Effect of a Neuropeptide Gene on Behavioral States in Caenorhabditis elegans Egg-Laying

Corresponding author: William R. Schafer, Department of Biology, University of California, 9500 Gilman Dr., San Diego, CA 92093-0349., wschafer{at}ucsd.edu (E-mail)

Communicating editor: R. K. HERMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Egg-laying behavior in the nematode Caenorhabditis elegans involves fluctuation between alternative behavioral states: an inactive state, during which eggs are retained in the uterus, and an active state, during which eggs are laid in bursts. We have found that the flp-1 gene, which encodes a group of structurally related neuropeptides, functions specifically to promote the switch from the inactive to the active egg-laying state. Recessive mutations in flp-1 caused a significant increase in the duration of the inactive phase, yet egg-laying within the active phase was normal. This pattern resembled that previously observed in mutants defective in the biosynthesis of serotonin, a neuromodulator implicated in induction of the active phase. Although flp-1 mutants were sensitive to stimulation of egg-laying by serotonin, the magnitude of their serotonin response was abnormally low. Thus, the flp-1-encoded peptides and serotonin function most likely function in concert to facilitate the onset of the active egg-laying phase. Interestingly, we observed that flp-1 is necessary for animals to down-regulate their rate of egg-laying in the absence of food. Because flp-1 is known to be expressed in interneurons that are postsynaptic to a variety of chemosensory cells, the FLP-1 peptides may function to regulate the activity of the egg-laying circuitry in response to sensory cues.

MANY aspects of behavior, including mood, aggression, sleep, and sexual arousal, involve discrete, alternative behavioral states. At the most basic level, these different behavioral states result from differences in the functional properties of the neurons and muscle cells in the circuits that produce the behavior. The regulation of switching between these functional states occurs largely through the action of molecules known as neuromodulators. In general, neuromodulators function by activating signaling pathways that regulate the activity of receptors and ion channels in excitable cells. A wide variety of molecules are known to function as neuromodulators, including biogenic amines (e.g., dopamine, serotonin, norepinephrine, and histamine), adenosine, glutamate, acetylcholine (through their action at muscarinic receptors), and a diverse array of neuropeptides. Identification of the mechanisms by which neuromodulators influence behavior at the molecular, cellular, and circuit levels is essential for understanding both simple and complex nervous systems.

We have employed a genetic approach to a simple animal, the nematode Caenorhabditis elegans, to investigate the molecular mechanisms by which neuromodulators control behavioral states. C. elegans is particularly well suited to molecular studies of nervous system function. It has a simple nervous system consisting of 302 neurons, and the position, cell lineage, and synaptic connectivity of each of these neurons is precisely known (SULSTON and HORVITZ 1977 ; SULSTON et al. 1983 ; WHITE et al. 1986 ). Because a particular neuron can be positively identified based on its position, it is possible to evaluate the function of an individual neuron or group of neurons through single cell laser ablation (BARGMANN and AVERY 1995 ). Moreover, because of their short generation time, small genome size, and accessibility to germline transformation, these animals are highly amenable to molecular and classical genetics (WOOD 1988 ). Thus, in C. elegans, it is relatively easy to identify genes involved in specific behaviors and to characterize the functions of their products using molecular, behavioral, and immunocytochemical analyses. Although its nervous system contains relatively few neurons, C. elegans makes use of a surprisingly broad array of neuromodulators, including a large number of putative neuropeptides and the biogenic amines serotonin, dopamine, and octopamine (Rand AND NONET 1997 ). In addition, it exhibits a number of easily assayed and quantifiable behaviors that are affected by a wide range of neuroactive substances (Rand AND JOHNSON 1995 ). For these reasons, it is an excellent model organism for studying the molecular and cellular basis of neuromodulator action.

The largest class of neuromodulators in C. elegans are the FMRFamide-related peptides (FaRPs). These peptides are characterized by a common carboxy-terminal motif of Arg-Phe-NH₂ and generally range in size from 4 to 20 amino acids. FaRPs are the predominant family of neuropeptides in invertebrates, where they have been shown to play a role in cardioregulation, learning, and the modulation of muscle contraction (MAULE et al. 1996 ). FaRPs have also been identified in vertebrates, where they have been implicated in the regulation of pain responses (YANG et al. 1985 ). In C. elegans, immunocytochemical experiments have shown that at least 30 neurons and gland cells contain peptides that have the C-terminal RFamide epitope characteristic of the FaRP family (SCHINKMANN and LI 1992 ). The C. elegans genomic sequence contains at least 20 genes (designated flp genes, for FMRFamide-like peptide deficient), which encode predicted polypeptide precursors of >50 distinct FaRPs (NELSON et al. 1998A ). Most of these flp genes have subsequently been shown to be expressed in larval and/or adult C. elegans, and in some cases the predicted peptide products have actually been purified from C. elegans extracts. At present, only one flp gene, flp-1, has been extensively characterized at the molecular and genetic level (ROSOFF et al. 1992 ; NELSON et al. 1998B ). flp-1 is expressed in a specific subset of head neurons and encodes two alternatively spliced products that can be processed to give rise to seven closely related FaRPs. flp-1 loss-of-function mutants exhibit a number of behavioral abnormalities, including hyperactive locomotion, nose touch insensitivity, and defective osmotic avoidance (NELSON et al. 1998B ). Although many questions remain concerning the molecular and cellular mechanisms through which the flp-1-encoded peptides influence these behaviors, it is clear that the peptides encoded by flp-1 have many specific effects on behavior that are at least partially distinct from the other C. elegans FaRPs.

