Collins, Kevin M. et al. (2013) International Worm Meeting "Understanding how neurotransmitter signaling drives two-state activity of the C. elegans egg-laying behavior circuit."

Collins, Kevin M., Koelle, Michael R.

[International Worm Meeting, 2013]

We are interested in understanding how neurotransmitter signaling allows a neural circuit to execute distinct behavior states. C. elegans regulates egg laying by alternating between an inactive phase and a ~3 minute "active" state during which clusters of 3-6 eggs are laid. Six VC motor neurons release acetylcholine to excite the vulval muscles, and two HSN motor neurons release serotonin which signals through vulval muscle receptors to promote the active phase. Four uv1 cells release tyramine to inhibit egg laying. We are using GCaMP calcium imaging in behaving animals to understand how the signaling events in the circuit produce its two state behavior. We recently reported that the vulval muscles are rhythmically excited during each locomotor body bend (1). We found the UNC-103 K+ channel depresses muscle excitability below the threshold that drives calcium transients and contraction during the inactive phase, while still allowing signals above threshold during the active phase. To understand how the HSN, VC, and uv1 neurons regulate vulval muscle excitability, we are manipulating neurotransmitter release from these cells and using GCaMP to record changes in neuron and vulval muscle activity. Activation of serotonin release from the HSNs using Channelrhodopsin induces rhythmic vulval muscle twitching and egg-laying behavior-hallmarks of the active phase. We find that VC neuron activity increases during the active phase, and preliminary results show that egg-laying events mechanically distort the uv1 cells and trigger calcium transients. We have previously shown that TRPV channels, which can be mechanically gated, are required for uv1 to inhibit egg laying (2). We propose that successive egg-laying events increase uv1 calcium signaling, promoting release of tyramine and neuropeptides which terminate the active phase of egg laying. (1) Collins and Koelle (2013). J. Neurosci. 33, 761-775. (2) Jose et al. (2007). Genetics 175, 93-105.

Brewer, Jacob et al. (2015) International Worm Meeting "The HSN command neuron co-releases serotonin and NLP-3 neuropeptides to coordinate egg-laying activity."

Brewer, Jacob, Koelle, Michael R.

[International Worm Meeting, 2015]

It remains unclear exactly how serotonin regulates neural circuit activity in the brain or how changes in serotonin signaling might cause mental disorders. The C. elegans egg-laying system is well-suited for studying the basics of how serotonin functions in a circuit. The serotonergic HSN motor neurons initiate ~2.5 minute egg-laying active phases that occur every ~20 minutes. The evidence for this is that rhythmic HSN Ca2+ activity measured using a GCaMP reporter always accompanies egg-laying active phases, and optogenetic activation of HSN neurons is sufficient to stimulate activity of the egg-laying circuit.We knocked out serotonin biosynthesis and showed that optogenetically stimulating the HSNs still caused animals to lay eggs. We hypothesized that the HSNs release a signal in addition to serotonin to stimulate egg laying. We found that neuropeptides encoded by the nlp-3 gene, which is known to be expressed in the HSNs, are such a signal. Knocking out either nlp-3 or serotonin biosynthesis alone caused mild egg-laying defects, but knocking out both caused a strong egg-laying defect that phenocopies that of animals lacking HSNs. Interestingly, co-release of serotonin and a neuropeptide also occurs in the mammalian brain, although the function of such co-release is unknown.Serotonin stimulates egg-laying through the G-protein coupled receptors (GPCRs) SER-1 and SER-7 that are expressed on the egg-laying muscles.1,2,4 Consistent with this previous work, we found that double-mutant worms lacking NLP-3 and either SER-1 or SER-7 show a strong synthetic egg-laying defect. NLP-3 neuropeptides were previously reported to act in an aversive response circuit through the GPCR NPR-17.3 Using a fosmid-based GFP reporter, we observed additional previously unreported expression of NPR-17 in cells of the egg-laying circuit.We found that animals lacking HSNs, serotonin, or NLP-3 neuropeptides, which all lay eggs less frequently than wild type, surprisingly all show increased activity of the egg-laying circuit as measured using GCaMP recordings in freely-behaving animals. We therefore hypothesize that co-release of serotonin and NLP-3 from the HSNs is not essential for activity of the circuit, but is required to coordinate muscle activity, allowing the animal to productively lay eggs.1Carnell et al. (2005) J. Neurosci. 25:10671-81; 2Hapiak et al. (2009) Genetics 181:153-63; 3Harris et al. (2010) J. Neurosci. 30:7889-99; 4Hobson et al. (2006) Genetics 172:159-69.

