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. 2003 Sep 3;23(22):8060-9.
doi: 10.1523/JNEUROSCI.23-22-08060.2003.

Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior

I Amy Bany  1 , Meng-Qiu Dong, Michael R Koelle
Affiliations

Genetic and cellular basis for acetylcholine inhibition of Caenorhabditis elegans egg-laying behavior

I Amy Bany et al. J Neurosci. .

Abstract

Egg-laying behavior in Caenorhabditis elegans is activated by signaling through the G-protein G(rho)q and inhibited by signaling through a second G-protein, G(rho)o. Activation of egg laying depends on the serotonergic hermaphrodite-specific neurons (HSNs), but the neurotransmitter(s) and cell(s) that signal to inhibit egg laying are not known. Mutants for G-protein signaling genes have well characterized defects in egg laying. Here we present an analysis of mutants for other genes reported to lack inhibition of egg laying. Of the nine strongest, six have morphological defects in the ventral-type C (VC) neurons, which synapse onto both the HSNs and the egg-laying muscles and are thus the third cell type comprising the egg-laying system. Laser-ablating VC neurons could also disrupt the inhibition of egg laying. The remaining three mutants (unc-4, cha-1, and unc-17) are defective for synthesis or packaging of acetylcholine in the VCs. The egg-laying defects of unc-4, cha-1, and unc-17 were rescued by VC-specific expression of the corresponding cDNAs. In addition, increasing synaptic acetylcholine by reducing acetylcholinesterase activity, with either mutations or the inhibitor aldicarb, decreased egg laying. Finally, we found that a knock-out for the HSN-expressed receptor G-protein-coupled acetylcholine receptor 2 (GAR-2) shows a partial defect in the inhibition of egg laying and fails to respond to aldicarb. Our results show that acetylcholine released from the VC neurons inhibits egg-laying behavior. This inhibition may be caused, in part, by acetylcholine signaling onto the HSN presynaptic terminals, via GAR-2, to inhibit neurotransmitter release.

