The C. elegans D2-Like Dopamine Receptor DOP-3 Decreases Behavioral Sensitivity to the Olfactory Stimulus 1-Octanol

We previously found that dopamine signaling modulates the sensitivity of wild-type C. elegans to the aversive odorant 1-octanol. C. elegans lacking the CAT-2 tyrosine hydroxylase enzyme, which is required for dopamine biosynthesis, are hypersensitive in their behavioral avoidance of dilute concentrations of octanol. Dopamine can also modulate the context-dependent response of C. elegans lacking RGS-3 function, a negative regulator of Gα signaling. rgs-3 mutant animals are defective in their avoidance of 100% octanol when they are assayed in the absence of food (E. coli bacterial lawn), but their response is restored when they are assayed in the presence of food or exogenous dopamine. However, it is not known which receptor might be mediating dopamine's effects on octanol avoidance. Herein we describe a role for the C. elegans D2-like receptor DOP-3 in the regulation of olfactory sensitivity. We show that DOP-3 is required for the ability of food and exogenous dopamine to rescue the octanol avoidance defect of rgs-3 mutant animals. In addition, otherwise wild-type animals lacking DOP-3 function are hypersensitive to dilute octanol, reminiscent of cat-2 mutants. Furthermore, we demonstrate that DOP-3 function in the ASH sensory neurons is sufficient to rescue the hypersensitivity of dop-3 mutant animals, while dop-3 RNAi knockdown in ASH results in octanol hypersensitivity. Taken together, our data suggest that dopaminergic signaling through DOP-3 normally acts to dampen ASH signaling and behavioral sensitivity to octanol

Sex-based Differences in C. elegans Responsiveness to Aversive Stimuli

Behavioral differences between sexes are evident across many species. The underlying mechanisms surrounding such differences are not fully elucidated, however, due to the complexities of animal behavior. The nematode Caenorhabditis elegans (C. elegans) is a well-characterized, genetically amenable species with two sexes, hermaphrodites (XX) and males (XO). This makes it an appropriate model system for investigating sex-based behavioral differences. Chemosensation in C. elegans is mediated by exposed ciliated sensory neurons, one of which is ASH. ASH is a polymodal nociceptor that elicits reversal when an animal encounters aversive stimuli. We hypothesized that hermaphrodite and male C. elegans worms respond differently to stimuli detected by ASH such as the bitter tastant quinine, the detergent sodium dodecyl sulfate (SDS), and the heavy metal copper (CuCl2). Wild-type assay-age hermaphrodites and males were picked from a nematode growth media (NGM) plate with E. coli OP50 and kept on an NGM plate without food for 10 minutes prior to assaying. A drop of aversive stimulus was placed in front of a forward-moving animal, and the animal’s response was recorded. A positive response is backwards movement within 4 seconds after contact with the stimulus. Our results reveal a quantifiable difference in how wild-type hermaphrodite and male C. elegans respond to aversive stimuli. Specifically, wild-type males are less responsive than hermaphrodites to quinine, SDS, and CuCl2. Further investigations will be conducted through experiments with C. elegans strains in which hermaphrodites have masculinized, and males have feminized nervous systems or subsets of neurons. Through these experiments, we aim to explore potential sites of difference that lead to these observable differences in responsiveness to aversive stimuli

G Protein-Coupled Receptor Kinase Function Is Essential for Chemosensation in C. elegans

AbstractG protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca2+ imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Gα subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation

Using a Robust and Sensitive GFP-Based cGMP Sensor for Real-Time Imaging in Intact Caenorhabditis elegans.

