Identification of avoidance genes through neural pathway-specific forward optogenetics

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.

Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans

“…Final expression plasmids were generated by Gateway cloning between promoters (slot 1), mNeonGreen fluorescent ORF (slot 2; dg353; Hostettler et al 2017 ), unc-54 3′ UTR (slot 3; pMH473, gift from Marc Hammarlund), and pDEST-R4-R3. The control localization/expression plasmid [ sdf-9p :: NLS :: wrmScarlet ] was generated through a Gateway recombination reaction between dg801 (slot 1), dg651 (slot 2; Marques et al 2019 ), pMH473 (slot 3), and pDEST-R4-R3. Finally, promoter :: mNeonGreen and sdf-9p :: NLS :: wrmScarlet plasmids were co-injected into N2 young adult hermaphrodites at 20 ng/μl each, with 20 ng/μl of dg9 ( unc-122p :: RFP ; red coelomocyte) as a co-injection marker (#8938; Addgene; Miyabayashi et al 1999 ).…”

Neuropeptidergic regulation of Compulsive Ethanol Seeking in C. elegans

“…Recent studies on multisensory integration of the worms have provided sophisticated experimental manipulation with complex behavioral decision paradigms in vivo animals where information from distinct sensory networks is concurrently processed to demonstrate the comprehensible representation of the environment ( 78 , 79 , 80 ). We introduced nociceptive stimuli, of which perception and mediation are well defined ( 38 , 81 , 82 , 83 ), as an obstacle to interfere with the animals' ethanol seeking behavior. Since ethanol pretreatment does not change the worm's sensitivity to the aversive stimulus (Fig.…”

Tissue-specific isoforms of the single C. elegans Ryanodine receptor gene unc-68 control specific functions

“…Videos of grinder movements of adult animals on food were recorded at a 160X magnification thanks to a stereomicroscope (Leica M2015FA) equipped with a camera (Leica DFC345FX), as previously described [ 42 ]. Grinder movements were scored manually over 20 s to determine pumping rate.…”