Identification of avoidance genes through neural pathway-specific forward optogenetics

Filipe Marques,Richard J Poole,Laurent Falquet,Dominique A Glauser,Gabriella Saro,Andrei-Stefan Lia,Kaveh Ashrafi

doi:10.1371/journal.pgen.1008509

Filipe Marques, Richard J Poole + Show 5 more

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https://doi.org/10.1371/journal.pgen.1008509

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Abstract

Understanding how the nervous system bridges sensation and behavior requires the elucidation of complex neural and molecular networks. Forward genetic approaches, such as screens conducted in C. elegans, have successfully identified genes required to process natural sensory stimuli. However, functional redundancy within the underlying neural circuits, which are often organized with multiple parallel neural pathways, limits our ability to identify ‘neural pathway-specific genes’, i.e. genes that are essential for the function of some, but not all of these redundant neural pathways. To overcome this limitation, we developed a ‘forward optogenetics’ screening strategy in which natural stimuli are initially replaced by the selective optogenetic activation of a specific neural pathway. We used this strategy to address the function of the polymodal FLP nociceptors mediating avoidance of noxious thermal and mechanical stimuli. According to our expectations, we identified both mutations in ‘general’ avoidance genes that broadly impact avoidance responses to a variety of natural noxious stimuli (unc-4, unc-83, and eat-4) and mutations that produce a narrower impact, more restricted to the FLP pathway (syd-2, unc-14 and unc-68). Through a detailed follow-up analysis, we further showed that the Ryanodine receptor UNC-68 acts cell-autonomously in FLP to adjust heat-evoked calcium signals and aversive behaviors. As a whole, our work (i) reveals the importance of properly regulated ER calcium release for FLP function, (ii) provides new entry points for new nociception research and (iii) demonstrates the utility of our forward optogenetic strategy, which can easily be transposed to analyze other neural pathways.

Highlights

One of the main functions of the nervous system is to perceive environmental stimuli, process them, and produce appropriate behavioral responses to limit threat and damage to the organism and maximize survival and reproductive prospects
Efficient sensory behavior often entails complex neural circuits, connecting various types of neurons, such as sensory neurons required for stimuli sensation, interneurons implicated in signal processing and propagation and motor neurons driving muscular activity and behavioral outcome
Our understanding of the molecular framework involved in sensory behaviors has strongly benefited from classical genome-wide mutagenesis genetic screens such as those conducted in invertebrates [1,2,3,4,5]

Summary

Introduction

One of the main functions of the nervous system is to perceive environmental stimuli, process them, and produce appropriate behavioral responses to limit threat and damage to the organism and maximize survival and reproductive prospects. The analysis of C. elegans sensory behavior neural circuits revealed in many instances a high level of degeneracy[6,7,8,9], i.e. that they contain “structurally different components that can perform a similar function with respect to context”[10, page 14]. This functional redundancy between parallel neural pathways, which may exploit different sets of regulatory molecules, improves the robustness of behavioral responses, but at the same time limits the outcomes of forward genetic screens (Fig 1A, 1B and 1C). Identifying neural pathway-specific genes is essential to obtain a comprehensive understanding of the molecular mechanisms underpinning sensory-behaviors

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