Molecular and functional characterization of GLR-3 and GLR-6 ionotropic glutamate receptor subunits. Craig S. Walker , Jerry E. Mellem, Nathalie Strutz, Andres V. Maricq. Biology, University of Utah, Salt Lake City, UT.

Understanding how C. elegans migrates towards favorable temperatures and away from those that are not is a fascinating problem of behavioral neurobiology. Laser ablation studies have identified the neural circuit that regulates this behavior; however the molecular mechanisms that control thermotaxis are not well understood. Using genetic, behavioral and electrophysiological analyses, we are characterizing the role of ionotropic glutamate receptors in this behavior. The RIA interneurons of the thermotatic circuit are major integrating neurons that receive input from several different sensory neurons including AIY, AIZ and ASH. Of the ten ionotropic glutamate receptor subunits identified in C. elegans, GLR-3 and GLR-6 are only expressed in RIA. Using the Tc1 based approach, we have generated deletion mutations in glr-3 and glr-6 . To determine the role of these subunits in thermotaxis we are assessing the ability of the mutants to track towards the appropriate temperature within a linear temperature gradient. In order to further study the function of RIA we have expressed the human caspase ICE in RIA under the regulation of glr-3 promoter sequences. Expression of ICE results in the death of the neurons as a result of an apoptotic cell death. We have begun to characterize the behavioral consequences of the loss of RIA. We would also like to determine which subunits constitute a functional heteromeric glutamate receptor in RIA. Using electophysiological recording techniques we have shown that application of glutamate activates a rapid current in RIA. Deletion of both glr-3 and glr-6 eliminates the majority of the glutamate-gated current. Moreover, co-expression of GLR-3 and GLR-6 in Xenopus lavis oocytes is sufficient to form a functional glutamate gated channel. These data suggest that GLR-3 and GLR-6 constitute the components of a functional glutamate receptor in RIA. Along with signals from the thermotatic circuit, RIA receives input from several other neurons as well. To determine which of these neurons form glutamatergic synapses with RIA, we are using cell specific promoters to express the presynaptic marker VAMP fused to CFP. By expressing VAMP::CFP and either GLR-3::YFP or GLR-6::YFP in transgenic worms, we hope identify glutamatergic synapses by colocalization of the CFP and YFP signals. These studies we allow us to better understand the role of glutamate receptors in RIA and the molecular mechanisms of thermotaxis in the worm.

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