PMC

Model of the cholinergic network regulating feeding rate. The MCs, the M2s, and M4 can directly stimulate pumping, and the I1s can stimulate pumping via the MCs and the M2s. The MCs and the M2s are connected by gap junctions. The serotonin receptor SER-7 is expressed in the MCs, the M2s, and M4. The MCs stimulate pumping via a nicotinic receptor containing EAT-2 and EAT-18 and also via the GAR-3 muscarinic receptor. NS, nervous system; nAChR, nicotinic ACh receptor.

Cholinergic motor neurons MC, M2, and M4, but not M1, are excitatory for pumping. A: MC stimulation excites pumping in an all-trans-retinal (ATR)-dependent manner. This effect is abolished when the MCs are ablated but not when the M2s are ablated. n = 10, 7, 10, 9 Animals for each pair of bars, respectively. B: M2 stimulation excites pumping in an ATR-dependent manner. This effect is abolished when the M2s are ablated but not when the MCs are ablated. n = 10, 7, 10, 9 Animals for each pair of bars, respectively. C: M4 stimulation excites pumping in an ATR-dependent manner. This effect persists when the MCs and the M2s are ablated. n = 10, 7, 7 Animals for each pair of bars, respectively. D: M1 stimulation does not excite pumping. n = 9, 7 Animals for each pair of bars, respectively. 5-HT (10 mM) was used for each experiment. Each bar represents mean ± SE. Statistical significance was calculated using the 2-tailed Student's t-test. *P < 0.05; **P < 0.01.

Optogenetic inhibition of pharyngeal cholinergic neurons inhibits 5-HT-stimulated pumping. A: optogenetic inhibition of the MCs, the M2s, and M4, but not M1, inhibits 5-HT-stimulated pumping. Simultaneous inhibition of the MCs and the M2s causes an inhibition of pumping greater than that observed during inhibition of either the MCs or the M2s alone, consistent with an endogenous role for the M2s in pumping regulation. “None” indicates that no neurons were inhibited. n = 15 For control, 14 for the MCs, 13 for the M2s, 9 for M4, and 4 for M1. B: inhibition of the I1s inhibits 5-HT-stimulated pumping. Simultaneous I1 inhibition increased the effects of individual MC inhibition or M2 inhibition, but not of combined MC and M2 inhibition, indicating that I1 stimulates pumping endogenously via the MCs and the M2s. n = 9 For each bar. 5-HT (10 mM) was used for each experiment. Each bar represents mean ± SE. Statistical significance was calculated using the 2-tailed Student's t-test. *P < 0.05; **P < 0.01.

The I1 interneurons are required for basal pumping and stimulate pumping via the MCs and the M2s. A: I1 ablation reduces pumping rate in the absence of food. n = 8, 3, 46, 7 Animals for each bar, respectively. See materials and methods for details. B: I1 stimulates pumping in an ATR-dependent manner and in the absence of either the MCs or the M2s but not both. n = 10, 7, 10, 9, 7 Animals for each pair of bars, respectively. 5-HT (10 mM) was used for each experiment in B. Each bar represents mean ± SE. Statistical significance was calculated using the 2-tailed paired Student's t-test. **P < 0.01.

The MCs can directly excite pumping via both nicotinic and muscarinic receptors. Optogenetic MC stimulation increases pumping in both eat-2 and eat-18 nicotinic receptor mutants but not in unc-17 (vesicular ACh transporter) mutants, demonstrating that MC can act via a nonnicotinic cholinergic pathway in addition to the known EAT-2/EAT-18 nicotinic pathway. Serial section electron microscopy suggests that the MCs and M2s form gap junctions; MC stimulation directly increases pumping in the absence of the M2s in eat-18 mutants. MC stimulation does not increase pumping in eat-18 mutants in the presence of the muscarinic antagonist atropine. GAR-3 is a muscarinic receptor expressed in pharyngeal muscle; MC stimulation does not increase pumping in eat-18; gar-3 double mutants. Injection of a transgene containing the gar-3 genomic region restores the ability of MC stimulation to excite pumping in eat-18; gar-3 mutants. n = 10, 10, 8, 9, 9, 10, 10 Animals for each pair of bars, respectively. 5-HT (10 mM) was used for each experiment except those with atropine, where we used 5 mM 5-HT and 5 mM atropine. Each bar represents mean ± SE. Statistical significance was calculated using the 2-tailed Student's t-test. *P < 0.05; **P < 0.01.

Optogenetic stimulation of the pharyngeal cholinergic nervous system and machine vision quantification of pumping. A: cholinergic pharyngeal neurons. Only 1 of each of the paired I1, MC, and M2 neurons is shown. In A–D, anterior is to the left, and ventral is toward the bottom. B: differential interference contrast (DIC) image of the pharynx. Red box denotes region used for velocity calculations. C: wide-field green fluorescent protein (GFP) fluorescence image of the same field of view as in B. The blue circle represents a stimulus region for an MC soma. D: GFP fluorescence image of the same field of view as in B and C during selective illumination of an MC soma. E: experimental schematic. A laser beam with 473-nm wavelength is shaped by a digital micromirror device (DMD) and enters a microscope to stimulate selectively neurons of interest expressing either GFP- and yellow fluorescent protein (YFP)-tagged blue-light-sensitive channelrhodopsin-2 (ChR2) or YFP-tagged blue-light-powered proton pump (Mac) from Leptoshaeria maculans. Pharyngeal behavior is imaged using a red filter and DIC optics. F: velocity from machine vision algorithm during ChR2-mediated stimulation of the MCs. Each peak in the velocity represents a pump. G: mean pump rate from 9 intervals of MC excitation in each of 10 worms. In F and G, the blue bar denotes timing of laser illumination.