA species, about half of which were novel. Thirty-eight of these 43 mRNAs (corresponding to 85 of the clones in the sample) exhibited enrichment in the hypothalamus; 23 were highly enriched. In situ hybridization studies revealed that one novel species was restricted to cells in a small bilaterally symmetric area of the paraventricular hypothalamus. Other novel mRNAs showed substantial enrichment in basal diencephalic structures, particularly the hypothalamus, without restriction to single hypothalamic nuclei. The data suggest that the hypothalamus utilizes at least two distinct strategies for employing its selectively expressed proteins. Secretory neuropeptides utilized for intercellular communication are produced by functionally discrete nuclei, while several other proteins are shared by structures that are unrelated in their physiological roles but may share biochemical systems.The hypothalamus is a phylogenetically ancient region of the mammalian brain. In contrast to laminar cortical structures such as the cerebellum and hippocampus whose final functions rely on innervation from the thalamus and brain stem, the hypothalamus is organized as a collection of distinct, autonomously active nuclei with discrete functions. Ablation and electrical stimulation studies and medical malfunctions have implicated several of these nuclei as central regulatory centers for major autonomic and endocrine homeostatic systems mediating processes such as reproduction, lactation, fluid balance, metabolism, and aspects of behaviors, such as circadian rhythmicity, basic emotions, feeding and drinking, mating activities, and responses to stress, as well as normal development of the immune system (1). Distinct hormones and releasing factors have been associated with some of these nuclei, but, at best, the organizations and molecular operations of these structures are only partially understood.A substantial portion of a mammal's genetic endowment is dedicated to the function of its central nervous system, as evidenced by the substantial number of mRNAs selectively expressed in the brain (2). Many of these have been observed to be selectively associated with distinct neural subsets. We hypothesized that ensembles of mRNAs selectively associated with discrete hypothalamic nuclei encode proteins singularly associated with the unique functions of those nuclei. The hypothesis must be at least partially valid given existing knowledge on the expression of specific hypothalamic hormones and releasing factors, but the extent to which it can account for the function of this organ is not known. To address this hypothesis further and to illuminate additional m...

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