Stephen Nurrish

Facilitation of synaptic transmission by EGL-30 Gqalpha and EGL-8 PLCbeta: DAG binding to UNC-13 is required to stimulate acetylcholine release.

We show that neurotransmitter release at Caenorhabditis elegans neuromuscular junctions is facilitated by a presynaptic pathway composed of a Gqalpha (EGL-30), EGL-8 phospholipase Cbeta (PLCbeta), and the diacylglycerol- (DAG-) binding protein UNC-13. Activation of this pathway increased release of acetylcholine at neuromuscular junctions, whereas inactivation decreased release. Phorbol esters stimulated acetylcholine release, and this effect was blocked by a mutation that eliminates phorbol ester binding to UNC-13. Expression of a constitutively membrane-bound form of UNC-13 restored acetylcholine release to mutants lacking the egl-8 PLCbeta. Activation of this pathway with muscarinic agonists caused UNC-13 to accumulate in punctate structures in the ventral nerve cord. These results suggest that presynaptic DAG facilitates synaptic transmission and that part of this effect is mediated by UNC-13.

Serotonin Inhibition of Synaptic Transmission

We show that serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and we describe a signaling pathway that mediates this effect. Release of acetylcholine from motor neurons was assayed by measuring the sensitivity of intact animals to the acetylcholinesterase inhibitor aldicarb. By this assay, exogenous serotonin inhibited acetylcholine release, whereas serotonin antagonists stimulated release. The effects of serotonin on synaptic transmission were mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 G(alpha)0 accumulated abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites.

Sensory Regulation of C. elegans Male Mate-Searching Behavior

How do animals integrate internal drives and external environmental cues to coordinate behaviors? We address this question by studying mate-searching behavior in C. elegans. C. elegans males explore their environment in search of mates (hermaphrodites) and will leave food if mating partners are absent. However, when mates and food coincide, male exploratory behavior is suppressed and males are retained on the food source. We show that the drive to explore is stimulated by male-specific neurons in the tail, the ray neurons. Periodic contact with the hermaphrodite detected through ray neurons changes the males behavior during periods of no contact and prevents the male from leaving the food source. The hermaphrodite signal is conveyed by male-specific interneurons that are postsynaptic to the rays and that send processes to the major integrative center in the head. This study identifies key parts of the neural circuit that regulates a sexual appetitive behavior in C. elegans.

The Gα12-RGS RhoGEF-RhoA signalling pathway regulates neurotransmitter release in C. elegans

In Caenorhabditis elegans adults, the single Rho GTPase orthologue, RHO‐1, stimulates neurotransmitter release at synapses. We show that one of the pathways acting upstream of RHO‐1 in acetylcholine (ACh)‐releasing motor neurons depends on Gα12 (GPA‐12), which acts via the single C. elegans RGS RhoGEF (RHGF‐1). Activated GPA‐12 has the same effect as activated RHO‐1, inducing the accumulation of diacylglycerol and the neuromodulator UNC‐13 at release sites, and increased ACh release. We showed previously that RHO‐1 stimulates ACh release by two separate pathways—one that requires UNC‐13 and a second that does not. We show here that a non‐DAG‐binding‐UNC‐13 mutant that partially blocks increased ACh release by activated RHO‐1 completely blocks increased ACh release by activated GPA‐12. Thus, the upstream GPA‐12/RHGF‐1 pathway stimulates only a subset of RHO‐1 downstream effectors, suggesting that either the RHO‐1 effectors require different levels of activated RHO‐1 for activation or there are two distinct pools of RHO‐1 within C. elegans neurons.

A network of G-protein signaling pathways control neuronal activity in C. elegans.

The Caenorhabditis elegans neuromuscular junction (NMJ) is one of the best studied synapses in any organism. A variety of genetic screens have identified genes required both for the essential steps of neurotransmitter release from motorneurons as well as the signaling pathways that regulate rates of neurotransmitter release. A number of these regulatory genes encode proteins that converge to regulate neurotransmitter release. In other cases genes are known to regulate signaling at the NMJ but how they act remains unknown. Many of the proteins that regulate activity at the NMJ participate in a network of heterotrimeric G-protein signaling pathways controlling the release of synaptic vesicles and/or dense-core vesicles (DCVs). At least four heterotrimeric G-proteins (Galphaq, Galpha12, Galphao, and Galphas) act within the motorneurons to control the activity of the NMJ. The Galphaq, Galpha12, and Galphao pathways converge to control production and destruction of the lipid-bound second messenger diacylglycerol (DAG) at sites of neurotransmitter release. DAG acts via at least two effectors, MUNC13 and PKC, to control the release of both neurotransmitters and neuropeptides from motorneurons. The Galphas pathway converges with the other three heterotrimeric G-protein pathways downstream of DAG to regulate neuropeptide release. Released neurotransmitters and neuropeptides then act to control contraction of the body-wall muscles to control locomotion. The lipids and proteins involved in these networks are conserved between C. elegans and mammals. Thus, the C. elegans NMJ acts as a model synapse to understand how neuronal activity in the human brain is regulated.

