The broad, long-term objectives are to combine knowledge from past research in behavior, pharmacology, and electrophysiology with newly acquired knowledge in cellular, molecular, and neurobiology obtained from attending courses and seminars, and to acquire the skills necessary to evaluate the cellular, anatomical, and molecular aspects of the mechanisms of action of drugs, specifically, the consequences of drug abuse in fetal development. The specific aims are to investigate the normal sequence of ontogenetic expression, distribution, and regulation of various nicotinic acetylcholine receptors (nAChR) and to clarify the role(s) of these receptors in the developing chick brain utilizing immunohistochemical and molecular biology techniques, including immunofluorescence, in situ hybridization, confocal microscopy, and calcium imaging. nAChRs are pentamers composed of various combinations of alpha and beta subunits. Several alpha and beta subunits have been identified, thus there is potentially a large number of nAChR subtypes. The present proposal, however, will examine only subtypes containing alpha7, alpha8 and/or beta2. In situ hybridization techniques will be used to evaluate the onset of expression of genes that encode these nAChR subunits. Since the presence of mRNA in a cell does not necessarily mean that physiologically meaningful amounts of protein are present, immunohistochemical procedures will be used to investigate the localization of the actual nAChR subunits in various structures of the embryonic and hatchling chick brain. Calcium imaging techniques will be used to evaluate the affinity, functional significance, and timing when the different types of nAChRs first manifest responses to specific agonists. These studies will provide a basis for investigating the mechanisms of pathological changes due to prenatal exposure to cholinergic ligands, such as nicotine. Since there is no intervening maternal-placental transport system, the embryo can be observed at all stages of development and the levels of pharmacological agents received by the embryo can be monitored. Therefore, the effects of early chronic exposure to nicotine on nAChRs in the developing nervous system of the chicken can be evaluated.