Serotonin activates both pharyngeal pumping and isthmus peristalsis. SER-7 in MC and M4 (and possibly M2) separately activates pumping and isthmus peristalsis, respectively, in response to bacteria. Despite the separate regulation, isthmus peristalsis is coupled to the preceding pump. A, A temporal representation of the onsets of pharyngeal pumping and isthmus peristalsis. Each black bar indicates onset of a pharyngeal pump. Each gray bar indicates the onset of an isthmus peristalsis. B, C, IP was selectively coupled to the preceding (B) but not to the following (C) PP in wild-type worms. Each filled circle indicates the interval between isthmus peristalsis and the indicated pump in one worm. ***p < 0.001; Pearson’s correlation test; n.s. indicates not significant (p ≥ 0.05); data are shown as mean ± SD. D, E, Serotonin activated both pumping and isthmus peristalsis. D, A scatterplot of feeding rates of wild type (wt) with or without serotonin treatment. Each dot represents one worm. Thus, the x and y values of each dot indicate pumping rate and isthmus peristalsis rate, respectively, of one worm. E, For simplicity, we transformed scatterplots into ellipse plots. The central dot is plotted at the mean pumping and isthmus peristalsis rate for each tested strain or condition. The large ellipse, a plot of root-mean-square deviation from the mean, shows the extent of variation from animal to animal. The width of the ellipse is times the SD of pumping rate, the height is times the SD of isthmus peristalsis rate, and narrowness or breadth is related to correlation. For normally distributed data, 63% of data points would be within the ellipse. The small ellipse similarly shows the likely range of the mean. F, Both feeding rates of the tph-1(mg280) null mutant decreased to75%of wild-type animals in response to bacteria, a physiologically relevant stimulant of feeding. G, The ser-7(tm1325) null mutant was defective in activating pharyngeal pumping in response to serotonin. H, The ser-7 null mutation decreased isthmus peristalsis rate in the gsa-1(ce81) gain-of-function mutant in response to serotonin. I–M, SER-7 in MC cell-autonomously activates pharyngeal pumping in response to serotonin. I, J, Expression of ser-7 cDNA using the flp-21 promoter (I) or the flp-2 promoter (J) fully restored pumping in the ser-7 null mutant in response to serotonin. pflp-2::ser-7(+) only partly restored isthmus peristalsis rate (J). This might be because the flp-2 promoter drives weak occasional expression in M4 (Kim and Li, 2004). K, Expression of ser-7 cDNA in M4 using the ser-7b promoter increased isthmus peristalsis but not pumping in the ser-7 null mutant in response to serotonin. L, The eat-2(ad465) null mutation, which specifically blocks cholinergic transmission from MC to pharyngeal muscles, suppressed the rescue effect of pflp-21::ser-7(+) in the ser-7 mutant in response to serotonin. M, SER-7 acts mainly in M4 to stimulate isthmus peristalsis in response to serotonin. N, Both feeding rates of the ser-7 null mutant decreased to 75% of wild-type animals in response to bacteria, a physiologically relevant stimulant of feeding. O, As seen in response to serotonin, expression of ser-7 cDNA from the flp-21 promoter fully restored pumping in the ser-7 null mutant in response to bacteria. P, Expression of ser-7 cDNA in M4 fully restored isthmus peristalsis rate in the ser-7 null mutant with a small effect on pharyngeal pumping in response to bacteria. ***p < 0.001; **p < 0.01; n.s., not significant (p > 0.05); unpaired t test (two-tailed test) was used for comparison of pumping rates, and the level of significance is indicated by green asterisks; isthmus peristalsis rates were compared as described in Materials and Methods, and the level of significance is indicated by asterisks in pink. ^#Comparison of isthmus peristalsis rates was infeasible due to lack of overlap in pumping rates between the two compared samples. The number of animals tested (n ≥ 3 independent experiments per each group) is shown in parentheses. For the comparisons with the transgenics expressing rescue constructs, transgenic animals expressing only co-injection marker(s) were used as controls.

SER-7 activates isthmus peristalsis by activating G₁₂α signaling in M4. A, Schematic of the G₁₂α signaling pathway in C. elegans. B, C, Expression of constitutively gpa-12(Q205L) (B) or constitutively active rho-1(G14V) (C) in cholinergic neurons was sufficient to activate isthmus peristalsis but not pharyngeal pumping in the absence of serotonin. C, The F25N mutation completely suppressed the increase in isthmus peristalsis rate caused by the constitutively active rho-1(G14V) mutation. D, The dgk-1(sy428) null mutation increased both feeding rates in response to serotonin. E, F, The G₁₂α and G_sα signaling pathways act redundantly to activate isthmus peristalsis in response to serotonin. The gsa-1(pk75); gpa-12(pk322) double-null mutant (F), but not the gpa-12(pk322) single-null mutant (E), decreased isthmus peristalsis in response to serotonin. G, dgk-1(sy428) fully restored isthmus peristalsis rate in the transgenic animals expressing rho-1(F25NG14V) in cholinergic neurons to the level of the transgenics expressing rho-1(G14V) in response to serotonin. H, Expression of constitutively active gpa-12(Q205L) in M4 (and occasionally in M2) significantly increased the isthmus peristalsis rate in gsa-1(ce81);ser-7(tm1325) in response to serotonin. I, Expression of constitutively active rho-1(G14V) inM4(and occasionally in M2) fully restored isthmus peristalsis in gsa-1(ce81);ser-7(tm1325) in response to serotonin. The number of animals tested (n ≥ 3 independent assays per each group) is shown in parentheses. ***p < 0.001; *p < 0.05; n.s., not significant (p > 0.05); unpaired t test (two-tailed test) was used for comparison of pharyngeal pumping rates, and the difference is marked in green; isthmus peristalsis rates were compared as described in Materials and Methods, and the level of significance is indicated by asterisks in pink; the number of animals tested (n ≥ 3 independent assays per each group) is shown in parentheses. For the comparisons with the transgenics expressing rescue constructs, the transgenic animals expressing only co-injection marker(s) were used as controls.

