Richard Y. Hwang

Pickpocket Is a DEG/ENaC Protein Required for Mechanical Nociception in Drosophila Larvae

Highly branched class IV multidendritic sensory neurons of the Drosophila larva function as polymodal nociceptors that are necessary for behavioral responses to noxious heat (>39 degrees C) or noxious mechanical (>30 mN) stimuli. However, the molecular mechanisms that allow these cells to detect both heat and force are unknown. Here, we report that the pickpocket (ppk) gene, which encodes a Degenerin/Epithelial Sodium Channel (DEG/ENaC) subunit, is required for mechanical nociception but not thermal nociception in these sensory cells. Larvae mutant for pickpocket show greatly reduced nociception behaviors in response to harsh mechanical stimuli. However, pickpocket mutants display normal behavioral responses to gentle touch. Tissue-specific knockdown of pickpocket in nociceptors phenocopies the mechanical nociception impairment without causing defects in thermal nociception behavior. Finally, optogenetically triggered nociception behavior is unaffected by pickpocket RNAi, which indicates that ppk is not generally required for the excitability of the nociceptors. Interestingly, DEG/ENaCs are known to play a critical role in detecting gentle touch stimuli in Caenorhabditis elegans and have also been implicated in some aspects of harsh touch sensation in mammals. Our results suggest that neurons that detect harsh touch in Drosophila utilize similar mechanosensory molecules.

The Ankyrin Repeat Domain of the TRPA Protein Painless Is Important for Thermal Nociception but Not Mechanical Nociception

The Drosophila TRPA channel Painless is required for the function of polymodal nociceptors which detect noxious heat and noxious mechanical stimuli. These functions of Painless are reminiscent of mammalian TRPA channels that have also been implicated in thermal and mechanical nociception. A popular hypothesis to explain the mechanosensory functions of certain TRP channels proposes that a string of ankyrin repeats at the amino termini of these channels acts as an intracellular spring that senses force. Here, we describe the identification of two previously unknown Painless protein isoforms which have fewer ankyrin repeats than the canonical Painless protein. We show that one of these Painless isoforms, that essentially lacks ankyrin repeats, is sufficient to rescue mechanical nociception phenotypes of painless mutant animals but does not rescue thermal nociception phenotypes. In contrast, canonical Painless, which contains Ankyrin repeats, is sufficient to largely rescue thermal nociception but is not capable of rescuing mechanical nociception. Thus, we propose that in the case of Painless, ankryin repeats are important for thermal nociception but not for mechanical nociception.

Optogenetic manipulation of neural circuits and behavior in Drosophila larvae

Optogenetics is a powerful tool that enables the spatiotemporal control of neuronal activity and circuits in behaving animals. Here, we describe our protocol for optical activation of neurons in Drosophila larvae. As an example, we discuss the use of optogenetics to activate larval nociceptors and nociception behaviors in the third-larval instar. We have previously shown that, using spatially defined GAL4 drivers and potent UAS (upstream activation sequence)-channelrhodopsin-2∷YFP transgenic strains developed in our laboratory, it is possible to manipulate neuronal populations in response to illumination by blue light and to test whether the activation of defined neural circuits is sufficient to shape behaviors of interest. Although we have only used the protocol described here in larval stages, the procedure can be adapted to study neurons in adult flies—with the caveat that blue light may not sufficiently penetrate the adult cuticle to stimulate neurons deep in the brain. This procedure takes 1 week to culture optogenetic flies and ∼1 h per group for the behavioral assays.

Balboa Binds to Pickpocket In Vivo and Is Required for Mechanical Nociception in Drosophila Larvae

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In this study, in a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, we identify a gene that we name balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led us to hypothesize that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, we directly detect in vivo physical interactions among PPK and Balboa subunits. Combined, our results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.