Collins K M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "ERG Potassium Channels Localize to and Function at Post-Synaptic Sites of Egg-Laying Muscles and May Mediate the Effects of G Protein Signaling on Egg-Laying Behavior"

Bany I, Bellemer A, Koelle M R, Jose A, Collins K M

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Serotonin released from the HSN motor neuron acts on the egg-laying muscles (ELMs) via two G protein coupled receptors, SER-1 and SER-7, which activate Gαq and Gαs, respectively. These G proteins promote muscle contraction, but the specific downstream effector molecules through which they act remain unclear. In two different screens for mutants with increased egg-laying behavior, we isolated mutations in the unc-103 gene, which encodes the Ether-a-go-go related gene (ERG) K+ channel. Human ERG channel activity has been shown to be inhibited by Gαq signaling, both via PLC-dependent depletion of membrane PI(4,5)P2 and upon phosphorylation by diacylglycerol-activated protein kinases. ERG may also be regulated by Gαs signaling since it bears a conserved binding domain for cAMP, the second messenger produced by Gαs signaling through adenylate cyclase. Thus, we are testing the hypothesis that serotonin signaling through Gαq and/or Gαs inhibits ERG potassium currents to promote ELM electrical excitability and contraction. Consistent with this model, we have found that UNC-103 function in the ELMs is both necessary and sufficient for normal inhibition of egg-laying behavior. These conclusions are based on our findings that expression of a dominant-negative UNC-103 mutant in muscle of wild-type animals causes strongly hyperactive egg-laying behavior, and expression of wild-type UNC-103 in muscle rescues the hyperactive egg-laying behavior of unc-103 mutants. To address the mechanism of UNC-103 regulation, we are exploiting single-copy MosSCI transgenes to express at physiological levels channel mutants lacking putative sites of regulation. Expression of UNC-103 fused to GFP in the ELMs restores normal egg-laying behavior to unc-103 mutants, and UNC-103::GFP is highly localized to muscle arms of the vm2 cells that form the postsynaptic sites apposed to the presynaptic varicosity of the HSN motor neurons. Deletion of a predicted PDZ interaction sequence at the C-terminus of UNC-103::GFP disrupts its synaptic localization and phenotypic rescue activity. Because SER-1 and SER-7 receptors also bear predicted PDZ interaction sequences, we are testing whether these receptors, their G proteins, and downstream effectors are all enriched at the postsynaptic site to shape post-synaptic electrical responses through local modulation of inhibitory UNC-103 potassium currents.

Kopchock, R. et al. (2019) International Worm Meeting "Dissecting the function of the cholinergic VC motor neurons in C. elegans egg-laying behavior."

Kopchock, R., Collins, K.

[International Worm Meeting, 2019]

During egg-laying behavior in the nematode C. elegans, the cholinergic Ventral C (VC) motor neurons become active coincident with vulval muscle contraction, but how these neurons become activated and how they signal during egg-laying behavior is not well understood. To determine the in vivo function of the VC neurons, we are using mutants and transgenes to manipulate the activity of the VC neurons and test how VC signaling affects other cells in the egg-laying behavior circuit. Optogenetic activation of the VCs using Channelrhodopsin-2 fails to induce egg laying and instead hyper-contracts the body wall muscles, slowing locomotion. Individual egg-laying events are accompanied by a slowing of locomotion, and this acute slowing is diminished when VC neurotransmitter release is blocked. The VCs make extensive synapses onto the ventral body wall muscles, suggesting acetylcholine release from the VCs is mediating body wall muscle contraction and slowing. VC-specific expression of histamine-gated Cl- channels or Tetanus Toxin has no gross consequences on steady-state egg accumulation but does prevent serotonin-induced egg laying in liquid. Because the HSN command neurons release both serotonin and NLP-3 neuropeptides, these results suggest that serotonin modulates VC neurotransmitter release to drive egg laying in liquid. Consistent with this model, when VC neurotransmission is blocked, the vulval muscles frequently fail to release eggs during strong Ca2+ transients, suggesting the VCs facilitate uniform opening of the vulva. To determine if the VCs are activated in response to vulval muscle contractility we optogenetically activated the vulval muscles and recorded VC Ca2+ activity. Optogenetic activation of the vulval muscles was sufficient to induce VC Ca2+ activity, suggesting a feed-forward mechanism enhances neurotransmitter release from the VCs that enables full opening of the vulva for egg laying. Consistent with a role for the VC neurons in locomotion behavior, worm locomotion speed is decreased during vulval muscle-induced VC Ca2+ activity. Together, our results suggest that the VCs are involved both in facilitating vulval muscle contraction for successful egg-laying events and in coordinating locomotor behaviors with egg laying.