Collins, Kevin M. et al. (2011) International Worm Meeting "The C. elegans ERG (Ether-a-Go-Go Related Gene) K+ channel is a synaptically localized inhibitor of vulval muscle electrical excitability."

Koelle, Michael R., Collins, Kevin M.

[International Worm Meeting, 2011]

Neurotransmitters signal through heterotrimeric G proteins to alter cell excitability, often by modulating ion channel activities, and we seek to define the mechanism by which this occurs. In C. elegans, G proteins regulate specific behaviors that can be conveniently scored and quantitatively measured. One such behavior is egg laying, in which the HSN motor neurons release serotonin to activate postsynaptic Gaq and Gas signaling, promoting vulval muscle contractility and egg laying. Gao inhibits egg-laying behavior, although how it acts remains unclear. I cloned the gene identified by new C. elegans mutants that display the hyperactive egg-laying phenotype characteristic of loss of Gao signaling. The mutations affect unc-103, the C. elegans ortholog of the ether-a-go-go related gene (ERG), encoding a voltage-gated K+ channel. Disregulation of human ERG causes Long QT syndrome, a sometimes fatal arrhythmia of the heart. ERG functions in neurons and muscle cells to regulate and limit their excitability. I found that single-copy transgene expression of ERG fused to GFP in the vulval muscles restores normal egg-laying behavior to unc-103 null mutants. This ERG::GFP protein specifically localizes to postsynaptic muscle arms in the vm2 cells apposed to HSN presynaptic varicosities. Deletion of a predicted PDZ interaction sequence disrupts ERG::GFP enrichment at synapses and decreases phenotypic rescue. Thus, ERG acts postsynaptically to inhibit egg-laying behavior. We hypothesize that synaptic localization has two roles in ERG function. It locally inhibits postsynaptic electrical activity, and it allows for local modulation by G protein signaling. To test this, we are using high-speed video recording and calcium imaging of freely moving animals to study how ERG mutants affect muscle contractility. We find that during locomotion, vulval muscles of wild-type animals show smooth contraction and relaxation with adjacent body wall muscles. During the active phase of egg laying, vulval muscles display occasional twitches that coincide with these ventral contractions. In animals lacking ERG, vulval muscles twitch during nearly all ventral contractions (even between egg-laying clusters), suggesting ERG regulates the egg-laying contraction program during locomotion. We are testing how disrupting ERG synaptic localization affects the kinetics of calcium transients and egg laying induced by HSN signaling. Together, our experiments will show how localization and G protein signaling regulate ERG activity to modulate synaptic electrical excitability and muscle contractility.

Olson, Andrew C et al. (2013) International Worm Meeting "The Role of Post-Translational Modifications in the Regulation of Serotonin Signalling."

Olson, Andrew C, Koelle, Michael R

[International Worm Meeting, 2013]

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications seem to regulate serotonin signaling. First, through large-scale genetic screens for mutants that fail to respond properly to serotonin (Gurel et al., 2012), we found that C. elegans strains carrying mutations in either of two subunits of the ELPC ELongator" Protein Complex are defective for response to serotonin. ELPC is highly conserved from C. elegans to humans and functions as a lysine acetylase to reversibly modify other proteins. This is first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling. Second, our lab has also shown that Gao, a G protein through which serotonin signals to slow locomotion, is post-translationally modified in a manner that alters its charge, which is a hallmark of lysine acetylation. We hypothesize that ELPC may reversibly acetylate Gao (and/or one of the other proteins that mediate serotonin signaling) to regulate serotonin response. Serotonin regulates worm movement in C. elegans by redundantly signaling through the MOD-1 serotonin-gated ion channel and the SER-4 G Protein Coupled serotonin Receptor (GPCR), the GPCR through which Gao signals (Gurel et al. 2012). It is likely that ELPC modifies signaling through one or both of these receptors. To test this genetically, we have developed an assay that optogenetically stimulates the release of serotonin from the NSM and ADF serotonergic neurons. This strongly decreases the speed of worms that have intact MOD-1 and SER-4 signaling pathways, but this serotonin response is defective in animals missing one or both of the receptors. With WormLab worm-tracking software we are able to monitor the speed of worms throughout the course of the experiment. Using this assay we will be able to genetically determine if ELPC affects signaling through the MOD-1 or SER-4 receptor. We are also working to identify the post-translational modifications of Gao using mass spectrometry. Analysis of Gao immunoprecipitated from both C. elegans lysates as well as mouse brain lysates will allow us to determine if worm and mammalian Gao are equivalently modified.

