We previously demonstrated that during mating, male cholinergic sex neurons induce spicule muscles protraction by activating nicotinic acetylcholine receptors (nAChRs). Here we suggest that a muscarinic acetylcholine receptor (mAChR) signaling pathway might work parallel to nAChRs to modulate spicules protraction and normalize male mating under non-optimal conditions. The mAChR pathway can be hyperactivated by high concentrations of oxotremorine M (a mAChR specific agonist), which causes spicules protraction in wild-type males. egl-30
encodes the only Gqα in C. elegans, and gar-3
encodes a mAChR. egl-30
(lf) and gar-3
(o) mutations fully suppress drug sensitivity, suggesting that oxotremorine M (oxo M) stimulates spicules protraction through gar-3
GAR-3 is expressed in both pre- and post-synaptic cells of the circuit that controls spicules protraction: SPC, PCB, PCA neurons and spicule protractor muscles. A reduction-of-function mutation in unc-64
(syntaxin) suppresses 57% of males from protracting spicules in the drug, suggesting that oxo M acts on neurons to cause spicule muscle contraction. Consistent with this, ablation of either SPC or PCB abolishes ~90% oxo M stimulated spicules protraction, indicating that GAR-3 functions through these neurons. Interestingly, ablation of both SPC and PCB results in 35.5% recovery of spicules protraction. We hypothesize that impairment in synapses might increase gar-3
expression in spicule muscles, and partially rescue drug sensitivity.
GAR-3 is also expressed in other cells that might control mating behavior. Moderate oxo M exposure or overexpression of gar-3
can cause males to display multiple mating-like behaviors, in addition to spicule protraction. Thus, GAR-3 might modulate multiple steps of behaviors during mating. Surprisingly, gar-3
(o) males can perform many of the steps of mating behavior without major defects. However, when mating with hermaphrodites whose vulvas are difficult to penetrate, these gar-3
(0) males tend to disengage from the vulva during spicule insertion attempts significantly more than wild type. We speculate that GAR-3 signaling is not essential for basic neuronal or muscle function, but might be used to upregulate spicule insertion and other male sub-behaviors when mating conditions are not optimal.