cGMP plays a role in sensory signaling and plasticity by regulating ion channels, phosphodiesterases, and kinases. Studies that primarily used genetic and biochemical tools suggest that cGMP is spatiotemporally regulated in multiple sensory modalities. FRET- and GFP-based cGMP sensors were developed to visualize cGMP in primary cell culture and Caenorhabditis elegans to corroborate these findings. While a FRET-based sensor has been used in an intact animal to visualize cGMP, the requirement of a multiple emission system limits its ability to be used on its own as well as with other fluorophores. Here, we demonstrate that a C. elegans codon-optimized version of the cpEGFP-based cGMP sensor FlincG3 can be used to visualize rapidly changing cGMP levels in living, behaving C. elegans We coexpressed FlincG3 with the blue-light-activated guanylyl cyclases BeCyclOp and bPGC in body wall muscles, and found that the rate of change in FlincG3 fluorescence correlated with the rate of cGMP production by each cyclase. Furthermore, we show that FlincG3 responds to cultivation temperature, NaCl concentration changes, and sodium dodecyl sulfate in the sensory neurons AFD, ASEL/R, and PHB, respectively. Intriguingly, FlincG3 fluorescence in ASEL and ASER decreased in response to a NaCl concentration upstep and downstep, respectively, which is opposite in sign to the coexpressed calcium sensor jRGECO1a and previously published calcium recordings. These results illustrate that FlincG3 can be used to report rapidly changing cGMP levels in an intact animal, and that the reporter can potentially reveal unexpected spatiotemporal landscapes of cGMP in response to stimuli

A Functional Nuclear Localization Sequence in the C. elegans TRPV Channel OCR-2

The ability to modulate gene expression in response to sensory experience is critical to the normal development and function of the nervous system. Calcium is a key activator of the signal transduction cascades that mediate the process of translating a cellular stimulus into transcriptional changes. With the recent discovery that the mammalian Cav1.2 calcium channel can be cleaved, enter the nucleus and act as a transcription factor to control neuronal gene expression, a more direct role for the calcium channels themselves in regulating transcription has begun to be appreciated. Here we report the identification of a nuclear localization sequence (NLS) in the C. elegans transient receptor potential vanilloid (TRPV) cation channel OCR-2. TRPV channels have previously been implicated in transcriptional regulation of neuronal genes in the nematode, although the precise mechanism remains unclear. We show that the NLS in OCR-2 is functional, being able to direct nuclear accumulation of a synthetic cargo protein as well as the carboxy-terminal cytosolic tail of OCR-2 where it is endogenously found. Furthermore, we discovered that a carboxy-terminal portion of the full-length channel can localize to the nucleus of neuronal cells. These results suggest that the OCR-2 TRPV cation channel may have a direct nuclear function in neuronal cells that was not previously appreciated

A putative bipartite NLS in the TRPV channel OCR-2.

<p>Amino acids 841–856 of OCR-2 contain two clusters of lysine/arginine residues, separated by 9 amino acids. (A) Alignment with the canonical bipartite nuclear localization sequence demonstrates that the OCR-2 sequence closely matches an NLS. (B) A diagram of the <i>C. elegans</i> OCR-2 channel, with the putative NLS in black, reveals that the sequence is located in the cytoplasmic carboxy-terminus of the channel. The cytoplasmic side is located below the horizontal lines that denote the plasma membrane. (Diagram was created using TOPO2, Transmembrane Protein Display software, <a href="http://www.sacs.ucsf.edu/TOPO2" target="_blank">http://www.sacs.ucsf.edu/TOPO2</a>).</p

The cytoplasmic carboxy-terminal fragment of OCR-2 can utilize the NLS to localize to the nucleus.

<p>The entire carboxy-terminus (amino acids 765–900, “OCR-2_C-term”) was fused upstream of and in frame with GFP and expressed under the control of the <i>ocr-2</i> promoter (<i>ocr-2p::ocr-2_C-term::gfp</i>). (A) The OCR-2_C-term::GFP fusion was detected in the nucleus of <i>ocr-2</i> expressing head and tail neurons in 91/91 transgenic animals examined. The PHA/PHB tail neurons are shown here. Furthermore, the basic residues of the NLS are required for the nuclear accumulation observed. (B) A GFP fusion to the carboxy-terminal tail in which the 6 previously identified essential lysine/arginine residues had been mutated to alanine (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025047#pone-0025047-g002" target="_blank">Figure 2C</a>) (<i>ocr-2p::ocr-2_{C-term(NLSmut)}::gfp</i>) resulted in a much less pronounced signal in the nucleus. The OCR-2_{C-term(NLSmut)}::GFP signal was observed evenly distributed in the cytoplasm and nucleus of <i>ocr-2</i> expressing neurons in 94/94 transgenic animals examined. The PHA/PHB tail neurons are shown here. Numbers represent the combined data of ≥3 independent transgenic lines for each construct.</p