Olson, Andrew C. et al. (2015) International Worm Meeting "The Role of Post-Translational Modifications in the Regulation of Serotonin Signaling."

Olson, Andrew C., Koelle, Michael R.

[International Worm Meeting, 2015]

C. elegans uses serotonin as a neurotransmitter to slow locomotion, and we have used this model system to discover that post-translational modifications appear to regulate serotonin signaling. Through large-scale genetic screens for mutants that fail to paralyze in response to exogenous serotonin, we found that C. elegans mutants for either of two subunits of the ELPC ELongator Protein Complex are defective for serotonin signaling. Conversely, transgenic animals overexpressing ELPC are hypersensitive to the effects of exogenous serotonin. ELPC is conserved from C. elegans to humans and functions as a cytoplasmic lysine acetylase to reversibly modify other proteins. This is the first time that ELPC or lysine acetylation has been implicated in regulating serotonin signaling.We used two-dimensional gel electrophoresis to show that in C. elegans lysates, Galphao, a neural G protein encoded by the goa-1 gene through which serotonin signals to slow locomotion, exists as a complex series of species of differing charge. Acetylation eliminates the positive charge on a lysine side chain, and differential acetylation on several lysines could produce the complex pattern of Galphao species we see. Our preliminary results suggest that the series of differentially charged Galphao species in wild-type lysates shifts to a less complex and more positively charged set of Galphao species in lysates of ELPC mutants, as would be predicted if Galphao is acetylated by ELPC.We isolated Galphao from both mouse brain and C. elegans lysates and analyzed the proteins for post-translational modifications using mass spectrometry. In both species, Galphao is acetylated on several conserved lysine residues. We note that the signaling defects in ELPC mutants are much more restricted than those of Galphao null mutants. Both ELPC and Galphao mutants are defective for response to exogenous serotonin, but Galphao null mutants have additional defects not seen in ELPC mutants, including defects in response to exogenous dopamine as well as in many behavioral assays. Thus we hypothesize that ELPC may reversibly acetylate Galphao to specifically regulate its ability to be activated by serotonin receptors, while not strongly affecting its ability mediate signaling by other receptors.We are using CRISPR-Cas9 technology to mutate the acetylated lysine residues of Galphao to the similar but non-acetylatable amino acid arginine to determine if acetylation at specific positions is responsible for regulating serotonin signaling.

Pepper, Judy S. et al. (2011) International Worm Meeting "Mutants that fail to respond to exogenous serotonin identify new serotonin signaling genes."

Koelle, Michael R., Pepper, Judy S.

[International Worm Meeting, 2011]

Signaling by the neurotransmitter serotonin is thought to be reduced in the brain during depression, but the mechanisms that alter serotonin signaling remain unclear. We are using a genetic approach in C. elegans to delineate the molecules that mediate serotonin signaling to shed light on this issue. Several C. elegans behaviors are regulated by serotonin signaling, including locomotion, egg-laying, and pharyngeal pumping. Serotonin released when food-deprived animals encounter food slows locomotion, and high concentrations of exogenous serotonin paralyzes animals, presumably by exaggerating this response. To identify additional signaling molecules that mediate the effects of serotonin, we carried out a screen for mutants resistant to the paralytic effects of exogenous serotonin. Individual F1 progeny of mutagenized animals were cultured for one generation in 96-well microtiter dishes and 30 mM serotonin was added. This concentration paralyzes nearly all wild-type animals within minutes, and we looked for wells in which ~25% of the F2 progeny (mutant homozygotes) continued to thrash. We recovered 13 serotonin resistant mutants from a screen of 12,000 mutagenized haploid genomes. Using single nucleotide polymorphism (SNP) mapping and sequencing we identified the mutations causing serotonin resistance in the five strongest mutants. Four mutations were in genes previously known to be required for serotonin response. One mutation was in the gene encoding G protein coupled receptor SER-4 and two were in the goa-1 G protein gene, suggesting serotonin acts through SER-4 to activate GOA-1 signaling and slow locomotion. An additional mutation was in the serotonin-gated chloride channel MOD-1, suggesting serotonin has an additional role in slowing locomotion through this receptor. We found that the fifth strong serotonin resistance mutation, vs119, changes a glycine to a glutamate in the enzymatic core of the catalytic subunit of a conserved protein complex whose only known biochemical function is to post-translationally modify other proteins on lysine residues by acetylating them. Two additional alleles for this lysine acetylase gene as well as a mutation that disrupts another subunit of the complex all cause strong resistance to exogenous serotonin. We hypothesize that vs119 may function in serotonin signaling by modifying one or more of the other proteins required for serotonin response: the SER-4 or MOD-1 serotonin receptors, or the G protein, GOA-1. We previously found that the GOA-1 protein runs a series of species of different charge on an isoelectric focusing gel, and we are currently testing the hypothesis that this is due to variable modifications of GOA-1 by the acetylase.

