Benzodiazepines are among the most widely prescribed medications in the world, While benzodazepines are both clinically efficacious and safe, there are nonetheless concerns about whether inappropriate or over prescription of these drugs contributes to abuse and dependence. The site(s) and mechanism of behavioral actions of benzodiazepines in the central nervous system (CNS) remain uncertain. The proposed research is designed to test a working hypothesis that behaviors initiated by drug action at the benzodiazepine/y aminobutyric acidA (GABAA) receptor complex are mediated by a nitric oxide (NO)/cyclic GMP (cGMP)/protein kinase G (PKG) signaling pathway, i.e., stimulation of the benzodiazepine/GABAA receptor complex activates NO synthase (NOS) to produce NO, which activates soluble guanylyl cyclase (GC-S) to produce cGMP, which, in turn, activates a cyclic GMP-dependent protein kinase, leading to the behavioral effects. This will be accomplished by comparing the behavioral effects of the benzodiazepine chlordiazepoxide, the GABAA agonist 4,5,6,7-tetrahydro-isoxazolo[5,4-c]pyridin-3-ol (THIP) and nitrous oxide, another drug of abuse that appears to work at least in part through benzodiazepine/GABAA receptors, in mice following pharmacological manipulation of NOS, NO, guanylyl cyclase, cyclic GMP, and PKG in the CNS. Drug pretreatments will result in reduction in brain NO by inhibition of NOS, restoration of NO in NOS-inhibited brain, inhibition of NO action by an NO scavenger, reduction in brain cGMP by inhibition of GC-S, increase in brain cGMP by inhibition of cGMP degradation, and selective inhibition of protein kinase G (PKG). Other drug challenges will include NO-donors, dibutyryl cGMP and PKG-activators. Methods to be used in this research include behavioral testing in mice (elevated plus-maze and light/dark exploration), central microinjection of pretreatment drugs, and assays to measure cGMP and NOS or PKG enzyme activities. The purpose of the proposed studies is to identify the signaling pathway that mediates benzodiazepine-induced behaviors and provide a progressively more complete understanding, at the molecular level, of the pharmacological and neurochemical mechanisms underlying acute benzodiazepine-induced behaviors.
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