We are studying the cell biology of nicotine adaptation in C. elegans . Previously, we have shown that the levels of the UNC-29 nicotinic acetylcholine receptor (nAChR) subunit, expressed in the vulval muscles, are reduced in response to elevated doses of nicotine. This process involves the function of the C. elegans PKC homolog TPA-1 1 . We have now extended our studies towards three additional nAChR subunits, namely UNC-38, LEV-1 and UNC-63, all of which, together with UNC-29, are involved in the control of egg-laying 2 . We have created translational fusions of the nAChR subunits with GFP and studied their expression patterns in vivo . LEV-1 is strongly expressed in head and body muscles, including muscle arms projecting to the nerve ring, and in several as yet unidentified head, tail and ventral cord neurons (including VC 4 and 5 that innervate the vulval muscles). LEV-1 may also be expressed in vulval muscles, however, this could only be detected in animals expressing GFP driven by the
lev-1 promoter. LEV-1 expression is already detected in embryos and remains throughout the whole lifespan, although it is strong only in young and well-fed worms. The expression levels of UNC-63 and UNC-38 are lower and we detect these proteins in head and body muscles, various head and tail neurons, the nerve ring, vulval muscles, ventral cord neurons (including VC 4 and 5), and also in post-synaptic specializations of the nerve cord. It is currently unclear, whether these post-synaptic sites represent neuromuscular junctions or interneuronal synapses (or both). The expression patterns of all four receptor subunits under study are in agreement with previous results by other labs showing that UNC-29, UNC-38 and LEV-1 can form a functional receptor 3,4 , since they are (at least in part) present in the same cells and tissues. However, UNC-38 is expressed in many more head neurons than the other nAChR subunits, suggesting that this a -subunit may form functional receptors also with other, as yet unidentified, non- a -subunits. We now try to express epitope-tagged versions of all four nAChR subunits in a single worm, in order to allow for co-localization and co-immunoprecipitation studies. Furthermore, we studied the effects of nicotine treatment on the expression of LEV-1. Similar as UNC-29, LEV-1::GFP expression levels can be dramatically and reversibly reduced by incubating the worms on 0.25 - 0.5 % nicotine. Initial results suggest that this regulation may be achieved, at least in part, on the transcriptional level, since also GFP expression driven by the
lev-1 promoter alone is reduced by nicotine treatment. In contrast, GFP driven by the
unc-29 promoter is not affected by nicotine, consistent with our results for the translational fusion of UNC-29::GFP, which is regulated in a promoter-independent fashion 1 . We are currently studying the effect of nicotine on the expression of
unc-38 and
unc-63 and we are investigating whether mutations in
dyn-1 5 ( C. elegans dynamin) and in
nic-1 (isolated in our lab;
nic-1 mutants are impaired in nicotine-adaptation, see abstract by Kim et al.) affect the nicotine-induced downregulation of LEV-1::GFP. ------------------------ 1 Waggoner et al. (2000) J. Neurosci. 20 p. 8802-8811 2 Kim et al. (2001) Genetics 154 (4) 3 Fleming et al. (1997) J. Neurosci. 17 p. 5843-5857 4 Richmond and Jorgensen (1999) Nature Neurosci. 2 p. 791-797 5 Clark et al. PNAS 94 p. 10438-10443