Although many psychopharmacological factors contribute to nicotine addiction, midbrain dopaminergic systems have received much attention because of their roles in reinforcement and associative learning. It is generally thought that the mesocorticolimbic dopaminergic system is important for the acquisition of behaviors that are reinforced by the salient drives of the environment or by the inappropriate stimuli of addictive drugs. Nicotine, as obtained from tobacco, can activate nicotinic acetylcholine receptors (nAChRs) and excite midbrain neurons of the mesocorticolimbic system. Using midbrain slices from rats, wild-type mice, and genetically engineered mice, we have found differences in the nAChR currents from the ventral tegmental area (VTA) and the substantia nigra compacta (SNc). Nicotinic AChRs containing the alpha7 subunit (alpha7* nAChRs) have a low expression density. Electrophysiological analysis of nAChR currents, autoradiography of [125I]-alpha-bungarotoxin binding, and in situ hybridization revealed that alpha7* nAChRs are more highly expressed in the VTA than the SNc. In contrast, beta2* nAChRs are move evenly distributed at a higher density in both the VTA and SNc. At the concentration of nicotine obtained by tobacco smokers, the slow components of current (mainly mediated by beta2* nAChRs) become essentially desensitized. However, the minority alpha7* component of the current in the VTA/SNc is not significantly desensitized by nicotine in the range < or =100 nm. These results suggest that nicotine, as obtained from tobacco, can have multiple effects on the midbrain areas by differentially influencing dopamine neurons of the VTA and SNc and differentially desensitizing alpha7* and non-alpha7 nAChRs.

Development of the anterior pituitary gland involves proliferation and differentiation of ectodermal cells in Rathke's pouch to generate five distinct cell types that are defined by the trophic hormones they produce. A detailed ontogenetic analysis of specific gene expression has revealed novel aspects of organogenesis in this model system. The expression of transcripts encoding the alpha-subunit common to three pituitary glycoprotein hormones in the single layer of somatic ectoderm on embryonic day 11 established that primordial pituitary cell commitment occurs prior to formation of a definitive Rathke's pouch. Activation of Pit-1 gene expression occurs as an organ-specific event, with Pit-1 transcripts initially detected in anterior pituitary cells on embryonic day 15. Levels of Pit-1 protein closely parallel those of Pit-1 transcripts without a significant lag. Unexpectedly, Pit-1 transcripts remain highly expressed in all five cell types of the mature pituitary gland, but the Pit-1 protein is detected in only three cell types--lactotrophs, somatotrophs, and thyrotrophs and not in gonadotrophs or corticotrophs. The presence of Pit-1 protein in thyrotrophs suggests that combinatorial actions of specific activating and restricting factors act to confine prolactin and growth hormone gene expression to lactotrophs and somatotrophs, respectively. A linkage between the initial appearance of Pit-1 protein and the surprising coactivation of prolactin and growth hormone gene expression is consistent with the model that Pit-1 is responsible for the initial transcriptional activation of both genes. The estrogen receptor, which has been reported to be activated in a stereotypic fashion subsequent to the appearance of Pit-1, appears to be capable, in part, of mediating the progressive increase in prolactin gene expression characteristic of the mature lactotroph phenotype. This is a consequence of synergistic transcriptional effects with Pit-1, on the basis of binding of the estrogen receptor to a response element in the prolactin gene distal enhancer. These data imply that both transcriptional and post-transcriptional regulation of Pit-1 gene expression and combinatorial actions with other classes of transcription factors activated in distinct temporal patterns, are required for the mature physiological patterns of gene expression that define distinct cell types within the anterior pituitary gland.

Although protein phosphorylation has been characterized more extensively, modulation of the acetylation state of signaling molecules is now being recognized as a key means of signal transduction. The enzymes responsible for mediating these changes include histone acetyl transferases and histone deacetylases (HDACs). Members of the HDAC family of enzymes have been identified as potential therapeutic targets for diseases ranging from cancer to ischemia and neurodegeneration. We initiated a project to conduct comprehensive gene expression mapping of the 11 HDAC isoforms (HDAC1-11) (classes I, II, and IV) throughout the rat brain using high-resolution in situ hybridization (ISH) and imaging technology. Internal and external data bases were employed to identify the appropriate rat sequence information for probe selection. In addition, immunohistochemistry was performed on these samples to separately examine HDAC expression in neurons, astrocytes, oligodendrocytes, and endothelial cells in the CNS. This double-labeling approach enabled the identification of specific cell types in which the individual HDACs were expressed. The signals obtained by ISH were compared to radiolabeled standards and thereby enabled semiquantitative analysis of individual HDAC isoforms and defined relative levels of gene expression in >50 brain regions. This project produced an extensive atlas of 11 HDAC isoforms throughout the rat brain, including cell type localization, providing a valuable resource for examining the roles of specific HDACs in the brain and the development of future modulators of HDAC activity.

Activation of brain ␣7 nicotinic acetylcholine receptors (␣7 nAChRs) has broad therapeutic potential in CNS diseases related to cognitive dysfunction, including Alzheimer's disease and schizophrenia. In contrast to direct agonist activation, positive allosteric modulation of ␣7 nAChRs would deliver the clinically validated benefits of allosterism to these indications. We have generated a selective ␣7 nAChR-positive allosteric modulator (PAM) from a library of GABA A receptor PAMs. Compound 6 (N-(4-chlorophenyl)-␣-[[(4-chlorophenyl)amino]methylene]-3-methyl-5-isoxazoleacet-amide) evokes robust positive modulation of agonist-induced currents at ␣7 nAChRs, while preserving the rapid native characteristics of desensitization, and has little to no efficacy at other ligand-gated ion channels. In rodent models, it corrects sensory-gating deficits and improves working memory, effects consistent with cognitive enhancement. Compound 6 represents a chemotype for allosteric activation of ␣7 nAChRs, with therapeutic potential in CNS diseases with cognitive dysfunction.cognition ͉ ion channels ͉ memory ͉ nicotine ͉ schizophrenia

Nicotine, acting at pentameric neuronal nicotinic acetylcholine receptors (nAChRs), is the primary addictive component in tobacco. At low doses, it affects attention, learning, memory, anxiety, cardiovascular responses, thermoregulation, and nociception. At high doses, nicotine produces more drastic behaviors and eventually induces tonic-clonic seizures in rodents. In mammals, several subunits of the nAChRs have been cloned, including eight ␣ and three ␤ subunits. To study the physiological role of the ␣5 subunit, we have generated ␣5-deficient mice. These mice have a generally healthy appearance and are normal in a standard battery of behavioral tests. However, the sensitivity of ␣5 mutant mice to nicotine-induced behaviors and seizures is dramatically reduced compared with their wild-type littermates. These animals have a normal brain anatomy and normal levels of mRNA for other nAChR subunits, namely ␣4, ␣6, ␣7, ␤2, and ␤4. In addition, 125 I-epibatidine and [ 125 I]␣-bungarotoxin binding in the brains of ␣5-deficient mice is normal. Together, these results suggest a direct involvement of the ␣5 subunit in the observed phenotypes.