NPR-9, a Galanin-Like G-Protein Coupled Receptor, and GLR-1 Regulate Interneuronal Circuitry Underlying Multisensory Integration of Environmental Cues in Caenorhabditis elegans

Abstract

C. elegans inhabit environments that require detection of diverse stimuli to modulate locomotion in order to avoid unfavourable conditions. In a mammalian context, a failure to appropriately integrate environmental signals can lead to Parkinson's, Alzheimer's, and epilepsy. Provided that the circuitry underlying mammalian sensory integration can be prohibitively complex, we analyzed nematode behavioral responses in differing environmental contexts to evaluate the regulation of context dependent circuit reconfiguration and sensorimotor control. Our work has added to the complexity of a known parallel circuit, mediated by interneurons AVA and AIB, that integrates sensory cues and is responsible for the initiation of backwards locomotion. Our analysis of the galanin-like G-protein coupled receptor NPR-9 in C. elegans revealed that upregulation of galanin signaling impedes the integration of sensory evoked neuronal signals. Although the expression pattern of npr-9 is limited to AIB, upregulation of the receptor appears to impede AIB and AVA circuits to broadly prevent backwards locomotion, i.e. reversals, suggesting that these two pathways functionally interact. Galanin signaling similarly plays a broadly inhibitory role in mammalian models. Moreover, our identification of a mutant, which rarely initiates backwards movement, allowed us to interrogate locomotory mechanisms underlying chemotaxis. In support of the pirouette model of chemotaxis, organisms that did not exhibit reversal behavior were unable to navigate towards an attractant peak. We also assessed ionotropic glutamate receptor GLR-1 cell-specifically within AIB and determined that GLR-1 fine-tunes AIB activity to modify locomotion following reversal events. Our research highlights that signal integration underlying the initiation and fine-tuning of backwards locomotion is AIB and NPR-9 dependent, and has demonstrated the suitability of C. elegans for analysis of multisensory integration and sensorimotor control.

Fig 1. On and off food reversal frequencies are regulated by NPR-9 and GLR-1 in the AIB.

(A) Reversal phenotypes over the course of 3 minutes on food in wildtype (N2), glr-1 knockdown in the AIB (glr-1 KD), npr-9(LF), npr-9(LF);glr-1 KD, glr-1(LF), and npr-9(GF). (B) Off food reversal frequencies of N2, glr-1 KD, npr-9(LF), npr-9(LF);glr-1 KD, and npr-9(GF). Data are presented as mean +/- standard error (SE) with at least 5 animals assayed in three independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction. *** p < .001, ** p< .01, significantly different from wild-type animals under identical conditions. Asterisks above error bars indicate significantly different between the two strains.

Fig 2. Glutamate is a key regulator of omega turns.

N2, glr-1(LF), glr-1 KD, npr-9(LF), npr-9(LF);glr-1 KD, and npr-9(GF) were tested for the frequency of reversals that terminate with an omega turn during off food conditions Data are presented as mean +/- standard error (SE) with at least 5 animals assayed in three independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction. *** p < .001, significantly different from wild-type animals under identical conditions.

Fig 3. Over-expression of npr-9 inhibits nose touch responses.

Wildtype, glr-1(LF), glr-1 KD, npr-9(LF), npr-9(LF);glr-1 KD, and npr-9(GF) were evaluated for ability to respond to nose touch stimulation. Data are presented as mean +/- standard error (SE) with at least 10 animals assayed in three independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction. *** p< .001, significantly different from wild-type animals under identical conditions.

Fig 4. NPR-9 and GLR-1 signaling are non-essential for on food 30% octanol behavior, but play a role in off food 30% octanol responses.

(A) N2, eat-4, glr-1 KD, npr-9(LF), npr-9(LF);glr-1 KD, and npr-9(GF) were assayed for responses to 30% octanol on food. (B) The aforementioned strains were evaluated for responsiveness to 30% octanol off food. Data are presented as mean +/- standard error (SE) with at least 10 animals assayed in three independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction. *** p < .001, significantly different from wild-type animals under identical conditions. Asterisks above error bars indicate significantly different between the two strains.

Fig 5. npr-9(GF) initiates reversals in response to harsh touch.

Wildtype and npr-9(GF) organisms do not differ in their responses to mechanostimulation via harsh touch. Data are presented as mean +/- standard error (SE) with at least 5 animals assayed in three independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction. NS = non-significant.

Fig 6. npr-9(GF) is not attracted to low concentrations of diacetyl.

N2, odr-10, glr-1 KD, npr-9(LF), npr-9(LF);glr-1 KD, and npr-9(GF) were assessed for chemotaxis to low concentration of diacetyl. Data are presented as mean +/- standard error (SE) with at least 50 animals assayed in three independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction. *** p < .001, significantly different from wild-type animals under identical conditions.

Fig 7. Inducing reversals with plate tap stimulation improves diacetyl responsiveness in npr-9(GF) organisms.

odr-10 and npr-9(GF) populations were evaluated for the diacetyl response, while plate taps were performed. Data are presented as mean +/- SE and analyzed with an unpaired two-tailed t test with Welsch’s correction. ** p< .01, significantly different from odr-10 animals under identical conditions. NS = non-significant. Asterisks above error bars indicate significantly different between the two strains.

Fig 8. Inducing reversals with plate tap stimulation does not negatively affect chemotaxis.

N2 and npr-9(GF) were evaluated for individual diacetyl responses, while plate taps were performed. Data are presented as mean +/- standard error (SE) with one animal assayed in 20 independent experiments analyzed via an unpaired two-tailed t test with Welsch’s correction NS = non-significant. Asterisks above error bars indicate significantly different between the two strains. (away) indicates that organisms were initially oriented away from the attractant peak before plate tap, while (towards) indicates that organisms were initially moving towards the attractant peak before plate tap.

Fig 9. npr-9(GF) does not integrate food cues to modulate locomotory and aversive behavior to copper.

(A) N2 and npr-9(GF) were evaluated for their ability to reach and stay on food during the copper-modified food race assay. (B) The aforementioned strains were evaluated for copper aversion in the absence of food in which positive responses are scored as crossing the copper barrier. Data are presented as mean +/- SE and at least 10 animals were assayed in three independent experiments analyzed with a two-way RM ANOVA. ** p< .01, significantly different from N2 animals under identical conditions.