Optogenetic Stimulation Uncovers a Role for Neuropeptide Signalling in Habituation of a C. Elegans Avoidance Response

The overall objective of this research is to elucidate mechanisms of non-associative learning (habituation, dishabituation, sensitization) in Caenorhabditis elegans. The advantage of using C. elegans is that high-throughput learning assays can be used to investigate behaviours mediated by intact and accessible neural circuits. Polymodal sensory neuron ASH mediates backward locomotion in response to a variety of nociceptive stimuli, including osmotic pressure, nose touch, and a variety of volatile and non-volatile compounds. Using a Multi-Worm Tracking system capable of quantifying the behavior of dozens of worms simultaneously, we simulate aversive stimuli by optogenetic activation of ASH. This allows for high throughput manipulations without bottlenecks from either data collection or data analysis. A short light pulse (200-ms) of low intensity blue light elicited a reversal response of about 200-µm in 80-90% animals expressing ChR2 in ASH. A longer light pulse (2-s) elicited a larger reversal response more likely to end in an omega-shaped turn. Previous work demonstrated that ASH-mediated responses to different stimuli are genetically dissociable downstream of sensory transduction. We monitored the responses of various mutants and found that the behavioural impairments associated with nose touch and osmo-avoidance assays were also apparent in response to the short and long light pulses, respectively. This suggests that a part of the neural code distinguishing nose touch and chemo-aversion is the duration of depolarization of ASH. We next tested habituation of ASH-mediated responses to our “nose touch” and “chemoaversive” blue light pulses. Although the size of the reversal response did not change over trials, the likelihood of responding decreased with repeated stimuli separated by 10, but not 60-s. The kinetics of the behavioural decrement was dependent upon the duration of the light pulse, with the proportion of worms responding to the longer light pulse decreasing faster and further than responses to the nose touch-like stimulus. A non-localized mechanical stimulus (tap to the side of the Petri plate) could facilitate responses from baseline (sensitization) or a habituated level (dishabituation). To investigate the cellular mechanisms underlying habituation, we began by testing mutants to assess the role of various neuromodulators. This candidate gene approach identified habituation phenotypes for neuropeptide processing mutants (egl-3 and egl-21), i.e. loss of neuropeptide signalling resulted in worms that maintained a high level of responding after repeated activation of ASH. Current work is aimed at identifying the cellular source of the peptide signal, as well as the relevant ligand(s) and receptor(s).