Neuronal nicotinic acetylcholine receptors (nAChR) dissected

Upon the release of the neurotransmitter acetylcholine, nicotinic acetylcholine receptors (nAChRs) mediate signal transduction between nerve and muscle cells on one hand (muscle-type receptors), and at nerve cells of the brain and the autonomic nervous system (neuronal-type receptors) on the other hand. Each receptor consists of 5 either identical (homo-pentamers) or differing (hetero-pentamers) subunits. It is important to note that the functional properties of these receptors are primarily determined by their subunit composition. Accordingly, knowledge of the subunit composition is essential. We will address this question in the mouse superior cervical ganglion (SCG), a popular model for neuronal type receptors. nAChR subunits consistently found in the ganglion are a3, a5, a7, ß2, and ß4. We have recently discovered that mRNA of the a4 subunit, a principal constituent of nicotinic receptors in the brain, is also transiently expressed in the SCG of mice after birth. We know that except for a7, these subunits assemble into hetero-pentameric receptors. The exact composition of these receptors is, however, not known. This question will be addressed by a combined use of antibodies and radioligand binding techniques. Our focus will be on mice rather than rats or chicken because we are studying mouse models with targeted deletions of distinct nAChR subunit genes in parallel. Two of these models are of particular interest: Mice that lack the two subunits a5 and ß2, and mice without the subunits a5 and ß4. For reasons discussed in the proposal, the two models leave SCG neurons that express definite a3ß2 or a3ß4 hetero-pentameric receptors, respectively. Interestingly, both genotypes are viable and show no gross behavioral changes. To date, such definite receptors could only be studied by heterologous expression in frog (Xenopus) oocytes or in cell lines, but not in a physiological environment. The a5/ß4 double knockout model offer a particularly fascinating view into the function and regulation of nAChRs. If nerve cells are removed from ganglia of such animals and kept in culture, these cells unexpectedly show almost no response to nicotine, indicating that they miss functioning receptors. Since animals are viable we must conclude that SCG neurons in vivo maintain a3ß2 receptors at levels sufficient to guarantee the vital function of the ganglia, but are downregulated if neurons are kept in cell culture. One possible explanation we are going to explore is that downregulation of receptors is prevented if neurons are exposed to nicotine. Such effects have been observed with recombinant a3ß2 (but not a3ß4) receptors expressed e.g. in human embryonic kidney (HEK) cells. In vivo, the role of nicotine added to the culture medium could be taken by the neurotransmitter ACh, which would then operate not only on signal transduction at pre-existing receptors but would also affect their surface expression. These mechanisms are of general interest for nAChRs in the brain, where ß2-containing receptors are widespread and involved in such diverse functions as addictive behavior, cognition, and neurodegeneration.