DOP-2 D2-Like Receptor Regulates UNC-7 Innexins to Attenuate Recurrent Sensory Motor Neurons during C. elegans Copulation.

Neuromodulation of self-amplifying circuits directs context-dependent behavioral executions. Although recurrent networks are found throughout the Caenorhabditis elegans connectome, few reports describe the mechanisms that regulate reciprocal neural activity during complex behavior. We used C. elegans male copulation to dissect how a goal-oriented motor behavior is regulated by recurrently wired sensory-motor neurons. As the male tail presses against the hermaphrodite's vulva, cholinergic and glutamatergic reciprocal innervations of post cloaca sensilla (PCS) neurons (PCA, PCB, and PCC), hook neurons (HOA, HOB), and their postsynaptic sex muscles execute rhythmic copulatory spicule thrusts. These repetitive spicule movements continue until the male shifts off the vulva or genital penetration is accomplished. However, the signaling mechanism that temporally and spatially restricts repetitive intromission attempts to vulva cues was unclear. Here, we report that confinement of spicule insertion attempts to the vulva is facilitated by D2-like receptor modulation of gap-junctions between PCB and the hook sensillum. We isolated a missense mutation in the UNC-7(L) gap-junction isoform, which perturbs DOP-2 signaling in the PCB neuron and its electrical partner, HOA. The glutamate-gated chloride channel AVR-14 is expressed in HOA. Our analysis of the unc-7 mutant allele indicates that when DOP-2 promotes UNC-7 electrical communication, AVR-14-mediated inhibitory signals pass from HOA to PCB. As a consequence, PCB is less receptive to be stimulated by its recurrent synaptic partner, PCA. Behavioral observations suggest that dopamine neuromodulation of UNC-7 ensures attenuation of recursive intromission attempts when the male disengages or is dislodged from the hermaphrodite genitalia. SIGNIFICANCE STATEMENT: Using C. elegans male copulation as a model, we found that the neurotransmitter dopamine stimulates D2-like receptors in two sensory circuits to terminate futile behavioral loops. The D2-like receptors promote inhibitory electrical junction activity between a chemosensory and a mechanosensory circuit. Therefore, both systems are attenuated and the animal ceases the recursive behavior.

Sensory perception of food and insulin-like signals influence seizure susceptibility.

Food deprivation is known to affect physiology and behavior. Changes that occur could be the result of the organism's monitoring of internal and external nutrient availability. In C. elegans, male mating is dependent on food availability; food-deprived males mate with lower efficiency compared to their well-fed counterparts, suggesting that the mating circuit is repressed in low-food environments. This behavioral response could be mediated by sensory neurons exposed to the environment or by internal metabolic cues. We demonstrated that food-deprivation negatively regulates sex-muscle excitability through the activity of chemosensory neurons and insulin-like signaling. Specifically, we found that the repressive effects of food deprivation on the mating circuit can be partially blocked by placing males on inedible food, E. coli that can be sensed but not eaten. We determined that the olfactory AWC neurons actively suppress sex-muscle excitability in response to food deprivation. In addition, we demonstrated that loss of insulin-like receptor (DAF-2) signaling in the sex muscles blocks the ability of food deprivation to suppress the mating circuit. During low-food conditions, we propose that increased activity by specific olfactory neurons (AWCs) leads to the release of neuroendocrine signals, including insulin-like ligands. Insulin-like receptor signaling in the sex muscles then reduces cell excitability via activation of downstream molecules, including PLC-gamma and CaMKII.

Food deprivation attenuates seizures through CaMKII and EAG K+ channels.

Accumulated research has demonstrated the beneficial effects of dietary restriction on extending lifespan and increasing cellular stress resistance. However, reducing nutrient intake has also been shown to direct animal behaviors toward food acquisition. Under food-limiting conditions, behavioral changes suggest that neuronal and muscle activities in circuits that are not involved in nutrient acquisition are down-regulated. These dietary-regulated mechanisms, if understood better, might provide an approach to compensate for defects in molecules that regulate cell excitability. We previously reported that a neuromuscular circuit used in Caenorhabditis elegans male mating behavior is attenuated under food-limiting conditions. During periods between matings, sex-specific muscles that control movements of the male's copulatory spicules are kept inactive by UNC-103 ether-a-go-go-related gene (ERG)-like K(+) channels. Deletion of unc-103 causes approximately 30%-40% of virgin males to display sex-muscle seizures; however, when food is deprived from males, the incidence of spontaneous muscle contractions drops to 9%-11%. In this work, we used genetics and pharmacology to address the mechanisms that act parallel with UNC-103 to suppress muscle seizures in males that lack ERG-like K(+) channel function. We identify calcium/calmodulin-dependent protein kinase II as a regulator that uses different mechanisms in food and nonfood conditions to compensate for reduced ERG-like K(+) channel activity. We found that in food-deprived conditions, calcium/calmodulin-dependent protein kinase II acts cell-autonomously with ether-a-go-go K(+) channels to inhibit spontaneous muscle contractions. Our work suggests that upregulating mechanisms used by food deprivation can suppress muscle seizures.