Li, C. et al. (2017) International Worm Meeting "The UNC-7 innexin coordinates vulval muscle contraction and egg release."

Li, C., Collins, K.

[International Worm Meeting, 2017]

Gap junctions are intercellular connections needed for cellular communication and exchange. We have been investigating how gap junctions drive coordinated activity between cells in developing and mature neural circuits using the egg-laying behavior circuit as a model system. Gap junctions are thought to facilitate the coordinated activity of neurons and the muscle cells they innvervate. Gap junctions are of particular interest because they are thought to be targets of neuromodulators like serotonin whose dysfunction is implicated in human depression. We hypothesize that gap-junction proteins regulate egg-laying circuit activity and behavior. Previous work has shown that unc-7 and unc-9 mutants habe uncoordinated (unc) locomotion and are resistant to serotonin-induced egg laying. I found that unc-7 or unc-9 mutants have hyperactive egg-laying behavior. The locomotion and egg-laying behavior phenotypes resemble mutants with defective acetylcholine signaling from the presynaptic VC motor neurons that innervate the vulval muscles. To test this hypothesis, I have begun using Ca2+ imaging to quantify changes in cell activity in behaving animals. Wild-type animals show coordinated vulval muscle activity with strong egg-laying Ca2+ transients that allow release of eggs from the uterus within ~1 second. In contrast, unc-7 mutants show uncoordinated vulval muscles, with eggs often getting stuck in the contracting vulva. Eggs are finally released after ~30 seconds of sustained, tetanic muscle contraction. unc-9 mutants do not show as dramatic of an egg-laying or vulval muscle Ca2+ defect. To show where unc-7 and unc-9 function. I will re-express UNC-7 or UNC-9 in the VC neurons and look for a restoration of normal circuit activity and behavior. I hope to also perform dominant-negative experiments to see if inactivation of UNC-7 in specific cells of the egg-laying circuits recapitulates the null mutant phenotype. This work will begin to address how gap junction proteins like UNC-7 promote the proper development and function of neural circuits to execute a coordinated behavior.

Dhakal, P. et al. (2019) International Worm Meeting "Biochemical consequences of Galphaq signaling in the modulation of a serotonin motor circuit of C. elegans."

Collins, K., Dhakal, P.

[International Worm Meeting, 2019]

The biochemical consequences of neuromodulatory signaling through the major heterotrimeric GTPase, G?q, are unclear. Mutations that reduce or eliminate signaling through G?q and its conserved effectors, the Phosphatidylinositol (4,5) P2-specific Phospholipase-C? (PLC?) and the Trio Rho GTPase exchange factor reduce synaptic transmission in the nematode worm, C. elegans, providing a genetically tractable model to understand how serotonin signaling through G?q modulates motor circuit function. Using established methods for ratiometric Ca2+ imaging, we found that reduction of G?q or Trio signaling nearly eliminates postsynaptic vulval muscle activity. Unlike Trio mutants, PLC? null mutants have grossly normal circuit activity, suggesting that G?q signals through Trio and Rho signaling independent of Phospholipase-C?. Our preliminary results show pan-neuronal rescue of PLC? but not Trio is sufficient to restore egg-laying behavior, suggesting PLC? acts in neurons to regulate neurotransmitter release pathway while Trio has additional postsynaptic functions in muscle. Consistent with this model, optogenetic activation of presynaptic neurons or postsynaptic muscles in PLC? null mutants still drives egg release, while activation of the presynaptic neurons fails to drive motor behavior in Trio RhoGEF mutants. G?q signaling mutant behavior and circuit activity defects can be restored by exogenous treatment with the DAG analog, PMA, suggesting that DAG production from PI(4,5)P2 is a major biochemical consequence of G?q, Trio, and PLC? signaling. Phorbol esters also drive behavior in UNC-13 and Protein Kinase C mutants suggesting G?q and Rho produce DAG which signals via new effectors to promote circuit activity and behavior. Together, our work tests how neuromodulators like serotonin signal through G?q and unique downstream effectors to drive distinct presynaptic and postsynaptic activity responses during the execution of a simple behavior.

