The long term objectives of this research are to understand the mechanisms of action and the functions of muscarinic and nicotinic acetylcholine receptors (AChRs) in the central nervous system (CNS). Both classes of transmitter receptor are found throughout the human nervous system but have been studied in detail primarily in peripheral tissues such as skeletal muscle and heart. Central and peripheral AChRs are exceedingly important in normal human physiology and have been implicated in a wide variety of pathologies and diseases. These include, motor and cardiovascular deficiencies, nicotine-addiction and its related health effects (usually through smoking tobacco), convulsion induced neuronal injury and higher cognitive functions such as learning (particularly of new information) and the loss of memory and brain functions in Alzheimer's disease. Both nicotinic AChRs (nAChRs) and muscarinic AChRs (mAChRs) are major targets for therapeutic and surgically useful drugs. Despite these important roles it has only recently become possible to study specific AChRs in the CNS. In the vertebrates these studies are complicated by the remarkable diversity of genes for AChRs (at least 9 for nAChR subunits and 5 for the mAChRs), consequently there is a gap between our extensive understanding of AChRs in expression systems or cell cultures and their functions in the CNS. This proposal will address these problems using and insect (Manduca sexta) model system to characterize the actions and functions of AChRs in identifiable cholinoreceptive neurons. To this end, mAChRs will be cloned and localized in the CNS using whole mount in situ hybridization (Specific Aims 1 and 2). By identifying individual neurons in this way the functions of mAChRs can be directly tested in living ganglia. Although it is known that Manduca mAChRs regulate neuronal excitability, the biochemical pathways leading to this modulation are only partially known. Therefore, Specific Aims 3 and 4 will use calcium imaging and electrophysiological techniques to determine the roles of two implicated messengers, Ca 2+ and nitric oxide, in mediating muscarinic functions. To address the issue of subunit composition of Manduca nAChRs, the expression of two recently isolated nAChR-related genes (MARA1 and MARB1) will be blocked in primary cultured neurons using antisense oligonucleotides (Specific Aim 5). Through patch clamp recording from these neurons, the contribution of MARA1 and MARB1 to normal mAChR channels will be assessed at the molecular level. Together, these studies will help to identify the attributes of natively expressed AChRs and through the use of identified neurons it will eventually be possible to analyze the functional role of these receptors in central neuronal processing.