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Figures

Figure 1.
Figure 1.
The hyperactive egg-laying phenotype, as illustrated in mutants with defects in Gρo signaling. A, Wild-type adult hermaphrodite. B, Loss-of-function mutant for goa-1, the C. elegans ortholog of the G-protein Gρo. Arrows indicate unlaid eggs; asterisks indicate the vulva (through which eggs are laid). Average numbers of unlaid eggs are above the animals. Wild-type animals retain fertilized eggs for ∼2.5 hr before laying them, whereas hyperactive egg-laying mutants, such as goa-1, engage in egg-laying behavior so frequently that few eggs are retained. C, Freshly laid multicellular egg from a wild-type animal. D, Freshly laid two-cell egg from a goa-1 mutant. Eggs laid by the wild type have developed for ∼2.5 hr and typically contain 50-100 cells, whereas eggs laid by hyperactive egg-laying mutants are much younger and thus often contain fewer than eight cells. E, Percentage of early-stage eggs (8 cells or fewer) laid by wild-type or mutant strains. The three G-protein signaling mutants shown fail to inhibit egg laying and thus exhibit the hyperactive egg-laying phenotype.
Figure 2.
Figure 2.
Quantitation of the hyperactive egg-laying phenotype in a panel of uncoordinated mutants reported to have defects in inhibition of egg laying. The mutants showed defects in the early-stage egg assay ranging from extremely mild to severe. We chose to further analyze only those mutants that laid >50% early-stage eggs (dotted line).
Figure 3.
Figure 3.
Representative morphological defects seen in the VC neurons of certain hyperactive egg-laying mutants. Animals shown express a GFP reporter transgene in the VCs and are seen at increasing objective magnifications in A-C to illustrate different types of defects. A, Wild-type control (top panel) and an unc-5 mutant (bottom panel). Arrows indicate VC cell bodies; asterisks indicate the vulva. Although six VC neurons are present in the wild type, only four can be visualized in the unc-5 mutant shown, two of which are dim. B, Wild-type control (top panel) and an unc-42 mutant (bottom panel). Brackets indicate VC axonal processes between two VC cell bodies. The unc-42 mutant shown has completely lost the processes between the VC5 and VC6 cell bodies. C, Ventral views of the vulval region of a wild-type control (top panel) and an unc-115 mutant (bottom panel). The wild type shows processes that completely circle the vulva and that display varicosities at the sites of synaptic connections. The unc-115 mutant shown has breaks in these processes, which trail off laterally. The defects shown are representative of those seen at varying penetrance in six different hyperactive egg-laying mutants.
Figure 4.
Figure 4.
Effects of VC-expressed unc-4 or cha-1 cDNAs in unc-4 or cha-1 mutants, respectively. A vector containing a VC-expressed promoter derived from the lin-11 gene was used to generate constructs for VC expression of the unc-4 and cha-1 cDNAs (VC::unc-4 and VC::cha-1). A, Percentage early-stage eggs laid by unc-4 mutants and by transgenic strains carrying the vector or VC::unc-4 construct. B, Percentage early-stage eggs laid by cha-1 mutants and by transgenic strains carrying the vector or VC::cha-1 construct. The wild type is included in A for comparison. Five independent lines were analyzed for each transgene, and averages and SDs for the five lines are shown. VC expression of the unc-4 or cha-1 cDNAs rescued the hyperactive egg-laying defects of the corresponding mutants.
Figure 5.
Figure 5.
Effects of increasing synaptic acetylcholine on egg-laying behavior. A, Rates of egg-laying of wild-type worms on plates containing varying concentrations of levamisole and aldicarb. For each condition tested, the number of eggs laid in 1 hr by 10 adult animals was determined, and this was repeated 12 times. The data presented are the average and SE of the 12 trials. Levamisole stimulates nicotinic acetylcholine receptors, whereas aldicarb, by inhibiting acetylcholinesterase activity, amplifies all endogenous acetylcholine signaling. Levamisole stimulates egg-laying rates, whereas aldicarb slows the rate of egg laying. B, Wild-type adult hermaphrodite. C, ace-2; ace-1 double mutant, lacking function of two acetylcholinesterase genes and therefore lacking most acetylcholinesterase activity. Arrows indicate unlaid eggs; asterisks indicate the vulva. Average numbers of unlaid eggs are indicated above the animals. The ace-2; ace-1 mutant has higher endogenous levels of acetylcholine because acetylcholine is not efficiently removed from synapses. The mutant shows increased accumulation of unlaid eggs, indicating increased inhibition of egg laying. D, Rate of egg laying of gar-2 mutants on plates containing varying concentrations of aldicarb. In contrast to the wild type, the gar-2 mutant does not respond to aldicarb, suggesting that acetylcholine inhibits egg laying by signaling, at least in part, through the muscarinic GAR-2 receptor.
Figure 6.
Figure 6.
Effects of loss of acetylcholine in animals with and without HSNs. A, One model for the inhibition of egg laying by VC-derived acetylcholine. In this model, acetylcholine acts on the HSN through the GAR-2 receptor to inhibit release of neurotransmitters, including serotonin (5-HT). B, Wild-type adult hermaphrodite. C, Gain-of-function egl-1 mutant. The egl-1 mutant lacks the HSN neurons; it cannot stimulate egg-laying and retains a large number of eggs. D, Null unc-4 mutant. The unc-4 mutant is defective for synthesis and packaging acetylcholine in its VC neurons. E, unc-4; egl-1 double mutant. The hyperactive unc-4 single mutant retained very few eggs, whereas the unc-4; egl-1 double mutant was extremely defective in egg laying. F, Reduction-of-function cha-1 mutant, containing <1% normal acetylcholine levels. G, cha-1; egl-1 double mutant. The hyperactive cha-1 single mutant retained very few eggs, whereas the cha-1; egl-1 double mutant retains five times as many eggs, including eggs that are of a late developmental stage. Arrows indicate unlaid eggs, asterisks indicate the vulva, and round-tipped arrows indicate late-stage eggs. Average numbers of unlaid eggs are indicated for each strain. These results show that animals fail to lay eggs in the absence of HSNs, regardless of the presence or absence of acetylcholine.
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