Model of regulation of feeding by serotonin. A, In response to serotonin, SER-7 in MC cell-autonomously activates its downstream G_sα signaling pathway, which subsequently stimulates pharyngeal pumping by activating cholinergic transmission from MC to the pharyngeal muscles. B, In response to serotonin, SER-7 in M4 (and possibly in M2) activates its downstream G₁₂α pathways in a cell-autonomous manner, which subsequently activates M4. The stimulus from active M4, along with dense core vesicle release controlled by an unidentified pathway, activates isthmus peristalsis. The G_sα signaling pathway and downstream cholinergic transmission also contribute to activating isthmus peristalsis, but their sites of action have not been characterized. Given that expression of SER-7 in M4 fully restored isthmus peristalsis rate in the ser-7 null mutant, it is plausible that release of dense core vesicles from the unidentified cells is constitutively active rather than triggered by serotonin. C, Stimuli from an active M4 neuron and from the anterior part of the pharynx, excited by pumping, are both required to activate isthmus peristalsis. In the absence of either stimulus, isthmus peristalsis does not occur.

SER-7 activates pharyngeal pumping by activating the G_sα signaling pathway in response to serotonin. Activation of G_sα signaling has a small effect in activating isthmus peristalsis. A, Schematic of the G_sα signaling pathway in C. elegans. B, The constitutively active gsa-1(ce81) mutation activated pumping as effectively as serotonin did. C, Activation of adenylyl cyclases by an acute forskolin treatment activated pumping as effectively as serotonin did. D, ce179, a hypomorphic allele of kin-2, increased both feeding rates in response to serotonin. E, The gsa-1(pk75) null mutation completely suppressed the serotonin-stimulated pharyngeal pumping. Expression of gsa-1 using the flp-21 promoter fully restored the pumping rate in the gsa-1(pk75) null mutant in response to serotonin. F, Anacy-1(pk1279) null mutation attenuated pumping in response to serotonin. G, H, Forskolin (G) and ce179 (H), a hypomorphic allele of kin-2, increased pumping in the gsa-1(pk75) null mutant in response to serotonin. I, J, Activation of G_sα signaling by constitutively active gsa-1(ce81) (I) or by forskolin treatment (J) fully restored pumping in the ser-7(tm1325) null mutant in response to serotonin. K, Acute expression of constitutively active GSA-1(Q227L) using heat shock was sufficient to activate pharyngeal pumping, but not isthmus peristalsis in absence of serotonin. L, Acute expression of constitutively active GSA-1(Q227L) using heat shock activated both feeding motions in response to serotonin. M, The gsa-1(pk75) null mutation failed to suppress serotonin-stimulated isthmus peristalsis. N, The constitutively active acy-1(ce2) mutation activated isthmus peristalsis but not pumping in the absence of serotonin. ***p < 0.001; *p < 0.05; n.s., not significant (p > 0.05); the unpaired t test (two-tailed test) was used for comparison of pumping rates, and the difference indicated in green; isthmus peristalsis rates were compared as described in Materials and Methods, and the level of significance is indicated by asterisks in pink. ^#Comparison of isthmus peristalsis rates was infeasible due to lack of overlap in pumping rates between the two samples compared. The number of animals tested (n ≥ 3 independent assays per each group) is shown in parentheses.

G_sα signaling activated pumping by activating acetylcholine transmission from MC to the pharyngeal muscles. G_sα signaling and G₁₂α signaling activated isthmus peristalsis. Active isthmus peristalsis induced by G_sα signaling and G₁₂α signaling required the release of acetylcholine and dense core vesicles, respectively. A, B, The hypomorphic cha-1(p1152) mutation attenuated pumping in response to serotonin (A) or forskolin (B). C, D, The eat-2(ad465) null mutation suppressed the increase in pumping induced by serotonin (C) or by forskolin (D). E, cha-1(p1152) attenuated the kin-2(ce179)-stimulated isthmus peristalsis. F, The unc-31(e928) null mutation attenuated isthmus peristalsis rate in response to serotonin. G, The unc-31(e928) null mutation completely suppressed the cholinergic GPA-12(Q205L)-stimulated isthmus peristalsis. H, Expression of unc-31 cDNA in M4 failed to restore isthmus peristalsis rate in the unc-31(e928) mutant. I, Pan-neuronal expression of unc-31 cDNA failed to restore isthmus peristalsis rate in the unc-31(e928) mutant. The number of animals tested (n ≥ 3 independent assays per each group) is shown in parentheses. ***p < 0.001; **p < 0.01; n.s., not significant (p > 0.05); unpaired t test (two-tailed test) was used for comparison of pharyngeal pumping rates, and the difference was marked in green; isthmus peristalsis rates were compared as described in Materials and Methods, and the level of significance is indicated by asterisks in pink; the number of animals tested (n ≥ 3 independent assays per each group) is shown in parentheses. For the comparisons with the transgenics expressing rescue constructs, the transgenic animals expressing only co-injection marker(s) were used as controls.