Quantitative trait loci (QTL) are chromosomal regions containing genes that influence a complex trait such as drug withdrawal severity. We have established that there is a great deal of common genetic influence on withdrawal from sedative-hypnotics including barbiturates, benzodiazepines, nitrous oxide and alcohol. During the current funding period, we confirmed several QTL that jointly have a major influence on the severity of pentobarbital (PB) withdrawal. The two QTL with the largest effects on withdrawal are on mouse chromosomes (Chr) 1 and 4. During the current funding period, both have been narrowed to regions containing only a few genes. QTL in these regions are also provisionally mapped for withdrawal from zolpidem (this grant), and definitively mapped for alcohol withdrawal (with support from AA06243). The Chr 1 QTL was also provisionally mapped for nitrous oxide withdrawal. ? Using congenics that isolate the two QTL on a uniform (inbred) genetic background, we will continue toward identification of the genes that underlie the PB withdrawal QTL and the neural circuits involved. We propose to: (1) Test the predicted genes in the intervals for the two strongest QTL for expression to identify novel candidate genes. (2) Test the known and novel candidate genes for genotype-dependent differences in expression and sequence (coding and regulatory). When feasible, the protein products of promising candidates will be assessed immunohistochemically to determine the anatomical distribution of their expression. Genotype-dependent differences in protein abundance will be assessed using Western blots. (3) Identify the brain region(s) and circuit(s) that modulate QTL-dependent activation in PB withdrawn mice. (4) Develop innovative, transgenic models to rigorously test the hypothesis that promising candidates (e.g., Mpdz) underlie the two largest QTL, and to test their role in withdrawal from more widely abused sedative-hypnotics (flunitrazepam, zolpidem, nitrous oxide, GHB and alcohol). An innovative feature of this proposal is to combine robust behavioral models of physical dependence on 6 sedative-hypnotics with sophisticated molecular techniques to identify candidate genes of high quality and the circuit(s) involved in their effects on drug withdrawal, and novel transgenic models that can prove that a candidate gene underlies a QTL. This will complete the journey from identifying genetic risk due to multiple anonymous genes to the identification of specific genes that confer sensitivity to, or protection from, drug withdrawal. This will set the stage for future translational and mechanistic studies ? ?
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