This work will more thoroughly examine the neurochemical basis of the neurobehavioral abnormalities observed following prenatal exposure to opioids with a long-term goal of preventing or treating these deficits in children exposed to methadone prenatally. This project will continue work begun previously in this area and specifically will [1] determine whether neurochemical (i.e. changes in acetylcholine content and/or turnover) and behavioral (i.e. changes in acquisition of the negative geotaxic reflex and vertical screen performance and in tail-flick response to morphine) effects observed following prenatal exposure to methadone result from a direct action of methadone on the developing nervous system or rather from neonatal withdrawal; [2] determine whether neurochemical and behavioral effects following prenatal exposure to methadone and/or withdrawal are related to changes in brain regional endogenous opioids or opioid receptor binding; and [3] determine whether neonatal changes in brain regional acetylcholine (ACh) content observed in the above animals are related to changes in the number of cholinergic neurons or to changes in enzymatic activity. It is postulated that some of the observed neurochemical and behavioral changes observed after prenatal exposure may result from neonatal withdrawal whereas others may result from changes in endogenous opiate systems as a direct result of the action of methadone on the developing nervous system. Female rats will be implanted with a 28 day osmotic minipump containing water of methadone on day 8 of pregnancy. Neonatal withdrawal will be prevented by postnatal exposure to methadone via maternal milk. Pups will be cross-fostered to mothers who have been implanted with water or methadone pumps. The effect of this treatment will be determined on brain regional ACh and met- and leu-enkephalin content and mu-and delta-opioid receptor binding in weaning and adult rats. ACh turnover will be assessed by a mass fragmentographic technique that measures the relative incorporation of deuterium label from infused phosphorylcholine precursor into choline and ACh. Brain regional enkephalin and beta-endorphin content will be measured by the use of radioimmunoassay. Mu-Opioid and delta-opioid receptor binding will be determined in homogenates of relevant brain regions. Choline acetyltransferase (ChAT) activity will be assessed by a radioenzymatic assay and the number of the cholinergic neurons identified by immunohistochemical localization of ChAT. Behavioral testing will consist of measuring a standard array of developmental milestones and measuring the antinociceptive response to opioid manipulations in the tail-flick test. Once the effect of in utero methadone exposure on neurochemistry is more completely understood, efficacious therapeutic management can be developed for pregnant addicts, the neurobehavioral deficits exhibited by those exposed to methadone prenatally, and prevention of drug abuse in the children of addicts.
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