Ravi, B. et al. (2017) International Worm Meeting "Development of patterned activity in the C. elegans egg-laying behavior circuit."

Collins, K., Ravi, B.

[International Worm Meeting, 2017]

Coordinated patterns of activity are required to drive efficient behaviors in mature neural circuits. We are interested in understanding how activity patterns emerge during development that allow for proper execution of behaviors in adults. The C. elegans egg laying circuit is a simple, well-characterized neural circuit which develops during the L4 stage and drives a two-state behavior in adult animals with a ~20 minute inactive state and ~2 minute active state where eggs are laid. The circuit comprises two types of motor neurons: the serotonergic Hermaphrodite Specific Neurons (HSNs) and the cholinergic Ventral Type C motor neurons (VC), as well as the tyraminergic uv1 neuroendocrine cells and the vulval muscles which contact to lay eggs. During the L4 larval stage, the cells of the circuit undergo morphological maturation and establish synaptic connections, providing a model system to understand the origin and function of cell activity during development. We are using GCaMP calcium imaging to record activity in cells of the egg laying circuit at the late L4 larval stage and in adults after development. We observe little or no activity in the VC neurons or the uv1 cells in late L4 animals. However, we find the HSN neurons show rhythmic Ca2+ transients at the late L4 larval stage even before eggs are produced. HSN L4 activity is rhythmic, occurring every 55 seconds and lacks the characteristic burst-firing pattern of adults during egg-laying active states where Ca2+ transients occur every 20s. The vulval muscles show an increase in Ca2+ transient strength and frequency as animals develop into adults. During egg laying, vulval muscle Ca2+ transients are coordinated with transients occurring in both the anterior and posterior muscle cells. We find this coordinated muscle activity appears at the late L4 stage and is independent of HSN presynaptic input. During the course of development, the ability of the HSN neurons to robustly drive vulval muscle Ca2+ activity is coincident with the accumulation of eggs within the uterus. The 'burst-firing' activity in the HSN neurons is missing after animal sterilization with Floxuridine (FUDR) or after acute electrical silencing of the vulval muscles. Together, our data suggests the vulval muscles integrate signals of uterine stretch in response to egg accumulation and promote HSN Ca2+ activity that induces the egg-laying active state in adults. Because HSN activity in L4 animals is dispensable for egg-laying circuit development, we are investigating whether HSN activity drives changes in L4-specific behaviors including the reduction of locomotion and the cessation of defecation that accompanies lethargus. Together, these results will help us to understand how mature patterns of cell activity develop and drive robust, stable behaviors.

Dhakal, P. et al. (2017) International Worm Meeting "Understanding how Galphaq signaling regulates egg-laying circuit activity and behavior of Caenorhabditis elegans."

Collins, K., Dhakal, P.