Ghosh, D. Dipon et al. (2013) International Worm Meeting "Neuropeptide modulation of C. elegans light avoidance circuitry."

Koelle, Michael R., Ghosh, D. Dipon, Nitabach, Michael N.

[International Worm Meeting, 2013]

Neuropeptides modulate neuronal circuit activity to regulate animal behavior. We used Caenorhabditis elegans as a model system to investigate the mechanism by which these neuromodulators fine tune cellular circuit outputs to optimize behavioral responses. Recently, homologs of an arthropod peptide Pigment Dispersing Factor (PDF) and its cognate receptor, PDF Receptor (PDFR), were identified in C. elegans. Secretion of PDF and its mammalian relative, Vasoactive Intestinal Peptide (VIP), is triggered by activation of light sensors and can modulate locomotor rhythms in insects and mammals, respectively. We found that null mutant worms lacking a PDF homolog exhibit aberrant light avoidance responses. Further analysis of these deficits indicates that this peptide could function in sensorimotor circuitry to modulate navigation in the presence of light. Our results suggest a link between circadian light entrainment in mammals and photophobic responses in the simpler worm, and a physiological mechanism by which neuromodulators regulate behaviors.

Kim, Seongseop et al. (2015) International Worm Meeting "An atlas of G protein-coupled neurotransmitter and neuropeptide receptor expression patterns in C. elegans."

Fernandez, Robert, Koelle, Michael R., Pepper, Judy, Kim, Seongseop

[International Worm Meeting, 2015]

Many neurotransmitters and neuropeptides signal by activating G protein-coupled receptors (GPCRs), but there is a limited understanding of the biological purpose of such signaling in large part because there are mutant phenotypes for only a tiny fraction of the GPCRs. The few C. elegans GPCRs that have been studied in detail are expressed in a very limited number of cells, and mutants show very specific defects in the behaviors controlled by those cells. Although it is now possible to get knockout mutations for any receptor, it has not been possible to identify any defects since we do not know what very specific defects to look for. If we knew the expression patterns for all the neural GPCRs, then we could analyze in detail knockouts of just the GPCRs expressed within the circuit that controls a behavior of interest for specific defects in that behavior. This strategy would provide a path to finally understand the functions of neurotransmitter/neuropeptide signaling through GPCRs.We are analyzing the cellular expression patterns of the ~100 neuropeptide receptor homologs and ~23 GPCRs for small molecule neurotransmitters that are found in C. elegans. We are using C-terminal translational gfp fusion transgenes generated by Sarov et al. (2012). These fosmid-based transgenes contain long stretches of 5' and 3' regulatory sequences and thus should show accurate and complete expression patterns. We are generating high-copy transgenes by microinjection to get bright GFP expression and also to generate possible over-expression phenotypes. We identify GFP-expressing cells by confocal microscopy, using several RFP transgenes to label specific cells as landmarks.Our pilot experiments have confirmed and added to previously known expression patterns of several neurotransmitter receptors. For example, our egl-6::gfp fosmid transgene showed expression in the HSN and GLR cells, as did a smaller transgene used by Ringstad and Horvitz (2008). However our fosmid transgene also showed additional expression in head, vulval, anal sphincter muscles, the gonadal sheath cells, and several additional neurons.Our receptor expression atlas will help identify neurotransmitter signaling events in all neural circuits, but we will first demonstrate its utility by focusing on the egg-laying circuit. This circuit consists of just four cell types, and we hope to identify all the neurotransmitter receptors that act in this circuit and to characterize their functions in generating the pattern of activity by which this circuit controls egg-laying behavior.

Patikoglou GA et al. (1998) East Coast Worm Meeting "Two regulators of G protein signaling with similar expression patterns have opposite effects on egg laying"

Koelle MR, Patikoglou GA, Sigler PB

[East Coast Worm Meeting, 1998]