The Mood Stabilizer Valproate Inhibits both Inositol- and Diacylglycerol-signaling Pathways in Caenorhabditis elegans

The antiepileptic valproate (VPA) is widely used in the treatment of bipolar disorder, although the mechanism of its action in the disorder is unclear. We show here that VPA inhibits both inositol phosphate and diacylglycerol (DAG) signaling in Caenorhabditis elegans. VPA disrupts two behaviors regulated by the inositol-1,4,5-trisphosphate (IP(3)): defecation and ovulation. VPA also inhibits two activities regulated by DAG signaling: acetylcholine release and egg laying. The effects of VPA on DAG signaling are relieved by phorbol ester, a DAG analogue, suggesting that VPA acts to inhibit DAG production. VPA reduces levels of DAG and inositol-1-phosphate, but phosphatidylinositol-4,5-bisphosphate (PIP(2)) is slightly increased, suggesting that phospholipase C-mediated hydrolysis of PIP(2) to form DAG and IP(3) is defective in the presence of VPA.

The RHO-1 RhoGTPase Modulates Fertility and Multiple Behaviors in Adult C. elegans

The Rho family of small GTPases are essential during early embryonic development making it difficult to study their functions in adult animals. Using inducible transgenes expressing either a constitutively active version of the single C. elegans Rho ortholog, RHO-1, or an inhibitor of endogenous Rho (C3 transferase), we demonstrate multiple defects caused by altering Rho signaling in adult C. elegans. Changes in RHO-1 signaling in cholinergic neurons affected locomotion, pharyngeal pumping and fecundity. Changes in RHO-1 signaling outside the cholinergic neurons resulted in defective defecation, ovulation, and changes in C. elegans body morphology. Finally both increased and decreased RHO-1 signaling in adults resulted in death within hours. The multiple post-developmental roles for Rho in C. elegans demonstrate that RhoA signaling pathways continue to be used post-developmentally and the resulting phenotypes provide an opportunity to further study post-developmental Rho signaling pathways using genetic screens.

An Overview of C. Elegans Trafficking Mutants

It is almost 40 years since Sydney Brenner introduced Caenorhabditis elegans as a model genetic system. During that time mutants with defects in intracellular trafficking have been identified in a diverse range of screens for abnormalities. This should, of course, come as no surprise as it is hard to imagine any biological process in which the regulated movement of vesicles within the cells is not critical at some step. Almost all of these genes have mammalian homologs, and yet the role of many of these homologs has not been investigated. Perhaps the protein that regulates your favorite trafficking step has already been identified in C. elegans? Here I provide a brief overview of those trafficking mutants identified in C. elegans and where you can read more about them.

Dense Core Vesicle Release: Controlling the Where as Well as the When

Ca2+/calmodulin-dependent Kinase II (CaMKII) is a calcium-regulated serine threonine kinase whose functions include regulation of synaptic activity (Coultrap and Bayer 2012). A postsynaptic role for CaMKII in triggering long-lasting changes in synaptic activity at some synapses has been established, although the relevant downstream targets remain to be defined (Nicoll and Roche 2013). A presynaptic role for CaMKII in regulating synaptic activity is less clear with evidence for CaMKII either increasing or decreasing release of neurotransmitter from synaptic vesicles (SVs) (Wang 2008). In this issue Hoover et al. (2014) further expand upon the role of CaMKII in presynaptic cells by demonstrating a role in regulating another form of neuronal signaling, that of dense core vesicles (DCVs), whose contents can include neuropeptides and insulin-related peptides, as well as other neuromodulators such as serotonin and dopamine (Michael et al. 2006). Intriguingly, Hoover et al. (2014) demonstrate that active CaMKII is required cell autonomously to prevent premature release of DCVs after they bud from the Golgi in the soma and before they are trafficked to their release sites in the axon. This role of CaMKII requires it to have kinase activity as well as an activating calcium signal released from internal ER stores via the ryanodine receptor. Not only does this represent a novel function for CaMKII but also it offers new insights into how DCVs are regulated. Compared to SVs we know much less about how DCVs are trafficked, docked, and primed for release. This is despite the fact that neuropeptides are major regulators of human brain function, including mood, anxiety, and social interactions (Garrison et al. 2012; Kormos and Gaszner 2013; Walker and Mcglone 2013). This is supported by studies showing mutations in genes for DCV regulators or cargoes are associated with human mental disorders (Sadakata and Furuichi 2009; Alldredge 2010; Quinn 2013; Quinn et al. 2013). We lack even a basic understanding of DCV function, such as, are there defined DCV docking sites and, if so, how are DCVs delivered to these release sites? These results from Hoover et al. (2014) promise to be a starting point in answering some of these questions.

Shank is a dose-dependent regulator of Cav1 calcium current and CREB target expression

Shank is a post-synaptic scaffolding protein that has many binding partners. Shank mutations and copy number variations (CNVs) are linked to several psychiatric disorders, and to synaptic and behavioral defects in mice. It is not known which Shank binding partners are responsible for these defects. Here we show that the C. elegans SHN-1/Shank binds L-type calcium channels and that increased and decreased shn-1 gene dosage alter L-channel current and activity-induced expression of a CRH-1/CREB transcriptional target (gem-4 Copine), which parallels the effects of human Shank copy number variations (CNVs) on Autism spectrum disorders and schizophrenia. These results suggest that an important function of Shank proteins is to regulate L-channel current and activity induced gene expression. DOI: http://dx.doi.org/10.7554/eLife.18931.001