In this study, we investigated the role of flp-1 in another behavior—egg-laying. C. elegans hermaphrodites are self-fertile and continuously produce embryos and retain them in their uterus for at least 2 days following the adult molt. Egg-laying in C. elegans occurs when embryos are expelled from the uterus through the contraction of 16 vulval and uterine muscles (WHITE et al. 1986 ). In the presence of abundant food, wild-type animals lay eggs in a specific temporal pattern: egg-laying events (i.e., contractions of the egg-laying muscles leading to the expulsion of one or more eggs) tend to be clustered in short bursts, or active phases, which are separated by longer inactive phases during which eggs are retained. Both the onset of the active phase and egg-laying within the active phase are aperiodic and model closely as Poisson processes with distinct rate constants (Fig 1, a–c). This egg-laying pattern can be accurately modeled as a three-state probabilistic process, in which animals fluctuate between discrete inactive, active, and egg-laying states (WAGGONER et al. 1998 ). This process has three parameters: the rate constant for the duration of the inactive phase (₂), the rate constant for egg-laying within the active phase (₁), and the probability of remaining in the active phase after an egg-laying event (p). Using a maximum-likelihood algorithm, it is possible to estimate these egg-laying parameters from real behavioral data and thereby compare the egg-laying patterns of wild-type and mutant strains (ZHOU et al. 1997 ).

View larger version (49K): In this window In a new window Download PPT slide   Figure 1. Behavioral states in C. elegans egg-laying behavior. A more detailed description of the egg-laying pattern of C. elegans and its analysis is described in WAGGONER et al. 1998 . (a) Temporal pattern of egg-laying. Shown is a representative egg-laying pattern, with egg-laying events indicated by hash marks. According to the model, the animal can exist in one of three states: an inactive state (I), an active state (A), and an egg-laying state (E). Eggs are laid upon entry into the E state. 1 is the rate constant for the egg-laying state, 2 is the rate constant for the inactive state, and p is the probability that after a given egg-laying event another egg will be laid before the animal enters the inactive phase. Short intervals (resulting from a single visit to the A state) are governed by the time constant 1/1. Long intervals result from one or more visits to the I (inactive) state (i.e., from E to A to I to A to E, or from E to A to I to A to I to A to E, etc.); the time constant for these intervals is equal to 1/p2 (for proof, see ZHOU et al. 1997 ). (b) Histogram of log interval times. Shown is a histogram of intervals between egg-laying events recorded from wild-type animals; the bins are on a log scale. According to the coupled-Poisson process model, the location of the two peaks corresponds to the intracluster and intercluster time constants. Values for these time constants were estimated using maximum-likelihood analysis (see MATERIALS AND METHODS). (c) Log-tail distribution of egg-laying intervals. The log-tail distribution of egg-laying intervals from wild-type animals is shown. The duration of each interval (on the x-axis) was plotted against the percentage of intervals greater than or equal to that interval (on the y-axis; logarithimic scale). The model predicts that this distribution should be biphasic, with the steep part of the curve corresponding to the intracluster intervals and the more gradual part to the intercluster intervals. The slope of the long intervals (i.e., the right part of the curve) is equal to the intercluster rate constant.

Genetic, pharmacological, and cell ablation studies have provided important insights into the roles of particular neurons and neurotransmitters in the control of egg-laying (HORVITZ et al. 1982 ; TRENT et al. 1983 ; WEINSHENKER et al. 1995 ). Two classes of motorneurons make extensive synapses with the vulval muscles: the two HSNs and six VCs, each of which expresses multiple neurotransmitters and neuromodulators. For example, the HSNs express serotonin, acetylcholine, and one or more FaRPs, while the VCs express acetylcholine, FaRPs, and possibly a biogenic amine (DESAI et al. 1988 ; SCHINKMANN and LI 1992 ; Rand AND NONET 1997 ). Both serotonin and acetylcholine have been shown pharmacologically to increase the overall rate of egg-laying (TRENT et al. 1983 ; WEINSHENKER et al. 1995 ). By characterizing the egg-laying patterns of mutant and ablated animals, it has been possible to distinguish neurons and genes that modulate the switching between behavioral states from those that promote egg-laying within the active phase. For example, in both HSN-ablated animals and serotonin-deficient mutants, the inactive egg-laying phase is abnormally long, whereas egg-laying within the active phase is unimpaired (WAGGONER et al. 1998 ). Thus, serotonin released from the HSNs apparently stimulates egg-laying by facilitating the switch from the inactive to the active egg-laying state. Similar experiments have implicated acetylcholine, released from both the HSNs and VCs, in the induction of egg-laying events within the active phase (WAGGONER et al. 1998 ).

In this study, we show that the peptides produced by the flp-1 gene function in the regulation of egg-laying behavior. Specifically, the flp-1-encoded peptides appear to promote the onset of the active phase of egg-laying, an activity that is at least partially independent of the HSN motorneurons. In addition, we provide evidence that these peptides may participate in the regulation of egg-laying by sensory cues.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and genetic methods:
Routine culturing of C. elegans was performed as described (BRENNER 1974 ). The chromosomal locations of the genes studied in these experiments are as follows: LGI, goa-1; LGIV, flp-1; LGV, cat-4, egl-1. The ynIs9 integrated array contains the wild-type flp-1 allele and the coinjection marker lin-11(+). Unless otherwise indicated, all mutant strains are in the N2 genetic background. Behavioral assays were performed at room temperature (~22°). Serotonin (creatinine sulfate complex) was obtained from Sigma (St. Louis). The flp-1(yn2) allele was chosen for use in double mutant constructions because its phenotype and behavior in genetic crosses suggests that it causes a more severe loss of gene function (NELSON et al. 1998B ). goa-1(n1134) was used for behavioral analysis and double mutant construction because it encodes a putatively nonfunctional product and because the near sterility conferred by the n363 deficiency allele (brood size is ~35; SEGALAT et al. 1995 ) makes embryo production rather than egg-laying muscle contraction limiting for egg-laying in n363 mutant animals.

