Defecation in the nematode Caenorhabditis elegans occurs rhythmically every ~45s and is thought to be timed by cell-autonomous Ca2+ oscillations generated in the intestine by activation of the inositol 1, 4, 5-trisphosphate receptor (InsP3R). To date, studies of Ca2+ oscillations during defecation have been performed using either isolated tissues or physical restraints such as cyanoacrylate glue that prevent normal movement. We have developed a system for studying Ca2+ oscillations in the intestine of intact, freely behaving animals. Fluorescent imaging of unrestrained worms expressing the fluorescent Ca2+ biosensor YC6.1 demonstrated that the anterior and posterior intestinal cells display rhythmic Ca2+ oscillations resulting in synchronous, convergent wave propagation that correlates with execution of the tripartite defecation motor program (DMP). Screening of arrhythmic mutants showed that both temporal and spatial constraints contribute to shaping the Ca2+ wave, and that Ca2+ wave dynamics control execution of the DMP, as follows: 1.) PLC<font face=symbol>b</font>, which has been shown to regulate defecation rhythm independently of InsP3R function, determines the site of normal Ca2+ wave initiation, and a loss-of-function (lf) mutation in the PLC<font face=symbol>b</font> gene
egl-8 causes calcium waves that can initiate in the wrong cell(s) and travel through the intestine in the wrong direction; 2.) KCNQ M-type K+ channels encoded by
kqt-2 and
kqt-3 facilitate normal calcium wave propagation; 3.) The
unc-43(
sa200) mutation causes variable shadow contractions that reflect cryptic intracycle Ca2+ oscillations which occur at three-times the frequency of the defecation period. This suggests that CaMKII normally suppresses a response to signals that act upstream of Ca2+ influx, and that this signal oscillates faster than the defecation frequency. In addition, we have used weak RNAi of the SERCA gene
sca-1 to demonstrate that Ca2+ can remain elevated significantly longer than normal, for up to two-thirds of the cycle period, without affecting the frequency of defecation. Moreover, chronically elevated Ca2+ hinders the coupling between successive steps of the DMP. These results suggest that Ca2+ oscillations may be an output of an upstream pacemaker, and may serve to coordinate the motor program, rather than determine its overall rhythm. Finally, we will discuss recent results obtained using pH and IP3 biosensors in the intestine. Our findings contribute to a better understanding of the role of Ca2+ waves in signaling rhythmic behaviors and address one of the current major challenges in integrative physiology by bridging the gap between molecular and systemic functions.