[International Worm Meeting, 2017]

Gaq protein signaling modulates many cellular processes including neural circuit activity and synaptic transmission to regulate switching between behavior states. However, many important questions remain to be solved: how does Gaq and its effectors regulate presynaptic and postsynaptic activity; what are the downstream biochemical consequences of Gaq activity; and how does altered Gaq signaling lead to alternate behavior states? Because mutations that reduce or eliminate signaling through Gaq (egl-30) or its effectors Phosphopholipase C beta (egl-8) and the Trio RhoGEF (unc-73) reduce egg laying, the Caenorhabditis elegans egg-laying circuit provides a well-characterized model system to investigate the conserved mechanisms of Gaq protein signaling in vivo. Gaq signaling regulates serotonin release from the serotonergic Hermaphrodite Specific Neurons (HSNs), acetylcholine release from the ventral type A, B, and C neurons, and postsynaptically in the vulval muscles to modulate the response to released serotonin and acetylcholine. In rescue experiments we find that Phosphopholipase C beta (PLC beta ) expression in neurons rescued egg laying and aldicarb sensitivity of the egl-8(sa47) null mutant indicating EGL-8 function in the presynaptic cholinergic neurons is sufficient. In order to further investigate how Gaq and its effectors regulate presynaptic and/ or postsynaptic activity, we used GCaMP5 calcium imaging to record presynaptic HSN and postsynaptic vulval muscle activity in freely behaving animals. In this experiment we reveal that egl-8(sa47) mutants have similar HSN activity to the wild type and both egl-30(n686) and egl-8(sa47) mutants have persistent vulval muscle activity during infrequent egg-laying events. In contrast, we find a complete loss of vulval muscle activity in animals bearing the unc-73(ce362) RhoGEF mutant. These results suggest the UNC-73 RhoGEF is required for postsynaptic muscle activity explaining the strong egg-laying behavior defects. Taken together, our results suggest a working model for how Gaq signaling regulates egg-laying behavior. We propose that PLC beta regulates acetylcholine release from the VA, VB, and VC neurons, rhythmically excite the vulval muscles during locomotion and for egg laying. Postsynaptic Gaq signaling through UNC-73 and Rho potentiates the vulval muscle response to released acetylcholine, driving their rhythmic excitation that triggers muscle contractility and egg release.

Medrano, E. et al. (2019) International Worm Meeting "C. elegans egg laying is controlled by a stretch-dependent homeostat."

Collins, K., Medrano, E.

[International Worm Meeting, 2019]

Egg laying in Caenorhabditis elegans, is a two-state behavior where ~20-minute inactive states are punctuated by ~2-minute active states during which eggs are laid. This behavior is controlled by a simple circuit wherein both the serotonergic HSNs and the cholinergic Ventral Cord (VC) neurons synapse onto the vulval muscles which contract to release eggs. While many external and internal sensory signals have been identified that inhibit the active state, the signaling mechanisms that promote the active state are not well understood. Our recent results show that egg accumulation in the uterus regulates burst-firing activity of the presynaptic HSNs, suggesting a stretch-dependent homeostat promotes the egg-laying active state. The purpose of this study is to test our hypothesis that changes in internal pressure caused by the accumulation of unlaid eggs in the uterus promotes circuit activity which drives the egg-laying active state. We have developed a microinjection technique to acutely increase the internal pressure which mimics the accumulation of eggs in the uterus. Our results show that acute injection induces vulval muscle contractility along with egg release. Using ratiometric calcium imaging, we find acute microinjection drives an immediate activation of every cell in the egg-laying circuit. Mutant and transgenic animals defective in synaptic transmission from the VC neurons and/or HSNs show a normal induction of vulval muscle Ca2+ activity and egg release, suggesting that the vulval muscles are the direct targets of the stretch-dependent mechanical stimulus. Together, these results show that mechanosensory feedback of egg accumulation by the post-synaptic muscle cells is sufficient to drive patterns of presynaptic circuit activity that sustain the active behavior state.

Chun-Yi Huang et al. (2007) International Worm Meeting "eif-3.K is a pro-apoptotic gene in C. elegans."