Egg-laying assays:
Unless otherwise stated, nematodes were grown and assayed at room temperature on standard nematode growth medium (NGM) seeded with Escherichia coli strain OP50 as a food source. For dose response experiments, individual young, gravid hermaphrodites were placed in microtiter wells containing liquid M9 and the indicated concentration of drug. After a 1-hr incubation at room temperature, the eggs laid by each animal were counted.

Experiments comparing egg-laying rates in the presence or absence of food were performed by transferring well-fed individual animals to NGM plates that were either seeded with a lawn of E. coli OP50 or were left unseeded. After 1 hr at room temperature, the eggs laid by each animal were counted. Plates in which the animal had crawled off the agar surface before the end of the assay period were not included in the analysis. We were unable to record the behavior of individual animals on plates completely lacking a bacterial lawn, because the animals were prone to crawl to the edge of the plate where our tracking system could not follow them. However, we were able to track wild-type (but not flp-1) animals on plates containing only a very small (1–2 mm diameter) bacterial spot; the egg-laying parameters of these animals are indicated in Table 1.

Egg-laying behavior of individual animals on solid media (NGM agar) was recorded at room temperature (22°) for 4–8 hr as described (WAGGONER et al. 1998 ) using an automated tracking system. The flp-1(yn2); ynIs9[hs:flp-1(+)] strain was grown at 15° until early adulthood, then shifted to room temperature for tracking. For tracking experiments on serotonin, 5-hydroxytryptamine (creatinine sulfate complex; Sigma) was added to NGM agar at 7.5 mM.

Analysis of egg-laying patterns:
Intervals between egg-laying events were determined from analysis of videotapes obtained using the automated tracking system. Quantitative analysis of the egg-laying pattern using this interval data was performed as described (ZHOU et al. 1997 ). Briefly, egg-laying events in C. elegans are clustered, with periods of active egg-laying, or active phases, separated by long inactive phases during which eggs are retained. Both the duration of the inactive phases ("intercluster intervals") and the duration of intervals between egg-laying events in a cluster ("intra-cluster intervals") model as exponential random variables with different time constants (WAGGONER et al. 1998 ). Thus, the probability density function for the intervals between events is

where the intracluster time constant is 1/₁ and the intercluster time constant is 1/p₂. The parameters were determined using the maximum-likelihood estimation technique described previously (ZHOU et al. 1997 ). The expected variance of estimated parameters and time constants was determined by generating 100 independent sets of simulated egg-laying data using the model probability density function, and computing the standard deviation of the parameters estimated from these simulations. All data in Table 1 were obtained and analyzed in this manner.

For analysis of egg-laying patterns on serotonin (Table 1), a single exponential time constant was estimated using a weighted least-squares linear regression to the log-tail distribution (WAGGONER et al. 1998 ). The expected variance of these time constants was determined by generating 100 independent sets of simulated egg-laying data using a simple exponential probability density function, and by computing the standard deviation of the parameters estimated from these simulations.

Construction of double mutant strains:
For flp-1; cat-4 double mutants, cat-4 and flp-1 single mutants were mated, and double mutant progeny were identified in the F₂ generation by scoring for bleach sensitivity (cat-4; LOER 1995 ) and the presence of a diagnostic PCR product using sequence-specific primers (flp-1; NELSON et al. 1998B ). For the goa-1; cat-4 and goa-1; flp-1 double mutants, single mutants were crossed as above, and goa-1 homozygotes were identified in the F₂ generation as hyperactive, egg-laying constitutive animals. These were picked individually and then allowed to self-fertilize; those F₂'s that were heterozygous for cat-4 or flp-1 segregated double mutant progeny that could be identified using the bleach or PCR assays described above.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

flp-1 affects the transition between behavioral states involved in egg-laying:
To test the possible involvement of FLP-1-encoded peptides in the modulation of egg-laying behavior, we analyzed the egg-laying patterns of flp-1 mutants. We first analyzed the egg-laying patterns of mutants carrying recessive loss-of-function mutations in the flp-1 gene. These animals were not grossly defective in the ability to lay eggs, and their egg-laying patterns were qualitatively similar to wild-type animals: egg-laying events were still clustered in active phases, and both the switch into the active phase and the laying of eggs within the active phase still modeled as Poisson processes. However, the duration of the inactive phase was substantially longer in the flp-1 mutants (₂ for flp-1(yn2) and flp-1(yn4) were 4.7 and 7.0 x 10^-4 sec^-1, respectively) than in wild-type animals (₂ was 1.4 x 10^-3 sec^-1; Table 1; Fig 2, a and b). In contrast, egg-laying within the active phase was unimpaired in flp-1 mutants, and the intracluster time constants were no slower in the flp-1(yn2) and flp-1(yn4) deletion mutants than in wild type (Table 1, Fig 2A). Expression of a wild-type flp-1 transgene in a flp-1(yn2) background rescued its egg-laying defect (Fig 2A legend, Table 1). Thus, loss of flp-1 function appeared to specifically decrease the probability of switching from the inactive to the active phase of egg-laying, suggesting that the function of the wild-type flp-1 gene products is to promote the onset of the active egg-laying state.

