The Caenorhabditis elegans neuromuscular junction (NMJ) contains three pharmacologically distinct ionotropic receptors: ␥-aminobutyric acid receptors, levamisole-sensitive nicotinic receptors, and levamisole-insensitive nicotinic receptors. The subunit compositions of the ␥-aminobutyric acid-and levamisole-sensitive receptors have been elucidated, but the levamisole-insensitive acetylcholine receptor is uncharacterized. To determine which of the ϳ40 putative nicotinic receptor subunit genes in the C. elegans genome encodes the levamisole-resistant receptor, we utilized MAPCeL, a microarray profiling strategy. Of seven nicotinic receptor subunit transcripts found to be enriched in muscle, five encode the levamisole receptor subunits, leaving two candidates for the levamisole-insensitive receptor: acr-8 and acr-16. Electrophysiological analysis of the acr-16 deletion mutant showed that the levamisole-insensitive muscle acetylcholine current was eliminated, whereas deletion of acr-8 had no effect. These data suggest that ACR-16, like its closest vertebrate homolog, the nicotinic receptor ␣7-subunit, may form homomeric receptors in vivo. Genetic ablation of both the levamisolesensitive receptor and acr-16 abolished all cholinergic synaptic currents at the NMJ and severely impaired C. elegans locomotion. Therefore, ACR-16-containing receptors account for all non-levamisole-sensitive nicotinic synaptic signaling at the C. elegans NMJ. The determination of subunit composition for all three C. elegans body wall muscle ionotropic receptors provides a critical foundation for future research at this tractable model synapse.

An organism's ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output.

The syntaxin-interacting protein tomosyn is thought to be a key regulator of exocytosis, although its precise mechanism of action has yet to be elucidated. Here we examined the role of tomosyn in peptide secretion in Caenorhabditis elegans tomosyn (tom-1) mutants. Ultrastructural analysis of tom-1 mutants revealed a 50% reduction in presynaptic dense-core vesicles (DCVs) corresponding to enhanced neuropeptide release. Conversely, overexpression of TOM-1 led to an accumulation of DCVs. Together, these data provide the first in vivo evidence that TOM-1 negatively regulates DCV exocytosis. In C. elegans, neuropeptide release is promoted by the calciumdependent activator protein for secretion (CAPS) homolog UNC-31. To test for a genetic interaction between tomosyn and CAPS, we generated tom-1;unc-31 double mutants. Loss of TOM-1 suppressed the behavioral, electrophysiological, and DCV ultrastructural phenotypes of unc-31 mutants, indicating that TOM-1 antagonizes UNC-31-dependent DCV release. Because unc-31 mutants exhibit synaptic transmission defects, we postulated that loss of DCV release in these mutants and the subsequent suppression by tom-1 mutants could simply reflect alterations in synaptic activity, rather than direct regulation of DCV release. To distinguish between these two possibilities, we analyzed C. elegans Rim mutants (unc-10), which have a comparable reduction in synaptic transmission to unc-31 mutants, specifically attributed to defects in synaptic vesicle (SV) exocytosis. Based on this analysis, we conclude that the changes in DCV release in tom-1 and unc-31 mutants reflect direct effects of TOM-1 and UNC-31 on DCV exocytosis, rather than altered SV release.

Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed innumerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-α. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence that the APC regulates transmission at inhibitory synapses and have implications for understanding nervous system pathologies, such as epilepsy, that are characterized by misregulated GABA signaling.

The SNARE proteins, syntaxin, SNAP-25 and synaptobrevin form a tertiary complex essential for vesicle fusion. Proteins that influence SNARE complex assembly are therefore likely to be important regulators of fusion events. In this study we have focused on tomosyn, a highly conserved, neuronally enriched, syntaxin-binding protein that has been implicated in the regulation of vesicle exocytosis. To directly test the role of tomosyn in neurosecretion we analysed loss-of-function mutants in the single Caenorhabditis elegans tomosyn gene, tom-1. These mutants exhibit enhanced synaptic transmission based on electrophysiological analysis of neuromuscular junction activity. This phenotype is the result of increased synaptic vesicle priming. In addition, we present evidence that tom-1 mutants also exhibit enhanced peptide release from dense core vesicles. These results indicate that tomosyn negatively regulates secretion for both vesicle types, possibly through a common mechanism, interfering with SNARE complex formation, thereby inhibiting vesicle fusion.