Nicotine acts on neuronal acetylcholine receptors (AChRs) and it is quite possible that this is the means whereby this drug expresses its addictive and psychoactive properties. Molecular studies of these receptors offer opportunities to identify possible structural and regulatory mechanisms which may underlie the processes of drug addiction, dependency or sensitivity. We propose the use of molecular techniques to elucidate the primary sequences of neuronal acetylcholine receptors (AChRs) from a marine mollusc, Aplysia californica. There are three distinct types of neuronal AChRs in Aplysia each of which has been well characterized physiologically and pharmacologically and each of which is expressed in identifiable neurons. The present proposal seeks an understanding of the molecular bases for the functional characteristics of the three types of receptors. Our studies will utilize polymerase chain reaction (PCR) techniques to selectively amplify AChR related portions of Aplysia genomic DNA. The selectivity will be generated with oligonucleotide primers, complimentary to highly conserved portions of neuronal AChRs from other species. Those PCR products exhibiting significant homology to other known AChRs will be used to screen Aplysia genomic and cDNA libraries. The recovered clones will be analyzed to determine the degree of homology that they have with each other and with those of other species at loci believed to be of functional importance (e.g. drug binding sites, ion channels). Northern analyses with probes derived from PCR and from cDNA clones will provide an indication of the size, number and relative abundance of mRNAs present in nervous tissues. Hybridization of the sequence with specific identified neurons will provide a direct correlation between a specific sequence and the functional type of receptor known to be expressed by that neuron. The full length cDNA clones will serve as templates for in vitro synthesis of cRNAs whose injection into oocytes will permit direct studies of function. These experiments would also provide a correlation between the physiological and pharmacological properties of the receptors expressed in oocytes and those of the native neuronal receptors.
| Ono, J K; McCaman, R E (1992) In situ hybridization of whole-mounts of Aplysia ganglia using non-radioactive probes. J Neurosci Methods 44:71-9 |