View larger version (43K): In this window In a new window Download PPT slide   Figure 2. Effect of flp-1 mutations on the pattern of egg-laying. flp-1 loss-of-function mutations increase the duration of the inactive phase. Two independent, extensively outcrossed alleles (yn2 and yn4) were analyzed. For these and other tracking experiments, the numbers of animals tracked, hours observed, and intervals analyzed, along with the estimated model egg-laying parameters, are in Table 1. (a) Effect of flp-1 recessive mutations on the pattern of egg-laying. Shown are histograms of intervals between egg-laying events (log scale); intracluster and intercluster time constants estimated using maximum-likelihood analysis are indicated. Both the alleles of flp-1 specifically lengthened the intercluster constant (indicated by the location of the right-most peak). The long intervals (>120 sec) were significantly longer in yn2 (P < 0.001) and yn4 (P < 0.05) than in wild type according to the Mann-Whitney rank sum test. The long intervals were also significantly longer (P < 0.001) in the flp-1(yn2) strain than in the strain expressing the wild-type transgene (genotype: flp-1(yn2); ynIs9[hs:flp-1(+)]). The short intervals (<120 sec) in the flp-1 mutant strains were not significantly different from wild type (P > 0.2 for yn2, P > 0.05 for yn4). (b) Effect of flp-1 recessive mutations on inactive phase duration. Shown are the log-tail distributions of egg-laying intervals for wild-type and flp-1 mutant animals. The more gradual slopes of the yn2 and yn4 curves indicate longer intercluster time constants for these mutants.

flp-1 and serotonin function in concert to promote the active egg-laying phase:
The egg-laying defect exhibited by flp-1 mutants—longer-than-normal inactive phase but rapid egg-laying within the active phase—was quantitatively and qualitatively similar to the defect seen in serotonin-deficient mutants. For example, a mutation in cat-4, a gene required for efficient biosynthesis of serotonin and dopamine (LOER and KENYON 1993 ), slows the rate of egg-laying and lengthens the inactive phase. In principle, serotonin and FLP-1 could function in a common biological pathway, or they could affect distinct parallel pathways. To examine these possibilities, we constructed a double mutant carrying loss-of-function mutations in both flp-1 and cat-4. Simple measurements of egg-laying rates indicated that the severity of the egg-laying defect in the double mutant was comparable to that of flp-1 or cat-4 single mutants (Fig 3A). Moreover, both the inactive phase rate constant ₂ and the intercluster time constant of the double mutant were essentially identical to those of either single mutant (Table 1). These results supported the hypothesis that serotonin and flp-1 most likely function in the same pathway to induce the active egg-laying state.

View larger version (53K): In this window In a new window Download PPT slide   Figure 3. Relationship between the effects of flp-1 and serotonin on egg-laying. (a) Egg-laying pattern of serotonin-deficient flp-1 animals. cat-4 mutants are defective in serotonin and dopamine biosynthesis; because dopamine inhibits rather than stimulates egg-laying, the slower egg-laying rate of cat-4 mutant animals is thought to be a consequence of serotonin deficiency. Shown are log-tail distributions of egg-laying interval times for flp-1, cat-4, and flp-1; cat-4 double mutant animals. The intercluster time constants (indicated by the curve tails) were essentially identical (see Table 1). The numbers of animals tracked, hours observed, and intervals analyzed, along with the estimated model egg-laying parameters, are in Table 1. Egg-laying rates (eggs per hour) for each strain were as follows: flp-1, 1.94 ± 0.50 (n = 6); cat-4, 2.35 ± 0.40 (n = 12); flp-1; cat-4, 3.03 ± 0.26 (n = 7). The slower intracluster egg-laying rate characteristic of the cat-4 mutant (WAGGONER et al. 1998 ) was not observed in flp-1; cat-4 animals (Table 1). (b) Serotonin response of flp-1 mutants. Egg-laying responses to serotonin were determined for wild type and flp-1 mutants by placing individual animals in liquid M9 at the indicated concentration of serotonin and counting the number of eggs laid by each animal after 1 hr. Individual points and error bars indicate the mean and SEM of the following numbers of trials: N2, 20; yn2, 58; yn4, 45. For both flp-1 mutants, the number of eggs laid at 10 mg/ml was significantly higher than in the no drug control (P < 0.001) and significantly lower than in wild type at the same concentration (P < 0.002) according to the Mann-Whitney rank sum test. The constitutive egg-laying observed in yn2 animals may result from a defect in osmosensation (NELSON et al. 1998B ), which could reduce the egg-laying inhibition produced by M9 salts. (c) Egg-laying patterns of flp-1 mutants on serotonin. The histograms show the distribution of interval times for wild type and flp-1 mutants on 7.5 mM serotonin; bins are on a log scale. A single exponential time constant for each data set (indicated by the dashed line) was estimated as described in MATERIALS AND METHODS. Intervals for flp-1(yn4) were significantly (P < 0.05) longer than for wild type according to the Mann-Whitney rank sum test. The intervals for the flp-1(yn2) allele were not statistically different from wild type.

To further explore the relationship between the effects of these two modulators on the regulation of egg-laying, we assayed the responses of flp-1 mutants to exogenous serotonin. We first measured the ability of serotonin to stimulate egg-laying under conditions that are normally inhibitory for egg-laying (i.e., in the hypertonic salt solution M9). In this assay, we observed that flp-1 loss-of-function mutants were still responsive to serotonin (Fig 3B). Moreover, the serotonin sensitivity of flp-1 mutants, as measured by the concentration of serotonin that gave half-maximal stimulation, was comparable to that of wild-type animals. However, the magnitude of their response (i.e., the number of eggs laid when stimulated by serotonin) was reduced relative to wild type. Thus, while a functional flp-1 gene did not appear to be essential for serotonin to stimulate egg-laying, neither was exogenous serotonin able to completely bypass the effect of flp-1 on egg-laying. To further investigate the effect of flp-1 mutations on the egg-laying response to serotonin, we analyzed the egg-laying patterns of wild-type and flp-1 mutants in the presence of exogenous serotonin. In wild-type animals, treatment with serotonin not only increased rate of egg-laying, it also caused eggs to be laid in a monophasic pattern resembling a simple Poisson process. This pattern implied that in the presence of exogenous serotonin, wild-type animals were mostly in the active egg-laying phase. In flp-1 mutants, we observed that serotonin treatment affected egg-laying behavior in a similar manner; eggs were laid at a higher rate and in a more monophasic pattern in the presence of serotonin than on drug-free medium, though the rate of egg-laying in the presence of serotonin was slower in one of the flp-1 mutants than that in wild type (Fig 3C). Taken together, these experiments led us to conclude that flp-1 is not necessary for the stimulation of egg-laying by serotonin, though a functional flp-1 product is required for a maximal serotonin response.

