Caenorhabditis elegans has emerged as a powerful model system for studying the biology of the synapse. Here we describe a widely used assay for synaptic transmission at the C. elegans neuromuscular junction. This protocol monitors the sensitivity of C. elegans to the paralyzing affects of an acetylcholinesterase inhibitor, aldicarb. Briefly, adult worms are incubated in the presence of aldicarb and scored for the time-course of aldicarb-induced paralysis. Animals harboring mutations in genes that affect synaptic transmission generally exhibit a change in their sensitivity to aldicarb (either increased sensitivity for enhancements in synaptic transmission or decreased sensitivity for blockage in synaptic transmission). This technique provides a simple assay for the accurate comparative analysis of synaptic transmission in multiple C. elegans strains. The protocol described can be performed relatively quickly and is a practical alternative to other techniques used to study synaptic transmission. This protocol can also be modified to follow the paralytic effects with other pharmacological reagents. The assay can be performed in about 3-6 hours depending on the severity of synaptic transmission defects.

Rab small GTPases are involved in the transport of vesicles between different membranous organelles. RAB-3 is an exocytic Rab that plays a modulatory role in synaptic transmission. Unexpectedly, mutations in the Caenorhabditis elegans RAB-3 exchange factor homologue, aex-3, cause a more severe synaptic transmission defect as well as a defecation defect not seen in rab-3 mutants. We hypothesized that AEX-3 may regulate a second Rab that regulates these processes with RAB-3. We found that AEX-3 regulates another exocytic Rab, RAB-27. Here, we show that C. elegans RAB-27 is localized to synapse-rich regions pan-neuronally and is also expressed in intestinal cells. We identify aex-6 alleles as containing mutations in rab-27. Interestingly, aex-6 mutants exhibit the same defecation defect as aex-3 mutants. aex-6; rab-3 double mutants have behavioral and pharmacological defects similar to aex-3 mutants. In addition, we demonstrate that RBF-1 (rabphilin) is an effector of RAB-27. Therefore, our work demonstrates that AEX-3 regulates both RAB-3 and RAB-27, that both RAB-3 and RAB-27 regulate synaptic transmission, and that RAB-27 potentially acts through its effector RBF-1 to promote soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) function. INTRODUCTIONNeurotransmitter release is accomplished by the fusion of neurotransmitter-filled synaptic vesicles at the presynaptic nerve terminal. This process occurs through a series of highly regulated steps that include synaptic vesicle transport, docking/tethering, priming, fusion, endocytosis, recycling, and neurotransmitter refilling (Sudhof, 2004). Several genes have been assigned roles in the various steps of the synaptic vesicle cycle. Rab3 (termed RAB-3 in Caenorhabditis elegans), a member of the Rab family of small GTPases, regulates synaptic transmission, possibly through the docking, priming, or fusion steps Schluter et al., 2004).Rabs act in a variety of cell types and regulate vesicular transport between organelles. Rabs cycle on and off membranes via a GTP-dependent mechanism (Zerial and McBride, 2001). Rab activity is regulated by two proteins, which act in an antagonistic manner. The guanine nucleotide exchange factor (GEF) exchanges GTP for GDP, and the GTPase activating protein activates the intrinsic GTPase activity of a Rab (Bernards, 2003). GDP bound Rabs are held off of the membrane by a GDP dissociation inhibitor (Wu et al., 1996). The GTP/membrane-bound form of Rabs typically binds to a variety of effectors that regulate particular steps of membrane transport and cell signaling (Zerial and McBride, 2001;Spang, 2004).Rab3 was once thought to play a central role in regulating release. However, recent work has shown that a quadruple knockout of all four isoforms of Rab3 in mice only results in a 30% reduction in evoked synaptic response (Schluter et al., 2004). This is consistent with work in C. elegans showing that mutations in the single rab-3 gene cause only mild defects in synaptic transmission . Surprisingly, more dramatic phe...

The Caenorhabditis elegans defecation motor program (DMP) is a highly coordinated rhythmic behavior that requires two GABAergic neurons that synapse onto the enteric muscles. One class of DMP mutants, called anterior body wall muscle contraction and expulsion defective (aex) mutants, exhibits similar defects to those caused by the loss of these two neurons. Here, we demonstrate that aex-2 encodes a G-protein-coupled receptor (GPCR) and aex-4 encodes an exocytic SNAP25 homologue. We found that aex-2 functions in the nervous system and activates a Gs␣ signaling pathway to regulate defecation. aex-4, on the other hand, functions in the intestinal epithelial cells. Furthermore, we show that aex-5, which encodes a pro-protein convertase, functions in the intestine to regulate the DMP and that its secretion from the intestine is impaired in aex-4 mutants. Activation of the Gs␣ GPCR pathway in GABAergic neurons can suppress the defecation defect of the intestinal mutants aex-4 and aex-5. Lastly, we demonstrate that activation of GABAergic neurons using the light-gated cation channel channelrhodopsin-2 is sufficient to suppress the behavioral defects of aex-2, aex-4, and aex-5. These results genetically place intestinal genes aex-4 and aex-5 upstream of GABAergic GPCR signaling. We propose a model whereby the intestinal genes aex-4 and aex-5 control the DMP by regulating the secretion of a signal, which activates the neuronal receptor aex-2. T he Caenorhabditis elegans defecation motor program (DMP)is a highly coordinated series of three muscle contractions that are executed every 45 sec [ Fig. 1A and supporting information (SI) Movie S1]. The cycle is initiated by a posterior body wall muscle contraction (pBoc), followed 2-3 sec later by an anterior body wall muscle contraction (aBoc). About 1 sec after the aBoc, enteric muscles contract, thus causing the expulsion (Exp) of intestinal contents. The process repeats itself Ϸ45 sec later with little variability in the timing of contractions (1). A genetic screen for mutants that displayed defects in the DMP isolated mutants defective in each of the three muscle contractions, known as pbo, abo, and exp (1). The screen also recovered mutants defective in the last two muscle contractions (aBoc and Exp [aex]) and mutants defective in the cycle periodicity (i.e., longer or shorter than normal DMP cycling times) (1). Molecular studies of these mutants have suggested that the behavior is orchestrated through the communication between the intestine, GABAergic neurons, and muscle.The periodicity of the DMP is regulated by the C. elegans intestine, a single-cell layer tube of polarized epithelial cells joined by gap junctions (2, 3). Intestinal Ca 2ϩ oscillations with Ϸ45-sec periodicity appear to play a central role in this timing. They consist of a posterior-to-anterior Ca 2ϩ wave whose levels peak in the posterior and anterior intestinal cells just before the pBoc and aBoc contractions, respectively (3-5). Mutations in genes involved in the maintenance of Ca 2ϩ oscillations or i...

How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming exposure. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild-population kin selection under resource-limiting and thermal stress conditions.