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1.
J Biol Methods ; 7(1): e129, 2020.
Article in English | MEDLINE | ID: mdl-32313814

ABSTRACT

Nociception and its plasticity are essential biological processes controlling adaptive behavioral responses in animals. These processes are also linked to different pain conditions in human and have received considerable attention, notably via studies in rodent models and the use of heat-evoked withdrawal behavior assays as a readout of unpleasant experience. More recently, invertebrates have also emerged as useful complementary models, with their own set of advantages, including their amenability to genetic manipulations, the accessibility and relative simplicity of their nervous system and ethical concerns linked to animal suffering. Like humans, the nematode Caenorhabditis elegans (C. elegans) can detect noxious heat and produce avoidance responses such as reversals. Here, we present a methodology suitable for the high-throughput analysis of C. elegans heat-evoked reversals and the adaptation to repeated stimuli. We introduce two platforms: the INFERNO (for infrared-evoked reversal analysis platform), allowing the quantification of the thermal sensitivity in a petri dish containing a large population (> 100 animals), and the ThermINATOR (for thermal adaptation multiplexed induction platform), allowing the mass-adaptation of up to 18 worm populations at the same time. We show that wild type animals progressively desensitize in response to repeated noxious heat pulses. Furthermore, analyzing the phenotype of mutant animals, we show that the mechanisms underlying baseline sensitivity and adaptation, respectively, are supported by genetically separable molecular pathways. In conclusion, the presented method enables the high-throughput evaluation of thermal avoidance in C. elegans and will contribute to accelerate studies in the field with this invertebrate model.

2.
Cell Rep ; 30(2): 397-408.e4, 2020 01 14.
Article in English | MEDLINE | ID: mdl-31940484

ABSTRACT

Pain sensation and aversive behaviors entail the activation of nociceptor neurons, whose function is largely conserved across animals. The functional heterogeneity of nociceptors and ethical concerns are challenges for their study in mammalian models. Here, we investigate the function of a single type of genetically identified C. elegans thermonociceptor named FLP. Using calcium imaging in vivo, we demonstrate that FLP encodes thermal information in a tonic and graded manner over a wide thermal range spanning from noxious cold to noxious heat (8°C-36°C). This tonic-signaling mode allows FLP to trigger sustained behavioral changes necessary for escape behavior. Furthermore, we identify specific transient receptor potential, voltage-gated calcium, and sodium "leak" channels controlling sensory gain, thermal sensitivity, and signal kinetics, respectively, and show that the ryanodine receptor is required for long-lasting activation. Our work elucidates the task distribution among specific ion channels to achieve remarkable sensory properties in a tonic thermonociceptor in vivo.


Subject(s)
Ion Channels/metabolism , Optogenetics/methods , Thermosensing/physiology , Animals , Animals, Genetically Modified , Caenorhabditis elegans , Nociceptors/metabolism , Temperature
3.
PLoS Genet ; 15(12): e1008509, 2019 12.
Article in English | MEDLINE | ID: mdl-31891575

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.


Subject(s)
Avoidance Learning , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/physiology , Optogenetics/methods , Animals , Caenorhabditis elegans/genetics , Calcium Signaling , Cytoskeletal Proteins/genetics , Gene Expression Regulation , Homeodomain Proteins/genetics , Intercellular Signaling Peptides and Proteins/genetics , Membrane Proteins/genetics , Nuclear Proteins/genetics , Receptors, Glutamate/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Vesicular Glutamate Transport Proteins
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