HSN independence of the flp-1 mutant phenotype:
The serotonergic neurons most strongly implicated in the control of egg-laying are the HSN motorneurons. Although the HSNs themselves do not express flp-1, a number of flp-1-expressing cells in the head lie in close proximity to, and in some cases actually make synapses with, the dendrite of the HSN in the nerve ring. This raised the possibility that the effects of flp-1-encoded peptides might be mediated through modulation of the HSNs. Alternatively, given the small size of C. elegans, it was also possible that flp-1 could regulate egg-laying through a neuroendocrine mechanism that bypassed the HSNs. To distinguish between these models, we tested the effect of a cell-specific ablation of the HSNs on the egg-laying behavior of flp-1 mutants. We observed that HSN-ablated flp-1 animals were no more severely egg-laying defective than HSN-ablated wild-type animals: both their overall egg-laying rates and their egg-laying patterns were essentially identical (Fig 4A). This result was consistent with our earlier observations indicating that serotonin, a neuromodulator known to be released from the HSNs, affects the same aspect of egg-laying behavior as flp-1. However, when we analyzed the behavior of HSN-ablated flp-1 animals in the presence of exogenous serotonin, we found that they laid eggs significantly more slowly than HSN-ablated wild-type animals under the same condition (Fig 4B). Thus, the ability of flp-1 to potentiate the stimulation of egg-laying by serotonin did not appear to require the HSNs. Thus, the effects of the FLP-1 peptides on egg-laying, in particular their ability to facilitate egg-laying in response to serotonin, were at least partially independent of the HSNs.

View larger version (46K): In this window In a new window Download PPT slide   Figure 4. Relationship between the effects of flp-1 and the HSN neurons on egg-laying. (a) Egg-laying pattern of HSN-ablated flp-1 animals. Shown are the log-tail distributions of egg-laying interval times for HSN-ablated wild-type and flp-1 mutant animals. The intercluster time constant (indicated by the slope of the curve tails) was no higher in the ablated mutant animals than in ablated wild type or unablated mutant animals (see Table 1). The numbers of animals tracked, hours observed, and intervals analyzed, along with the estimated model egg-laying parameters, are in Table 1. Egg-laying rates were also measured independently on NGM agar by counting eggs laid by individual animals in an hour; mean and SEM of these experiments were as follows (in eggs/hr): HSN-ablated wild type, 1.35 ± 0.25 (n = 4); unablated flp-1, 1.94 ± 0.50 (n = 6); HSN-ablated flp-1, 1.05 ± 0.41 (n = 11). (b) Egg-laying pattern of HSN-ablated flp-1 mutants on serotonin. The histograms show the distribution of interval times for HSN-ablated wild type and flp-1 mutants on 7.5 mM serotonin; bins are on a log scale. A single exponential time constant for each data set was estimated as described in MATERIALS AND METHODS. Intervals for HSN-ablated flp-1(yn2) were significantly (P < 0.001) longer than for HSN-ablated wild type according to the Mann-Whitney rank sum test.

flp-1 is necessary for regulation of egg-laying by food signals:
What functional role might the FLP-1 peptides play in the control of egg-laying behavior? Among the neurons that express flp-1 are several pairs of interneurons, which are major recipients of synaptic input from sensory cells and have been implicated in processing and relaying integrated sensory information to motor circuits. The expression of flp-1 within these cells raised the possibility that flp-1-encoded peptides might be involved in the regulation of egg-laying behavior by sensory cues. Egg-laying is affected by a number of environmental conditions, including the presence or absence of a bacterial food source. To determine if flp-1 affects the regulation of egg-laying by food, we tested the effects of flp-1 loss-of-function mutations on the ability of animals to control their egg-laying rate in response to the presence or absence of a bacterial lawn. We observed that wild-type animals maintained on agar plates seeded with E. coli laid eggs at a significantly higher rate than animals maintained on agar plates that lacked food. Strikingly, however, we observed that flp-1 loss-of-function mutants laid eggs at essentially the same rate in the presence of a bacterial lawn as in the absence of a lawn (Fig 5). This defect in food regulation of egg-laying was fairly specific to flp-1 mutants and was not merely a consequence of their general egg-laying defect. For example, egg-laying behavior in egl-1 mutants, which lacked HSN neurons as a result of inappropriate programmed cell death, was strongly regulated by food, even though their egg-laying rate and pattern in the presence of food was similar to that of flp-1 mutants. Thus, flp-1 appeared to function specifically in mediating the control of egg-laying behavior in response to the availability of food.

View larger version (31K): In this window In a new window Download PPT slide   Figure 5. Effect of flp-1 mutations on regulation of egg-laying by food. The histogram shows the mean egg-laying rates of wild type, egl-1, and flp-1 mutant animals in the presence and absence of food. Error bars indicate the SEM; the numbers of independent trials under each condition are indicated. The wild type and egl-1 mutant strains showed a significantly lower (P < 0.001 for wild type, P < 0.02 for egl-1) egg-laying rate in the absence of food according to the Mann-Whitney rank sum test. In contrast, there was no statistically significant difference between the egg-laying rates in the presence or absence of food for either flp-1 mutant (P > 0.5 for yn2, P > 0.2 for yn4).

goa-1 functions independently from flp-1 and serotonin in the control of egg-laying:
What genes might function downstream of flp-1 and serotonin in the control of egg-laying? One possible candidate is goa-1, which encodes a G_o homologue that has been hypothesized to mediate the effects of serotonin (SEGALAT et al. 1995 ) and the FLP-1 peptides (NELSON et al. 1998B ) on locomotion. goa-1 is expressed in the egg-laying neurons as well as the vulval muscles, and mutations in goa-1 have been shown to enhance (in the case of recessive alleles) or inhibit (in the case of dominant gain-of-function alleles) egg-laying behavior (MENDEL et al. 1995 ; SEGALAT et al. 1995 ). Thus, the goa-1 gene product was a plausible candidate for a gene that might function downstream of flp-1 and/or serotonin as a negative regulator of egg-laying.

