The long-term objectives of this proposal are to determine how transmitters transfer information between neurons, how individual neurons integrate and respond to this information, and how this processing contributes to behaviors and behavioral changes. Specifically, these studies concentrate on two types of cholinergic receptors that are expressed by an identified motoneuron (PPR) in the tobacco hornworm, Manduca sexta. One of these receptors, the nicotinic acetylcholine receptor (nAChR), is a member of the family of acetylcholine-gated channels found both peripherally and centrally in most mammals. In humans these receptors are targets for the actions of nicotine (usually absorbed from smoking tobacco) and a variety of clinically important drugs. Based on the pharmacological responses of PPR, nAChRs in Manduca appear to have several extraordinary properties. First, perhaps as an adaptation to Manduca's diet of tobacco, PPR has a low sensitivity to nicotine while retaining a normal responsiveness to other agonists. Second, since PPR remains depolarized during exposure to nicotinic agonists, there appears to be no desensitization of these NACHRS. This proposal will investigate whether these unusual properties are intrinsic to Manduca NACHRS by using patch clamp electrodes to isolate single receptors in membrane patches from PPR. Agonist sensitivity of the NACHRS will be measured in outside-out patches and their desensitization kinetics monitored in cell-attached patches. In view of the considerable health risks associated with nicotine addiction in humans, understanding the nicotine resistance and other special adaptations of Manduca NACHRS is of obvious medical importance. The other cholinergic receptor expressed by PPR in a muscarinic acetylcholine receptor (mAChR) that appears to modulate PPR's excitability. In mammals, mAChRs are of enormous importance in a wide range of cellular roles, but it is rare to able to study their actions in individual neurons in situ. This is easily achieved in the nervous system of Manduca. Hence, another of my specific aims is to characterize Manduca MACHRS and to determine their effects in identified neurons such as PPR. The pharmacology, distribution and possible heterogeneity of Manduca MACHRS will be established using radioligand binding assays in conjunction with immunological approaches. The cellular function of MACHRS in particular neurons will then be identified using electrophysiological methods. Because MACHRS can control diverse effects in different cells, an important part of this proposal will use intracellular injections to determine which G-proteins and second messenger pathways are employed by MACHRS in individual neurons. These proposed studies will establish how Manduca NACHRS may be specialized to tolerate nicotine, and further, will identify mechanisms of neuronal modulation by MACHRS.
