Prenatal opiate exposure is associated with a variety of neurobehavioral disturbances. For instance, we have demonstrated that adult male and female rats exposed prenatally to morphine exhibit sexually dimorphic perturbations in reproductive behavior, as well as hypothalamic norepinephrine (NE) levels and turnover in young adulthood. Moreover, these alterations in behaviors and hypothalamic NE systems correlate with sexually dimorphic alterations in mu opioid receptor binding in the hypothalamus, a receptor known to inhibit NE release. We now propose to test the hypotheses, in Specific Aims 1-4, that these sexually dimorphic effects of prenatal morphine on reproductive behavior and hypothalamic neurochemistry reflect sexually dimorphic effects of prenatal morphine on: (1) NE innervation of the hypothalamus; (2) local mechanisms regulating hypothalamic NE release; (3) hypothalamic opioid receptors; and (4) gene expression of endogenous opioid peptides in the hypothalamus.
Specific Aim 5 will determine whether there are causal relationships between the neurochemical and behavioral effects of prenatal morphine. We will test the hypothesis that the reduction of reproductive behavior in female rats exposed to morphine prenatally is functionally related to the changes in hypothalamic NE and endogenous opioid neurotransmission.
Specific Aim 1. To assess the NE innervation of the hypothalamus, the distribution of tyrosine hydroxylase and dopamine beta-hydroxylase immunoreactive fibers will be evaluated by immunocytochemistry, and the density of NE uptake sites labeled by [3H]-nisoxetine will be quantified by autoradiography. Thus this aim will test the hypothesis that prenatal morphine has sexually dimorphic effects on the density of NE terminals in the hypothalamus of adult rats.
Specific Aim 2. In vitro brain slice studies will be conducted to assess the sexually dimorphic effects of prenatal morphine on local mechanisms governing hypothalamic NE release.
Specific Aim 3. In vitro opioid receptor autoradiography will be used to investigate the sexually dimorphic effects of prenatal morphine on the density of mu opioid receptors in the adult brain and their regulation by gonadal steroids. To evaluate the specificity of prenatal morphine action on opioid receptor subtypes, delta and kappa opioid receptor binding sites will also be examined.
Specific Aim 4. Endogenous opioid gene expression will be examined by in situ hybridization to assess the sexually dimorphic effects of prenatal morphine on endogenous opioid peptide gene expression. Since both endogenous (beta-endorphin) and exogenous (morphine) opiates act via mu receptors to inhibit NE transmission and adult reproductive behavior, the expression of beta-endorphin mRNA will be analyzed using molecular probes for the precursor molecule proopiomelanocortin (POMC). To evaluate whether prenatal morphine influences only POMC gene expression, we will also use molecular probes for proenkephalin and prodynorphin.
Specific Aim 5. In vivo brain microdialysis will be employed to test the hypothesis that the inhibition of reproductive behavior in adult female rats exposed to morphine prenatally is functionally related to the changes in hypothalamic NE and endogenous opioid neurotransmission.