To investigate this possibility, we analyzed the egg-laying patterns of goa-1 mutants. We observed that the inactive phase was substantially shorter in goa-1 recessive mutant animals than in wild type (Table 1; see also Fig 6, a and b), implicating GOA-1 as a negative regulator of the switch into the active phase. Because this effect was roughly converse to the effect of mutations in flp-1 and cat-4, one possible interpretation of this result was that GOA-1 activity might be negatively regulated by serotonin and/or flp-1. Alternatively, goa-1 could function independently from, and antagonistically to, the pathway(s) activated by serotonin and flp-1. To distinguish these possibilities, we constructed double mutants carrying recessive mutations in goa-1 and either flp-1 or cat-4 and analyzed their egg-laying behavior. In each case, the double mutant showed a phenotype intermediate between that of the two single mutants (Fig 6, a and b; Table 1). For example, in the case of flp-1, both the inactive phase rate constant ₂ (0.0014 sec^-1) and the intercluster time constant (1950 sec) for the goa-1; flp-1 double mutant were intermediate between those of the goa-1 single mutant (0.0031 sec^-1; 890 sec) and the flp-1 single mutant (0.0005 sec^-1; 3840 sec). Similarly, both ₂ and the intercluster time constant for the cat-4; goa-1 double mutant were intermediate between the goa-1 single mutant and the cat-4 single mutant. Pharmacological experiments also supported the hypothesis that serotonin, flp-1, and goa-1 functioned independently. For example, goa-1 loss-of-function mutants still responded to serotonin, though weakly and at abnormally low threshold concentrations. In contrast, the goa-1; flp-1 double mutant's serotonin dose response was similar though not identical to that of the flp-1 single mutant (Fig 6C). Together, these results suggested that goa-1 defined a new pathway, independent of the ones activated by flp-1 and serotonin, regulating the switch into the active egg-laying phase.

View larger version (56K): In this window In a new window Download PPT slide   Figure 6. Effect of goa-1 on egg-laying behavior. (a) Independence of flp-1 and goa-1 egg-laying phenotypes. Shown are the log-tail distributions of egg-laying intervals for flp-1 and goa-1 mutant animals. The intercluster time constant of the goa-1; flp-1 double mutant curve (indicated by the slope of the curve) is intermediate between that of the flp-1 and goa-1 single mutants (see Table 1). The long intervals (>120 sec) in the double mutant were significantly shorter than in the flp-1 single mutant (P < 0.05) and significantly longer than in the goa-1 single mutant (P < 0.002) according to the Mann-Whitney rank sum test. (b) Independence of goa-1 and cat-4 egg-laying phenotypes. Shown are the log-tail distributions of egg-laying intervals for cat-4 and goa-1 mutant animals. The intercluster time constant of the goa-1; cat-4 double mutant curve (indicated by the slope of the curve) is intermediate between that of the cat-4 and goa-1 single mutants. The long intervals (>120 sec) in the double mutant were significantly shorter than in the cat-4 single mutant (P < 0.001) and significantly longer than in the goa-1 single mutant (P < 0.02) according to the Mann-Whitney rank sum test. (c) Serotonin responses of goa-1 mutants. Egg-laying responses to serotonin were determined for goa-1 and goa-1; flp-1 mutants as described in Fig 3. Individual points and error bars indicate the mean and SEM of the following numbers of trials: goa-1(n1134), 40; goa-1(n1134); flp-1(yn2), 25. (d) HSN dependence of the goa-1 hyperactive egg-laying phenotype. Shown are the log-tail distributions of egg-laying intervals for HSN-ablated and goa-1 mutant animals. The intercluster time constant of the HSN-ablated goa-1 mutant curve (indicated by the slope of the curve) is as long or longer than that of HSN-ablated wild type.

Pharmacological experiments have suggested that GOA-1 functions in both neurons and muscle cells to inhibit egg-laying (MENDEL et al. 1995 ; SEGALAT et al. 1995 ). In principle, GOA-1 might control the onset of the active egg-laying phase by negatively regulating the activity of the HSNs; alternatively, it might negatively regulate the response of the vulval muscles to modulatory inputs from neurons. To distinguish between these models, we analyzed the egg-laying behavior of HSN-ablated goa-1 mutant animals. Surprisingly, we observed that the inactive phase in HSN-ablated goa-1 mutants was no shorter than in HSN-ablated wild-type animals (Fig 6D; Table 1). This indicated that the shortening of the inactive phase by goa-1 mutations was dependent on the HSNs and suggested that GOA-1 controls the switch into the active phase by directly or indirectly modulating HSN function. HSN ablation was not completely epistatic to mutations in goa-1; the number of eggs laid within a given active phase (a function of the clustering parameter p) was higher in HSN-ablated goa-1 mutants than in HSN-ablated wild type (Table 1). Because goa-1 recessive mutants appeared to have longer active phases, this implied that the function of GOA-1 in the vulval muscles may be to promote the switch from the active egg-laying phase back to the inactive phase.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Modulation of egg-laying behavioral states by FaRPs:
Egg-laying behavior involves switching between two alternative behavioral states: an active state, during which eggs are laid in bursts, and an inactive phase, during which eggs are retained in the uterus. We observed that loss-of-function mutants defective in the gene flp-1, which encodes a set of FMRFamide-related peptides, displayed a specific abnormality in their temporal pattern of egg-laying: the inactive phase was abnormally long, whereas egg-laying within the active phase was normal. Thus, the flp-1 gene products appeared to function specifically to facilitate the switch from the inactive to the active egg-laying phase. Previous work had shown that the serotonergic HSN motorneurons were also specifically required to promote the onset of the active phase (WAGGONER et al. 1998 ). Nonetheless, at least some of the effects of flp-1 on egg-laying appeared to be HSN independent because flp-1 mutant animals whose HSNs had been eliminated through laser ablation responded less strongly to serotonin than HSN-ablated wild-type animals. These results were perhaps surprising because all the chains of synaptic connections between the flp-1-expressing neurons in the head and the vulval muscles involve the HSNs (WHITE et al. 1986 ). In fact, flp-1 mutations only slightly enhanced the egg-laying defect of HSN-ablated animals, suggesting that some of the effects of the FLP-1 peptides are likely to be HSN dependent. Thus, the FLP-1 peptides may regulate the egg-laying muscles both through modulation of the HSNs as well as through an HSN-independent humoral mechanism (Fig 7).

