Behavioral analyses of C.elegans have revealed that the ASE chemosensory neurons play a major role in mediating chemotaxis toward water-soluble attractants. Laser ablation of both left- and right-ASE neurons reduced chemotaxis to Na+, Cl-, cAMP, biotin [1]. Interestingly, though the pair of neurons share homologous morphology and cell position, they show a different gene-expression pattern: ASE-Left expresses the putative receptor guanylyl cyclase GCY-6 and -7, and ASE-Right expresses GCY-5[2]. Moreover, another laser ablation study showed the asymmetry extends to the functional level. Namely, the ablation of ASE-Left reduced the chemotaxis to Na+ selectively, whereas the ablation of ASE-Right almost abolished the chemotaxis to Cl- and K+ [3]. Direct measurements of the neuronal activities of the individual ASE neurons are desired for further analysis, but applications of high concentrations of Na+, K+, Cl- make electrophysiology difficult. We have developed optical imaging methods to monitor in vivo calcium transients in C.elegans neurons by using a gene-encodable calcium indicator, cameleon. The observed transients correspond to the neuronal activities in the mechanosensory neurons [4] and the ASH chemosensory neurons [5]. To apply this technique to the ASE chemosensory neurons, we generated a transgenic animal expressing cameleon specifically in ASE-Left and Right by using
flp-6 promoter. The transgenic worms were mounted on agarose-pad and exposed to a constant flow of a bath solution. The flow of a chemical stimulant was delivered to the nose through a glass needle. We could observe the increase in intracellular calcium level in response to NaCl and NaOAc in ASE-Left and to KCl and KOAc in ASE-Right. These responses were abolished when we used a cameleon mutant that has mutations in all the calcium binding sites. An advantage of this imaging technique is that we can simultaneously monitor the activities of multiple cells on the same focal plane. Although it is technically very challenging to mount a worm at a certain angle to have the tiny neurons on the same focal plane, we are getting successful imaging simultaneously from ASE-Left and Right. Our preliminary data suggests both ASE-Left and Right are responsive to KOAc and high osmolarity stimulation. Further analyses and the use of mutant animals (
egl-19,
osm-9,
lim-6) are in progress. [1] Bargmann, C.I. and Horvitz, H.R.(1991) Neuron 7, 729-42 [2] Yu, S. et al. (1997) PNAS 94, 3384-7 [3] Pierece-Shimomura, J.T., et al. (2001) Nature 410, 694-7. [4] Suzuki, H. et al. (2002) WCWM
ab35 [5] Hilliard, M.A., et al. (2002) WCWM
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