G alpha(q)-coupled muscarinic acetylcholine receptors enhance nicotinic acetylcholine receptor signaling in Caenorhabditis elegans mating behavior

Abstract

In this study, we address why metabotropic and ionotropic cholinergic signaling pathways are used to facilitate motor behaviors. We demonstrate that a G alpha(q)-coupled muscarinic acetylcholine receptor (mAChR) signaling pathway enhances nicotinic acetylcholine receptor (nAChR) signaling to facilitate the insertion of the Caenorhabditis elegans male copulatory spicules into the hermaphrodite during mating. Previous studies showed that ACh (acetylcholine) activates nAChRs on the spicule protractor muscles to induce the attached spicules to extend from the tail. Using the mAChR agonist Oxo M (oxotremorine M), we identified a GAR-3(mAChR)-G alpha(q) pathway that promotes protractor muscle contraction by upregulating nAChR signaling before mating. GAR-3(mAChR) is expressed in the protractor muscles and in the spicule-associated SPC and PCB cholinergic neurons. However, ablation of these neurons or impairing cholinergic transmission reduces drug-induced spicule protraction, suggesting that drug-stimulated neurons directly activate muscle contraction. Behavioral analysis of gar-3 mutants indicates that, in wild-type males, GAR-3(mAChR) expression in the SPC and PCB neurons is required for the male to sustain rhythmic spicule muscle contractions during attempts to breach the vulva. We propose that the GAR-3(mAChR)/G alpha(q) pathway sensitizes the spicule neurons and muscles before and during mating so that the male can respond to hermaphrodite vulva efficiently.

Figure 1.

Anatomy, circuit diagram, and model of GAR-3(mAChR)-mediated cell interactions. A, Cutaway representation of muscles and neurons used in spicule protraction behavior. B, Partial circuit diagram of connections between the SPC neuron, protractor muscles, and the p.c.s. neurons [adapted from Sulston et al. (1980) and S. W. Emmons, D. H. Hall, and M. Xu, personal communication] (www.wormatlas.org). The SPC and p.c.s. neurons make more extensive connections to other cells not covered in this report. C, Before mating, activated GAR-3(mAChR)/Gα_q on the SPC and PCB neurons promotes low levels of acetylcholine release. The low-level release of acetylcholine also indirectly results in an increased sensitivity of postsynaptic nAChR signaling potential on the spicule protractor muscle cells and possibly also in the SPC and PCB neurons.

Figure 2.

Levamisole-induced spicule protraction requires GAR-3(mAChR)/Gα_q signaling and ACh synaptic transmission. The numbers on the vertical axis represent the percentage of males that protract their spicules at each concentration of LEV. The numbers above or within the bars are the actual percentage of males that protracted in the drug. The numbers of males assayed at each concentration are listed below the bars.

Figure 3.

Male tail expression of unc-38 and gar-3 promoters. Fluorescence images of the right lateral tail region of an adult male expressing the unc-38 promoter::YFP construct (A), early L4 male expressing the unc-38 promoter::YFP construct (B), and adult (C) and late L4 (D) males expressing the P_gar-3B::YFP construct pYL1. Scale bar, 10 μm.

Figure 4.

Dose–response of wild-type males to oxotremorine M. The numbers on the x-axis represent the concentrations of Oxo M at which the wild-type males were treated. The numbers on the y-axis represent the percentage of males that protracted their spicules within 10 min. The black squares represent the percentage of males that protracted their spicules. Approximately 30 males were assayed for each drug concentration.

Figure 5.

Locations of the gar-3 promoters and the gk305 deletion. The genomic position of gar-3 is indicated on top; the scale bar is in kilobases. Two promoters used in this study, P_gar-3A and P_gar-3B, are also depicted. Four transcriptional isoforms of gar-3 are also shown [adapted from Hwang et al. (1999) and http://www.wormbase.org, stable release WS160]. The open boxes are exons, the gray boxes are UTRs, and the lines are introns; the arrows and arrows merged with gray boxes depict the translation directions. The deletion in the gar-3(lf) allele is indicated by a double arrowed line, and the positions of putative transmembrane domains (I–VII) are indicated with horizontal lines [adapted from Park et al. (2003)].

Figure 6.

Native and heterologous expression of the gar-3::YFP translational fusion protein. A, B, DIC (A) and fluorescence (B) images of the left lateral tail region of a mid L4 male expressing both P_gar-3A::gar-3::YFP (pYL8) and P_gar-3B::gar-3::YFP (pYL9). Nuclei of the neurons are labeled with arrows. In B, positions of these neurons are indicated with circles, and localizations of GAR-3::YFP on the cell membranes are denoted by arrows. C, D, DIC (C) and fluorescence (D) images of left lateral tail region of an adult male expressing P_{unc-103 E}::gar-3::YFP (pYL10). E, F, Merged DIC and fluorescence (E) and fluorescence (F) images of left lateral tail region of a mid L4 male expressing the P_{unc-103 F} ::gar-3::YFP (pYL11). In E, nuclei of neurons are labeled with arrows. GAR-3::YFP localization on cell membranes can be seen as white puncta. In F, neuronal positions are indicated with circles, and localizations of GAR-3::YFP on the cell membranes are denoted by arrows. Scale bar, 10 μm.

Figure 7.

gar-3(lf) males use more spicule insertion attempts to achieve vulva penetration. The spots represent the total number of vulval stops (insertion attempts) used by individual males. Twenty-six wild-type males and 29 gar-3(lf) males were assayed. The horizontal bar indicates the sample median. The p value was calculated using the Mann–Whitney test.