View larger version (19K): In this window In a new window Download PPT slide   Figure 7. Model for neural and molecular regulation of egg-laying. Egg-laying in C. elegans is affected by at least two parameters: the rate of switching from the inactive to the active phase and the rate of egg-laying within the active phase. FLP-1 peptides, released from neurons in the head, specifically regulate the switch into the active phase, a process also regulated by serotonin and possibly another neuromodulator whose release from the HSNs is controlled by GOA-1. The FLP-1 peptides may also modulate the activity of HSNs. The activity of the FLP-1 pathway may be sensitive to chemosensory cues indicating the abundance of food.

Many questions remain about the cellular mechanism through which FLP-1 peptides regulate egg-laying. flp-1 expression has been detected in a number of neurons in the head, including AIA, AIY, AVA, AVK, AVE, RIG, and RMG (NELSON et al. 1998B ). Based on the results presented here, the simplest hypothesis is that humoral release of FLP-1 peptides from one or more of these neuronal classes modulates the egg-laying muscles directly. Alternatively, it is possible that some or all of the effects of the FLP-1 peptides on egg-laying could be indirect. For example, FLP-1 peptides could modulate the activity of other neurosecretory cells in the head, affecting the release of a hypothetical neurohormone that modulates the egg-laying muscles. Some of the effects of flp-1 on egg-laying might also involve the VC neurons, although the fact that flp-1 mutations lengthen the inactive phase much more than ablations of the VCs do (WAGGONER et al. 1998 ; L. WAGGONER, unpublished results) argues that the VCs are not the primary target of the FLP-1 peptides. Laser ablations of various combinations of flp-1-expressing neurons, as well as neurons postsynaptic to these cells, may provide more detailed information about the cellular basis for the effect of flp-1 on egg-laying behavior.

Interactions between flp-1 and serotonin in the control of egg-laying:
The effects of flp-1 on egg-laying are quite similar to the effects of another neuromodulator, serotonin. We observed here that loss of flp-1 function did not confer resistance to the effects of serotonin on egg-laying, though it did significantly reduce the magnitude of the serotonin response. Therefore, the flp-1-encoded peptides appear to stimulate egg-laying at least in part by enhancing the response of the egg-laying neuromusculature to serotonin. This hypothesis is consistent with previously published work, which demonstrated that synthetic peptides identical to the shared carboxy terminus of the seven flp-1 peptides (FLRF-NH₂) increased the average number of eggs laid in response to serotonin (SCHINKMANN and LI 1992 ). Serotonin and FLP-1 appear to function in concert not only in the stimulation of egg-laying but in other C. elegans behaviors as well. For example, both serotonin and FLP-1 inhibit locomotion, and the ability of serotonin to inhibit movement has been shown to require a functional flp-1 gene (NELSON et al. 1998B ). Thus, for locomotive behavior, FLP-1 peptides appear to be necessary to potentiate the effects of serotonin. Although the molecular pathways through which serotonin and flp-1 control egg-laying are likely to differ in some respects from those involved in locomotion, it is tempting to speculate that the parallel actions of these two modulators on these two different behaviors might depend on a conserved molecular mechanism. Because comodulation of neuromuscular activity by biogenic amines and FaRPs is observed in many organisms (KLEIN et al. 1986 ; SCOTT et al. 1997 ), the molecular interactions between the flp-1 and serotonin-activated signaling pathways in the egg-laying cells may provide a useful model for similar processes in other animals.

Insights into the regulation of egg-laying by sensory information:
The analysis of the flp-1 mutants also revealed a role for the FLP-1 peptides in the control of egg-laying behavior by sensory cues. We observed that whereas wild-type worms laid eggs at a much slower rate in the absence of a bacterial food source, flp-1 loss-of-function mutants laid eggs at the same rate in either presence or absence of food. This insensitivity to the presence of bacteria was not merely a consequence of the flp-1 animals' generally slower egg-laying rate, as other egg-laying-defective animals (e.g., egl-1 mutants that lacked the HSN motorneurons) still showed significant regulation of egg-laying by food availability. Therefore, the flp-1-encoded peptides may be specifically dedicated to relaying signals of food abundance to the egg-laying circuit. In the absence of bacteria, levels of FLP-1 release could be low, leading to long inactive phases and slow egg-laying, whereas abundant food would lead to increased FLP-1 release and more active egg-laying. Consistent with this model, we found that the egg-laying patterns of wild-type animals in the presence of low levels of food resembled that of flp-1 animals (Table 1). Other aspects of the flp-1 mutant phenotype are consistent with FLP-1 functioning as an indicator of food availability. For example, when nematodes, maintained in the absence of food, encounter a lawn of bacteria, they slow their rate of movement (SAWIN 1996 ). Both the hyperactive locomotion and the "wandering" behavior previously noted in flp-1 recessive mutants (NELSON et al. 1998B ) could plausibly stem from a defect in this response to food. Thus, flp-1 may function quite generally to facilitate a variety of behavioral patterns that are appropriate for conditions of food abundance.

