This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During the reporting period we used single cell RT-PCR to complement our physiological study of 5HT receptor function in the BNST. We now have data suggesting that individual BNST neurons can express multiple 5HT receptors, and that chronic restraint stress can dramatically alter the expression patterns of specific 5HT receptor mRNA in these same neurons. In part, the stress response can be mimicked by repeated cortisol injections suggesting that the alteration may be part of a feedback response to regulate BNST neuronal excitability in the face of prolonged stress. Significantly, we also have Western blot- and behavioral data to show that the mRNA change has a corresponding change in peptide expression in the BNST and that loss of a particular peptide (Kv4.2) is positively correlated with an increase in anxiety-like behavior in chronic restraint stress animals. We also continued our single cell RT-PCR examination of specific ion channel subunit mRNA expression in order to determine which ion channel subunits contribute to the intrinsic membrane currents that are expressed by distinct cell types in the BNST (Types I - III). To date we have characterized at least 5 IA channel subunits, 3 IT channel subunits and 4 IH channel subunits, and we hope to submit a paper on the molecular dissection of Type II BNST neurons early in 2009. We have also developed a novel transgenic mouse in which the CRF promoter is used to drive the expression of green fluorescent protein (GFP). We have shown that GFP expression is restricted to those areas predicted to contain high numbers of CRF neurons, including the BNST. Preliminary physiological characterization of the properties of the CRF-containing neurons suggests that they correspond to the Type III subtype of BNST neuron seen in rat tissue.
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