In the previous funding period, we have demonstrated that nicotine interacts directly with nicotinic receptors (nAChRs) on catecholamine neurons to stimulate norepinephrine (NE) and dopamine (DA) release in developing rat brain. We now propose a series of studies to examine the physiological and clinical relevance of these observations. We will study the species specificity of nAChR-mediated catecholamine release in C57BL/6J mice, and will use null mutant mouse strains to examine the subunit composition of nAChRs on developing NE and DA terminals. Two types of approach will be used to examine whether nAChRs on developing noradrenergic locus coeruleus (LC) terminals may be activated by endogenous ligand. We will use immunohistochemistry to establish the developmental appearance of cholinergic cells and fibers in relationship to LC and its forebrain and hindbrain terminal fields. Antibodies specific for the vesicular acetylcholine transporter (VAChT) and for dopamine beta hydroxylase (DBH) will be used to visualize developing cholinergic and noradrenergic elements, respectively. We will also determine whether nAChR activation or blockade can modify early learning in paradigms that have been shown to require activation of the LC, including early olfactory learning and somatosensory associative conditioning. Learning in each task has been shown to necessitate LC activation by tactile stimulation and electrical shock, respectively. We will test the hypothesis that peripheral administration of nicotine induces sufficient LC stimulation to facilitate learning in the absence of sensory stimuli. We will also use the nAChR antagonist, mecamylamine, to determine whether endogenous acetylcholine (ACh) influences sensory stimulus-induced LC activation on these tasks. Having established the functional roles of nAChRs in acute regulation of catecholamine release, we will examine the effects of both tonic and phasic modes of chronic prenatal nicotine exposure on the development of central catecholamine systems. Osmotic minipumps will be used for constant infusion and self- administration for pulsatile delivery of nicotine to pregnant rats. Offspring of varying ages will be used for functional and neuroanatomical studies to determine whether prenatal nicotine exposure after the overall excitability, or nicotine-sensitivity, of NE and DA terminal fields. Behavioral tests that involve both central NE (early olfactory learning, associative somatosensory conditioning) and DA systems (locomotion, conditioned place preference, self-administration) will be used to determine whether chronic nicotine-induced changes in catecholamine system development are associated with long-term alterations in behavioral response. These combined in vitro and in vivo approaches are designed to elucidate mechanisms underlying adaptive responses of central catecholamine neurons to chronic prenatal nicotine exposure.
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