Determination of the precise mechanisms of ethanol's actions on CNS neurotransmitter function has been the focus of much ongoing investigative effort. Use of an animal model of genetic differences in initial CNS sensitivity to ethanol (LS and SS mice) has provided evidence that certain of the behavioral actions of ethanol may be mediated by this agent's differential effects on monoaminergic neuronal systems in specific brain regions and the adrenal gland. It has also been determined that LS and SS mice differ substantially in the extent of the analeptic response to thyrotropin-releasing hormone (TRH) following ethanol administration. Although controversy exists as to the exact sites and mechanism of TRH's analeptic action it is believed to involve activation of central cholinergic and/or monoaminergic neurons. A thorough examination of TRH effects on monoamine function in LS and SS mice in the presence and absence of ethanol should provide further insight into the neurochemical mechanisms mediating both the CNS depression by ethanol and its antagonism by TRH. Similar experiments will be conducted with pentobarbital, a CNS depressant to which LS and SS mice do not differ in initial CNS sensitivity, and the results compared to those obtained with ethanol. Initial studies will include a comparison of the effects of TRH with various TRH analogs (possessing improved pharmacokinetic and non-endocrine profiles) on behavioral actions of ethanol or pentobarbital, i.e., narcosis and hypothermia. Subsequently, the effects of TRH and TRH analogs on these behavioral measures of ethanol and pentobarbital intoxication will be correlated with their effects on ethanol-induced alterations of monoamine neurochemistry. Aspects of monoamine function to be evaluated in brain regions and adrenal gland include: (1) monoamine synthesis by performing in vitro kinetic analysis of the enzymes tyrosine and tryptophan hydroxylase as well as in vivo estimates of enzyme activity by measuring the accumulation of dopa and 5-hydroxytryptophan, respectively, following treatment with a decarboxylase inhibitor, (2) levels of the enzyme substrates, tyrosine and tryptophan, (3) availability of the enzyme cofactor by measuring biopterin levels and the activity of its synthetic enzyme, GTP-cyclohydrolase, and (4) monoamine release (overflow) in vivo in specific brain regions by employing both electrochemical and microdialysis techniques. Finally, in order to assess the possible contribution of the pituitary-thyroid axis to the analeptic and thermogenic actions of acute TRH and TRH analog administration, we will evaluate their effects on thyroid function, including serum thyrotropin (TSH) triiodothyronine (T3), and thyroxine (T4) levels, % hormone uptake, and free hormone index, in the presence and absence of ethanol and pentobarbital.
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