Molecular signaling involved in regulating feeding and other motivated behaviors.

The metabolic and nutritional status of an organism influences multiple behaviors in addition to food intake. When an organism is hungry, it employs behaviors that help it locate and ingest food while suppressing behaviors that are not associated with this goal. Alternatively, when an organism is satiated, food-seeking behaviors are repressed so that the animal can direct itself to other goal-oriented tasks such as reproductive behaviors. Studies in both vertebrate and invertebrate model systems have revealed that food-deprived and -satiated behaviors are differentially executed and integrated via common molecular signaling mechanisms. This article discusses cellular and molecular mechanisms for how insulin, neuropeptide Y (NPY), and serotonin utilize common signaling pathways to integrate feeding and metabolic state with other motivated behaviors. Insulin, NPY, and serotonin are three of the most well-studied molecules implicated in regulating such behaviors. Overall, insulin signaling allows an organism to coordinate proper behavioral output with changes in metabolism, NPY activates behaviors required for locating and ingesting food, and serotonin modulates behaviors performed when an organism is satiated. These three molecules work to ensure that the proper behaviors are executed in response to the feeding state of an organism.

Behavioral genetics of caenorhabditis elegans unc-103-encoded erg-like K(+) channel.

The Caenorhabditis elegans unc-103 gene encodes a potassium channel whose sequence is most similar to the ether-a-go-go related gene (erg) type of K+ channels. We find that the n 500 and e 1597 gain-of-function (gf) mutations in unc-103 cause reduced excitation in most muscles, while loss-of-function (lf) mutations cause mild muscle hyper-excitability. Both gf alleles change the same residue near the cytoplasmic end of S6, consistent with this region regulating channel activation. We also report additional dominant-negative and lf alleles of unc-103 that can antagonize or reduce the function of both gf and wild-type alleles. The unc-103 locus contains 6 promoter regions that express unc-103 in different combinations of body-wall and sex-specific muscles, motor-, inter- and sensory-neurons. Each promoter drives transcripts containing a unique first exon, conferring sequence variability to the N-terminus of the UNC-103 protein, while three splice variants introduce variability into the UNC-103 C-terminus. unc-103(0) hermaphrodites prematurely lay embryos that would normally be retained in the uterus and lay eggs under conditions that inhibit egg-laying behavior. In the egg-laying circuit, unc-103 is expressed in vulval muscles and the HSN neurons from different promoters. Supplying the proper UNC-103 isoform to the vulval muscles is sufficient to restore regulation to egg-laying behavior.

Integration of male mating and feeding behaviors in Caenorhabditis elegans.

The Caenorhabditis elegans male must integrate various environmental cues to ensure proper execution of mating. One step of male mating, the insertion of the male copulatory spicules into its mate, requires UNC-103 ERG (ether-a-go-go-related gene)-like K+ channels. unc-103(lf) alleles cause males to protract their spicules spontaneously in the absence of mating cues. To identify proteins that work with UNC-103, we suppressed unc-103(lf) and isolated lev-11(rg1). LEV-11 (tropomyosin) regulates the spicules directly by controlling the male sex muscles and indirectly by controlling the pharyngeal muscles. lev-11-mediated suppression requires the pharyngeal NSM neurosecretory motor neurons; ablating these neurons in lev-11(rg1); unc-103(lf) males restores spontaneous spicule protraction. Additionally, unc-103-induced spicule protraction can be suppressed by reducing a pharyngeal-specific troponin T. These observations demonstrate that non-genitalia cells involved in feeding also mediate male sexual behaviors.

Structural analysis of sterol distributions in the plasma membrane of living cells.

Although plasma membrane (PM) cholesterol-rich and -poor domains have been isolated by subcellular fractionation, the real-time arrangement of cholesterol in such domains in living cells is still unclear. Therefore, dehydroergosterol (DHE), a naturally occurring fluorescent sterol, was incorporated into cultured L-cell fibroblasts. Two PM markers, the enhanced cyan fluorescent protein (ECFP-Mem) and 3'-dioctadecyloxacarbocyanine perchlorate [DiOC(18)(3)], were used to distinguish DHE localized at the PM of living cells. Spatial enrichment of DHE in the PM of living cells was visualized in real time by multiphoton laser scanning microscopy (MPLSM). Quantitative models and image-processing techniques were developed for statistical analysis of the distribution of DHE within the PM. The PM was resolved from the cytoplasm in a two-step process, and a smooth trajectory reference of the PM was refined by statistical regression and moments-based techniques. Thus, DHE intensities over the PM were measured following the major DHE intensity distributions. Spatial distributions of DHE within the PM were examined by a statistical inference technique, complete spatial randomness (CSR). For PM regions densely populated with DHE, the distributions of DHE exhibited statistical arrangements that were not spatial random (i.e., homogeneous Poisson process) or regular but, instead, exhibited strong cluster patterns. In effect, real-time MPLSM imaging data for the first time demonstrated that sterol enrichment occurred in clustered regions in the PM, consistent with the existence of cholesterol-rich domains in the plasma membrane of living cells.