Crosstalk with the GAR-3 receptor contributes to feeding defects inCaenorhabditis elegans eat-2mutants

Abstract

Article SummaryAcetylcholine stimulates different contractions in adjacent muscle cells in the C. elegans pharynx called pumping and peristalsis. The signaling mechanisms stimulating pumping have been characterized, but how these mechanisms affect peristalsis is unknown. Here we examined muscle contractions and Ca 2+ transients during peristalsis in wild-type animals and acetylcholine signaling mutants. Surprisingly we found that while mutants affecting the eat-2 nicotinic acetylcholine receptor exhibited reduced pumping, they also hyperstimulated peristalses. This hyperstimulation depends on crosstalk with the GAR-3 muscarinic acetylcholine receptor in adjacent cells, and it contributes to the well-characterized dietary restriction and extended adult lifespan observed in eat-2 mutants. AbstractPrecise signaling at the neuromuscular junction (NMJ) is essential for proper muscle contraction.In the C. elegans pharynx, acetylcholine (ACh) released from the MC and M4 motor neurons stimulates two different types of contractions in adjacent muscle cells, termed pumping and isthmus peristalsis. MC stimulates rapid pumping through the nicotinic ACh receptor EAT-2, which is tightly localized at the MC NMJ, and eat-2 mutants exhibit a slow pump rate.Surprisingly, we found that eat-2 mutants also hyperstimulated peristaltic contractions, and these are characterized by increased and prolonged Ca 2+ transients in the isthmus muscles.This hyperstimulation depends on crosstalk with the GAR-3 muscarinic acetylcholine receptor as gar-3 mutation specifically suppressed the prolonged contraction and increased Ca 2+ observed in eat-2 mutant peristalses. Similar GAR-3 dependent hyperstimulation was also observed in mutants lacking the ace-3 acetylcholinesterase, and we suggest that NMJ defects in eat-2 and ace-3 mutants result in ACh stimulation of extrasynaptic GAR-3 receptors in isthmus muscles. gar-3 mutation also suppressed slow larval growth and prolonged lifespan phenotypes that result from dietary restriction in eat-2 mutants, indicating that crosstalk with the GAR-3 receptor has a long-term impact on feeding behavior and eat-2 mutant phenotypes.

An hourglass circuit motif transforms a motor program via subcellularly localized muscle calcium signaling and contraction

“…The myo-2p promoter drives expression in the pm3s and in no other cell in the anterior pharynx (Okkema et al, 1993;Okkema and Fire, 1994). We obtained animals that express GCaMP3 under this promoter (Kozlova et al, 2019) and used confocal microscopy to confirm that these myo-2p::GCaMP3 animals expressed GCaMP in the pm3s but not in pm1 or the pm2s (Figure 2-figure supplement 2B). After stimulation with light, 16 of the 19 myo-2p::GCaMP animals examined responded with subcellular calcium responses at the anterior tips of the pm3s (Video 13); 11 of these 16 animals also showed a subsequent calcium increase in the posterior myo-2-expressing regions of the pharynx (Figure 2K-2L).…”

“…Despite these examples of subcellularly localized muscle calcium function in physiological settings, how subcellular muscle calcium signaling contributes to behavior is poorly understood. The C. elegans pm5 pharyngeal muscle cells function to swallow food from the anterior pharynx to the terminal bulb via a peristaltic wave of subcellular contraction (Avery and Horvitz, 1987), and subcellular calcium signals in the pm5s likely underlie swallowing behavior (Shimozono et al, 2004;Kozlova et al, 2019). These transients have been reported to resemble calcium waves in cardiac muscle (Kozlova et al, 2019), a phenomenon driven by the propagation of calcium-induced calcium release from internal stores (Cheng et al, 1993).…”

“…The C. elegans pm5 pharyngeal muscle cells function to swallow food from the anterior pharynx to the terminal bulb via a peristaltic wave of subcellular contraction (Avery and Horvitz, 1987), and subcellular calcium signals in the pm5s likely underlie swallowing behavior (Shimozono et al, 2004;Kozlova et al, 2019). These transients have been reported to resemble calcium waves in cardiac muscle (Kozlova et al, 2019), a phenomenon driven by the propagation of calcium-induced calcium release from internal stores (Cheng et al, 1993). Subcellular signals have also been observed in murine enteric muscles, which can contract peristaltically, but the functional significance of these signals is unknown (Yamazawa and Iino, 2002).…”

“…Calcium changes in pharyngeal muscle were monitored using GCaMP3 (Tian et al, 2009) expressed from one of three transgenes: nIs686 (this study), driven by the gpa-16 promoter (Jansen et al, 1999); cuIs36 (Kozlova et al, 2019), driven by the myo-2 promoter (Okkema and Fire, 1994;Okkema et al, 1993); or nEx3045 (this study), driven by the C32F10.8 promoter (McKay et al, 2003;Hunt-Newbury et al, 2007;this study). Calcium changes in M1 were monitored using GCaMP6s (Chen et al, 2013) expressed in M1 using the transgene nIs678 driven by the glr-2 promoter (Brockie et al, 2001).…”