Intestinal Ca²⁺ wave dynamics in freely moving C. elegans coordinate execution of a rhythmic motor program

Abstract

Defecation in the nematode worm Caenorhabditis elegans is a highly rhythmic behavior that is regulated by a Ca(2+) wave generated in the 20 epithelial cells of the intestine, in part through activation of the inositol 1,4,5-trisphosphate receptor. Execution of the defecation motor program (DMP) can be modified by external cues such as nutrient availability or mechanical stimulation. To address the likelihood that environmental regulation of the DMP requires integrating distinct cellular and organismal processes, we have developed a method for studying coordinate Ca(2+) oscillations and defecation behavior in intact, freely behaving animals. We tested this technique by examining how mutations in genes known to alter Ca(2+) handling [including egl-8/phospholipase C (PLC)-beta, kqt-3/KCNQ1, sca-1/sarco(endo)plasmic reticulum Ca(2+) ATPase, and unc-43/Ca(2+)-CaMKII] contribute to shaping the Ca(2+) wave and asked how Ca(2+) wave dynamics in the mutant backgrounds altered execution of the DMP. Notably, we find that Ca(2+) waves in the absence of PLCbeta initiate ectopically, often traveling in reverse, and fail to trigger a complete DMP. These results suggest that the normal supremacy of the posterior intestinal cells is not obligatory for Ca(2+) wave occurrence but instead helps to coordinate the DMP. Furthermore, we present evidence suggesting that an underlying pacemaker appears to oscillate at a faster frequency than the defecation cycle and that arrhythmia may result from uncoupling the pacemaker from the DMP rather than from disrupting the pacemaker itself. We also show that chronic elevations in Ca(2+) have limited influence on the defecation period but instead alter the interval between successive steps of the DMP. Finally, our results demonstrate that it is possible to assess Ca(2+) dynamics and muscular contractions in a completely unrestrained model organism.

Analysis of Ca2+ Signaling Motifs That Regulate Proton Signaling through the Na+/H+ Exchanger NHX-7 during a Rhythmic Behavior in Caenorhabditis elegans*

“…In general, when young adult worms were subjected to cmd-1 RNAi for 18 h, they began to exhibit weak reiterations of the DMP. This has been observed previously in unc-43 CaMKII loss-of-function mutants (20,25), where these reiterations were referred to as "shadows" or "echos." The interval between the principal, or strong, DMP and the shadows was much shorter (ϳ16 -18 s) in cmd-1 RNAi worms than the average defecation period (ϳ45 s), consistent with cmd-1 normally functioning to suppress inappropriate intracycle execution of the DMP.…”

“…To circumvent this limitation, we turned to cell-specific RNAi. The RDE-1 protein is required for effective RNAi to occur, and an rde-1 mutant strain can be complemented with a transgene expressing recombinant RDE-1 in a limited set of cells such as the intestine (25).…”

“…Genetic analyses have shown that intestinal cell-autonomous oscillatory Ca 2ϩ signaling is central to the defecation pacemaker (22). The first motor step of the DMP, pBoc, depends on Ca 2ϩ signals initiating in the posterior cells of the intestine (17,18), whereas a subsequent wave of Ca 2ϩ that propagates posterior-to-anterior is necessary for the coupling and timing of the remaining motor steps (23)(24)(25).…”

A Regulatory Module Controlling Pharyngeal Development and Function in Caenorhabditis elegans

“…The mosaic analysis also raised the possibility that sup-37 has a nonessential function in the intestine. Notably, the intestine, the pharynx, and the somatic gonad share a common feature, rhythmic contractions, that are achieved via electrical coupling (Avery and Thomas 1997;McCarter et al 1999;Nehrke et al 2008;Altun et al 2002Altun et al -2010. Taken together, our data suggest that SUP-37 may play a role in promoting rhythmic contractions within an organ.…”

A calcineurin homologous protein is required for sodium-proton exchange events in the C. elegans intestine

“…A critical component of the defecation cycle timing is 1,4,5 inositol trisphosphate receptor calcium release that initiates a calcium wave (14,20,43,50,59). Therefore proton release, like the release of many traditional neurotransmitters, must be signaled directly or indirectly by transient calcium spike.…”