With recent data indicating an increased incidence of cigarette use among young women, the potential consequences of maternal smoking on fetal outcome is an issue of increasing clinical significance. In vivo data indicate that prenatal nicotine exposure has significant effects on the development of brain catecholamine (CA) systems, and that this may underlie some observed behavioral deficits. However, the exact mechanism by which nicotine affects central CA development is unclear. The purpose of the present application is to determine whether brain CA neurons are directly regulated by nicotinic receptors (nAChRS) during early brain development. Unlike the majority of previous studies, we propose to use in vitro methodologies so that experimental variables can be better controlled and indirect effects minimized. Quantitative anatomical techniques will be used to examine the developmental expression of nAChR mRNA and high affinity binding sites within developing norepinephrine (NE) and dopamine (DA) cells of the locus coeruleus (LC) and substantia nigra/ventral tegmental area (SN/VTA), respectively. Combined isotopic and non-isotopic in situ hybridization will be used to quantitate the developmental expression of nAChR subunit mRNAs within cells which also express the CA synthetic enzyme, tyrosine hydroxylase. High affinity nAChR binding in these developing cell groups will also be examined using [3H]cytisine and [3H]epibatidine as radioligands. Animals to be used for the study will be analyzed by age and sex, and will encompass the full range of CA development from embryonic day 15 through adult. The functional role of nAChRs in controlling NE and DA release in developing brain will be evaluated using transmitter release assays. Using brain slices, the effects of nicotine will be examined on NE and DA release from selected terminal fields of the LC and SN/VTA. The age of onset of nAChR regulation will be examined, as will the pharmacological characteristics of the receptor in immature brain. In particular, we will evaluate the hypothesis that the properties of nAChRs regulating LC function change with age. A primary neuronal cell culture model will also be used to analyze the properties of nAChRs on developing LC neurons, and to examine the developmental consequences of chronic nAChR activation. It is anticipated that the proposed in vitro analysis will provide a substantial framework for interpreting data on the effects of chronic perinatal nicotine administration in vivo.
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