Jia-Yun Chen, Yi-Chun Wu, Chun-Yi Huang

[International Worm Meeting, 2007]

Programmed cell death (or apoptosis) is an important feature of C. elegans development. Previous studies have identified pro-apoptotic genes egl-1, ced-3 and ced-4 and anti-apoptotic genes ced-9 and icd-1 that control programmed cell death.. We have identified and characterized a novel pro-apoptotic gene eif-3.K. Loss-of-function by mutation or RNAi inactivation in eif-3.K resulted in a decrease of cell corpses, whereas heatshock-induced over-expression of eif-3.K weakly but significantly increased cell corpses. Interestingly, the eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 or ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the programmed cell death pathway. Using a cell-specific promoter to express eif-3.k in touch neurons, we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. To further explore EIF-3.K function, we generated antibodies against bacterially expressed EIF-3.K protein. We found that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. As human eif-3.K can functionally substitute C. elegans eif-3.K in an eif-3.K mutant, the function of eif-3.K in apoptosis is likely conserved in evolution.

Chun-Yi Huang et al. (2006) East Asia C. elegans Meeting "eif-3.K is a pro-apoptotic gene in C. elegans"

Teng-Wei Huang, Chun-Yi Huang

[East Asia C. elegans Meeting, 2006]

Programmed cell death or apoptosis is an important feature during C. elegans development. The pro-apoptotic genes egl-1, ced-4 and ced-3 are required for the execution of cell death. We have identified and characterized a novel pro-apoptotic gene eif-3.K. A loss-of-function mutation or inactivation by RNA interference in eif-3.K resulted in a reduction of cell corpse number during embryogenesis, whereas heatshock-induced over-expression of eif-3.K weakly but significantly promoted programmed cell death. In addition, eif-3.K mutation partially suppressed ectopic cell deaths caused by over-expression of egl-1 and ced-4. This result suggests that eif-3.K may act downstream of or in parallel to egl-1 and ced-4 in the genetic pathway during programmed cell death. Using a cell-specific promoter we showed that eif-3.K likely promoted cell death in a cell-autonomous manner. We generated antibodies against bacterially expressed EIF-3.K protein. The immunostaining result showed that EIF-3.K was ubiquitously expressed during embryogenesis and localized to the cytoplasm. To better understand the cell-death defect of eif-3.K mutants, we are currently performing a 4D microscopic analysis of the cell death process in wild-type and eif-3.K mutants.

Nassar, L.M. et al. (2017) International Worm Meeting "Regulation of female reproductive behavior states in C. elegans."

Bode, A., Nassar, L.M., Collins, K., Scheetz, M.

[International Worm Meeting, 2017]

We are interested in understanding how females make reproductive behavior decisions using Caenorhabditis elegans as a model system. Specifically, we are identifying the neural signaling mechanisms that drive two mutually exclusive vulval motor behaviors: mating with males or the release of progeny during egg laying. We hypothesize that circuit activity that drives egg-laying behavior is inhibited during mating while mating behavior is inhibited when females have sufficient sperm to fertilize oocytes. To investigate this hypothesis, we are using calcium imaging to record changes in cell activity in the egg-laying behavior circuit during mating and using histamine-gated Cl- channels to test the functional importance of cell activity during female mating behavior. We find that fertile hermaphrodites and sperm-deficient fog-2 mutant females show low vulval muscle activity before mating that resembles the egg-laying inactive state. Consistent with this observation, fog-2 females display low activity in the presynaptic HSN motor neurons and the cholinergic VC motor neurons whose activity coincides with egg release. Vulval muscle activity increases before spicule insertion and silencing of muscle activity with histamine delays mating, suggesting vulval muscle twitching contractions facilitate male spicule insertion. We recently showed that the uv1 neuroendocrine cells are mechanically activated by passage of eggs through the vulva, and we find they are similarly activated upon male spicule insertion during mating. Based on this data, we predict that male prodding of the vulva mechanically activates the VC motor neurons that slow female locomotion and allow mating. During male spicule insertion, the vulval muscles and uv1 neuroendocrine cells are mechanically deformed, with the activated uv1 cells releasing tyramine and neuropeptides that silence the HSN motor neurons to prevent female escape and egg laying that would interrupt mating. Sperm and seminal fluid deposited into the uterus activates the uterine and vulval muscles, allowing for ejection of the male spicules and inactivation of the uv1 cells, signaling mating is complete. Together, our comparative analysis of C. elegans reproductive behaviors will show how altered activity in the same neural circuit can be used to drive distinct reproductive behavior states.