The expression pattern of flp-1 in the C. elegans nervous system is well suited for a gene that encodes a food signal. The presence of bacteria in the environment is thought to be detected primarily through olfactory or chemosensory cues (BARGMANN and MORI 1997 ). The primary route through which nematodes gather chemosensory information is by using a pair of polymodal sense organs known as amphids. Synaptic output from the amphid sensory neurons is relayed to four pairs of amphid interneurons: AIA, AIB, AIY, and AIZ (WHITE et al. 1986 ). In thermotaxis behavior, the amphid interneurons have been shown to be an important site for integrating and processing sensory information that is used to modulate behavioral outputs (MORI and OHSHIMA 1995 ). Both AIA and AIY express flp-1; thus a simple model for how egg-laying behavior could be controlled by food signals is that under conditions favorable to egg-laying (i.e., abundant food) the AIA and AIY neurons release FLP-1 peptides, switching the animal into the active egg-laying state. This release of FLP-1 peptides from the amphid interneurons could likewise switch the animal into a more inactive state with respect to locomotion.

Evidence that goa-1 modulates neural states involved in egg-laying behavior:
In addition to the pathways activated by FLP-1 and serotonin, a third pathway, defined genetically by the goa-1 gene, also appears to regulate the onset of the active phase of egg-laying in a manner antagonistic to and apparently independent of both flp-1 and serotonin. A recessive mutation in goa-1, which encodes the C. elegans homologue of the G_o alpha subunit (MENDEL et al. 1995 ; SEGALAT et al. 1995 ), increased the rate of egg-laying by shortening the inactive phase. Genetic analysis indicated that goa-1 probably functions to regulate egg-laying in a pathway distinct from the ones activated by flp-1 and serotonin. Interestingly, the effect of goa-1 on the onset of the active phase appeared to be completely dependent on the HSNs. Thus, goa-1 may function by negatively regulating release from the HSNs of a neuromodulator that facilitates the switch from the inactive to the active egg-laying state.

An interesting implication of this hypothesis is that the behavioral states involved in egg-laying may correspond not only to functional states of the egg-laying muscles themselves, but also to distinct functional states of neurons (such as the HSNs) dedicated to egg-laying. Our earlier studies led to the hypothesis that the active egg-laying state depends on a functional activation of the vulval muscles, which allows the excitatory transmitter acetylcholine to readily induce muscle contraction. Our analysis of goa-1 mutants suggests that the active and inactive egg-laying states may also correspond to functional states of the HSNs—an inactive state in which the HSNs release neurotransmitter with low probability, and an active state in which the probability of neurotransmitter release is high. According to this model, activated GOA-1 may inhibit the switch of the HSNs into this active state; thus, when GOA-1 is inactive or absent, the switch into the active state becomes more frequent. The likely involvement of GOA-1 in controlling HSN activity implies that additional neuromodulators, possibly released from neurons in the head, may regulate egg-laying behavior by controlling GOA-1 activity.

Regulation of egg-laying muscle activity by multiple neuromodulators:
Although the effect of goa-1 on the switch into the active phase was dependent on the HSNs, it was not completely dependent on the wild-type cat-4 gene. The cat-4 mutation is not a precise serotonin knockout: cat-4 mutants are defective in the synthesis of other amines, and it is possible that they may contain very low levels of serotonin. However, despite these caveats, the simplest explanation for the difference between the egg-laying patterns of HSN-ablated goa-1 and cat-4; goa-1 animals is that GOA-1 has HSN-dependent effects on egg-laying that are serotonin independent. This implies that the HSNs may contain another neuromodulator in addition to serotonin that facilitates the switch into the active phase, whose release may be regulated by GOA-1.

Although at present we can only speculate as to the identity of such a molecule, there appear to be a number of candidates. For example, the HSNs almost certainly contain one or more non-flp-1-encoded FaRPs, because they contain FaRP immunoreactivity, which is not eliminated by deletions of the flp-1 gene (SCHINKMANN and LI 1992 ; NELSON et al. 1998B ). In addition, pharmacological experiments indicate that muscarinic acetylcholine agonists stimulate egg-laying (WEINSHENKER et al. 1995 ). Because the HSNs are cholinergic, acetylcholine released from the HSNs might modulate egg-laying through muscarinic receptors. In principle, GOA-1-regulated release of any of these molecules could facilitate the onset of the active egg-laying state. The diversity of neurotransmitter usage in the HSNs is also a hallmark of other neurons and gland cells that participate in the control of egg-laying. For example, non-flp-1-encoded FaRPs are present in both the VC motorneurons and the uv1 uterine gland cells (SCHINKMANN and LI 1992 ). In addition, the VCs contain acetylcholine and, because they express the vesicular monoamine transporter, possibly an unidentified biogenic amine as well (DUERR et al. 1999 ). Thus, egg-laying behavior is likely to be regulated by a surprisingly diverse array of neurotransmitters and neuromodulators, which are likely to activate complex, interacting signaling pathways in the vulval muscle cells. The elucidation of these signaling mechanisms represents an important challenge for future studies and may be a useful model for the functional interaction of neuromodulatory pathways in other organisms.

	ACKNOWLEDGMENTS

The authors especially thank Chris Li for generously providing strains, reagents, and advice. We also thank members of our lab for discussions, Kit Pogliano, Stanley Shyn, and Jinah Kim for comments on the manuscript, and the Caenorhabditis Genetics Center for strains. This work was supported by grants from the National Science Foundation (IBN-9723250), the National Institutes of Health (DA11556), the Joseph and Esther Klingenstein Foundation, the Alfred P. Sloan Foundation, the Arnold and Mabel Beckman Foundation (to W.R.S.), a predoctoral training grant from the National Institutes of Health (to L.E.W.), and an undergraduate fellowship from the Porter Beach Foundation (L.A.H.).

Manuscript received September 13, 1999; Accepted for publication November 12, 1999.

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