RGS (Regulator of G protein Signaling) proteins were identified through genetic studies as negative regulators of G protein signaling (reviewed in Koelle, 1997). Degenerate PCR and genomic sequence analyses predict the existence of many RGS proteins both in mammals and in C. elegans. In vitro studies using randomly chosen pairs of RGS and Ga proteins show that RGS proteins are potent GTPase Activating Proteins (GAPs), accelerating the slow intrinsic rate of GTP hydrolysis by Ga proteins and thus converting them to their inactive GDP-bound forms. Surprisingly, these assays show that a number of RGS proteins have promiscuous GAP activity for the same set of Ga proteins in vitro. However, little is known about the specificity of RGS proteins in vivo. One possibility is that each RGS protein is expressed in a restricted set of cells and interacts with many Ga proteins in those cells. Another possibility is that RGS proteins are widely expressed but are not promiscuous in vivo, and have specificity for only one or a few physiologic Ga targets. While all RGS proteins contain a core RGS domain that is required for GAP activity, a subset of RGS proteins also contain an additional N-terminal conserved region of unknown function. In C. elegans, this subset consists of the EGL-10 and C16C2 RGS proteins. EGL-10 was the first C. elegans RGS protein to be genetically characterized and was shown to inhibit the Ga protein GOA-1, which in turn inhibits egg laying (Koelle and Horvitz, 1996). We overexpressed all worm RGS proteins by generating 11 types of transgenic worm strains carrying multiple copies of the genomic DNA for each of the 11 RGS genes. We found that overexpression of EGL-10 increases the frequency of egg laying, while overexpression of C16C2 decreases it. These results suggest that C16C2 may inhibit signaling by the Ga protein EGL-30, which is an activator of egg laying. Furthermore, GFP reporter transgenes for EGL-10 and C16C2 indicate that they are both expressed in most or all neurons, as are the G proteins EGL-30 and GOA-1. Thus, it is possible that EGL-10 and C16C2 function in the same cells but are biochemically specific for different Ga protein subunit partners to achieve different functions. Our hypothesis is that the N-terminal conserved region may confer specificity for the correct Ga protein and thus direct GAP activity only to the appropriate protein target. To test this idea, we are conducting domain swapping experiments between C16C2 and EGL-10 to see if they result in a specificity swap in vivo. We are also analyzing the functions of the two conserved RGS protein domains by expressing them individually in C. elegans to assess their effect on egg laying. In addition, biochemical and X-ray crystallographic methods will be used to determine how the two conserved regions of the EGL-10 protein contribute to its interaction with GOA-1. Koelle, M. R. (1997) A new family of G-protein regulators - the RGS proteins. Curr Opin in Cell Biol, 9, 143-7. Koelle, M. R. and Horvitz, H. R. (1996) EGL-10 Regulates G Protein Signaling in the C. elegans Nervous System and Shares a Conserved Domain with Many Mammalian Proteins. Cell, 84, 115-125.

Nassar, Layla et al. (2015) International Worm Meeting "Understanding how sex modulates the female nervous system to drive distinct reproductive behavior states."

Collins, Kevin, Nassar, Layla, Bode, Addys

[International Worm Meeting, 2015]

We are interested in understanding how mating and reproductive behaviors are coordinated in the female nervous system. Specifically, we are identifying the neural signaling systems that drive two mutually exclusive vulval motor behaviors: mating with males or the release of progeny during egg laying [1]. We hypothesize that: 1. female vulval muscle twitching contractions facilitate male spicule insertion during mating; 2. specific mechanical and chemical signals report successful copulation and insemination; and 3. mating initiates reproductive behaviors including oocyte production and egg release. To investigate these hypotheses, we are using calcium imaging techniques to record vulval signaling events during mating with males, after successful insemination, and during the resumption of normal egg laying behavior. We have found that hermaphrodites with sperm have reduced vulval muscle twitching behaviors resulting in inefficient mating. In contrast, hermaphrodites depleted of sperm have increased vulval muscle twitching that facilitates male spicule insertion and mating. After mating is complete, hermaphrodites have sustained vulval muscle twitching that can result in release of sperm from the uterus. This behavior may act as a mechanism for competition with self-sperm. We are now examining how the other cells in the egg-laying circuit, including the HSN and VC neurons and uv1 neuroendocrine cells, respond during steps of male mating. During egg laying behavior, we found that the uv1 cells are mechanically activated by passage of eggs through the vulva, driving release of tyramine that inhibits egg laying [2]. We predict that mechanical stimulation by male spicules may similarly activate the uv1 cells to inhibit egg release during mating. However, once mating is complete, we expect egg laying to resume or even increase. Together, these results will explain how internal and external signals modulate activity in the same neural circuit to drive distinct behavior states.1. Collins, KM, and Koelle, MR (2013). Postsynaptic ERG Potassium Channels Limit Muscle Excitability to Allow Distinct Egg-Laying Behavior States in Caenorhabditis elegans. J. Neurosci. 33, 761-775.2. Collins KM, Bode A, Fernandez R, Tanis JE, Creamer M, Koelle MR (2015). Unpublished results.