Cross Talk with the GAR-3 Receptor Contributes to Feeding Defects in Caenorhabditis elegans eat-2 Mutants

Abstract

Precise signaling at the neuromuscular junction (NMJ) is essential for proper muscle contraction. In the Caenorhabditis 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 that they were characterized by increased and prolonged Ca²⁺ transients in the isthmus muscles. This hyperstimulation depends on cross talk with the GAR-3 muscarinic ACh receptor as gar-3 mutation specifically suppressed the prolonged contraction and increased Ca²⁺ 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 life span phenotypes that result from dietary restriction in eat-2 mutants, indicating that cross talk with the GAR-3 receptor has a long-term impact on feeding behavior and eat-2 mutant phenotypes.

Keywords: C. elegans; GCaMP3; feeding; life span; muscarinic acetylcholine receptor; nicotinic acetylcholine receptor; peristalsis; pharynx.

Figure 1

Pharyngeal anatomy and contractions. (A) DIC micrograph of an adult pharynx indicating anatomical regions (white bars) and colored to indicate the location of pharyngeal muscle cells (pm). The pharynx exhibits threefold rotational symmetry, and there are three of the pm3–pm7 muscle cells surrounding the central lumen (Albertson and Thomson 1976). pm5 cells extend the length of the isthmus and are the primary focus of this work. (B and C) Diagrams indicating pharyngeal muscle contractions during pumping and peristalsis. The black regions indicate open lumen, and the arrow indicates the direction of the peristaltic contraction.

Figure 2

Muscarinic and nicotinic receptor agonists stimulate pharyngeal muscle contractions in cha-1 mutants. The percentage of wild-type (wt) or cha-1(ok2253) L1 animals exhibiting pharyngeal muscle pumping and peristalses, either untreated or treated with the indicated concentrations of arecoline or nicotine. Animals were visualized for 5 min each and the number of animals observed (n) is indicated.

Figure 3

eat-2 mutants are insensitive to exogenous nicotine. Percentage of wild-type or eat-2(ok3528) L1 animals exhibiting pharyngeal muscle pumping and peristalses, either untreated or treated with the indicated concentrations of nicotine. ** indicates significantly different from untreated animals (P < 0.0001), and the bar indicates significant difference between wild-type animals treated with increasing concentrations of nicotine. Animals were visualized for 5 min each and the number of animals observed (n) is indicated.

Figure 4

MC neuron morphology in wild-type and eat-2 mutant adults. Fluorescence (top), and merged fluorescence and DIC images of adult animals of the indicated genotypes expressing ceh-19b^Prom::gfp in the MC neuron. Anterior is left, and the metacorpus and anterior isthmus are shown. The MC cell body (arrowhead) and varicosities in the MC process (small arrows) are indicated. Fluorescence images are maximal intensity Z-projections of images through one MC cell and process.

Figure 5

MC neuron synapses in wild-type and eat-2 mutant adults. Fluorescence (top), DIC (middle), and merged images (bottom) of the adult animals of the indicated genotypes expressing ceh-19b^Prom::snb-1::gfp in the MC neuron. Anterior is left, and the metacorpus and anterior isthmus are shown. Synaptic vesicle clusters marking en passant synapses in the axon of one MC neuron are marked in the fluorescence and merged images (arrowheads). Nonspecific autofluorescence at the edges of the pharyngeal lumen are marked in the fluorescence and DIC images (asterisks). Fluorescence images are false colored using the look-up table at the bottom right.

Figure 6

Dynamic changes in Ca²⁺ levels in the isthmus muscles. Time-lapse fluorescence images of the pharyngeal isthmus of a wild-type (top) or eat-2(ok3528) (bottom) adult expressing the genetically encoded Ca²⁺ indicator GCaMP3 in the pharyngeal muscles. Images are false colored as indicated at the lower left, and one pump and peristalsis are shown. Boxes indicate the regions where fluorescence levels were quantified in the center (C) and posterior (P) isthmus. The amounts of time after fluorescence begins to increase are indicated, and the frames indicating maximum fluorescence in wild-type and eat-2(ok3528) are shown at 0.279 and 0.713 sec, respectively.

Figure 7

Characterization of GCaMP3 fluorescence in the pharyngeal isthmus. (A) False-colored fluorescence image of a wild-type animal expressing GCaMP3 in the pharyngeal muscles (anterior is left). (B) Kymograph of maximum GCaMP3 fluorescence intensity in the isthmus region [indicated by brackets in (A and B)]. Two pumps with peristalsis marked with asterisks are followed by a pump without a peristalsis. (C) Fluorescence levels (F) in the center and posterior isthmus plotted vs. time (seconds) for the contractions in (B). Time points for time-lapse imaging (circles) and example measurements for ∆F, peak duration, and rise time are indicated.

Figure 8

Quantification of GCaMP3 fluorescence in wild-type animals and mutants. Box and whisker plots comparing values measured from time-lapse imaging of GCaMP3 fluorescence during pumps followed by peristalses: (A) normalized GCaMP3 fluorescence levels (∆F/F_o), (B) peak duration, (C) peak rise time, and (D) peak delay between the center and posterior isthmus. Genotypes and measurements in the center (cen) and posterior (post) isthmus are indicated. The central bars (red) denote the median values with notches indicating the 95% C.I.s for the median, the boxes indicates the interquartile ranges (IQRs, 25th to 75th percentile), and the whiskers indicate values within 1.5 times the IQR. Suspected outlier values are indicated as red “+.”

Figure 9

gar-3 mutation suppresses the slow growth and prolonged life span of eat-2 mutants. (A) Bar graphs indicating the percent animals of the indicated genotypes reaching adulthood by day 5 or day 6 at 20° (n = 100 for each genotype). (B) Adult survival curves for animals of the indicated genotypes. Averages of representative triplicate assays performed on plates containing UV-killed E. coli as a food source. Nearly identical results were obtained in assays with living E. coli.