Habituation is a simple form of non-associative learning, in which multiple repetitions of a stimulus cause a reduction in the response .amplitude.. Habituation of the C. elegans withdrawal reflex in response to gentle touch or a tap of the culture dish is well characterized. Yet, only little is known about the (molecular) mechanisms of habituation and the exact point where habituation is effected within the signalling cascade leading to withdrawal. This could occur at the level of the primary sensory receptors, signalling within the sensory neurons, at their output synapses or in the downstream interneuron network. Previously, we could demonstrate that optogenetic, Channelrhodopsin-2 (ChR2) mediated in vivo stimulation of mechanosensory neurons can induce withdrawal behaviour and that multiple optical stimuli also induce habituation [1]. Here we used the
mec-4 promoter, which is exclusievely expressed in touch neurons. Expression of ChR2 in the downstream (.backward.) command interneurons also could induce withdrawal behaviour, however, promoters active in these cells are not highly specific for the AVA neuron. Thus the optogenetic stimulation will also affect other cells (e.g. PVC) and so it is difficult to draw firm conclusions. We would like to achieve highly specific expression of ChR2 for the AVA neuron, and thus enable a specific photostimulation, with the assistance of the cre-lox system that was recently introduced for C. elegans [2], or, alternatively, the FLP recombinase system [3]; see poster by Christian Schultheis. This method provides the ability to use two different promotors for gene expression, with expression patterns that include the cell of interest, but no additional overlap in other cells in which each promoter may be active. Thus, expression of ChR2 would occur only at the intersection of the two expression patterns. We like to establish cre-lox driven expression of ChR2 in AVA, then test these transgenic animals for occurrence of photoinduced withdrawal, and, importantly, habituation of the response, just as in mec-neurons. An additional sensory neuron inducing withdrawals is the polymodal aversive neuron ASH, which detects harmful or toxic chemicals. We will establish single-cell expression of ChR2 in ASH, test for photoinduced withdrawal and habituation. If this is also found, potential cross-habituation of the two sensory pathways (mec-neurons and ASH) leading to withdrawal will be tested by combined photo-stimuli and .natural. stimuli, which should occur at the level of the interneurons. This procedure will provide the possibility to find the synapses which are responsible for habituation of the reflex. 1. Nagel, Brauner, Liewald, Adeishvili, Bamberg and Gottschalk (2005) Curr. Biol. 15: 2279-2284 2 Macosko, Pokala, Feinberg, Chalasani, Butcher, Clardy and Bargmann (2009) Nature 58:1171-1175 3. Davis, Morton, Carroll and Jorgensen (2008) PLoS Genetics 4:
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