Eastman CL et al. (1997) International C. elegans Meeting "Regulation of unc-47 by unc-30"

Eastman CL, Jin YS

[International C. elegans Meeting, 1997]

We are interested in the development and function of the GABAergic D neurons in the ventral cord. These neurons mediate the coordinated sinusoidal motion of the worm by inhibiting body wall muscle contractions. D neurons in unc-30 mutants do not produce GABA, are defective in axonal pathfinding, and fail to make proper synaptic connections(J.White, personal communication). unc-30 encodes a homeodomain transcription factor expressed in all D neurons (Jin Y, Hoskins R and Horvitz HR Nature 372: 780), suggesting that UNC-30 controls transcription of genes that are involved in D neuron differentiation and function. unc-47 mutant worms express higher levels of GABA than wild type animals and display a similar locomotion phenotype to that of unc-30 (McIntire SL, Jorgensen E and Horvitz HR Nature 364:334). unc-47 has been shown to encode the vesicular pump for GABA and is expressed in all GABAergic neurons (K. Schuske and E. Jorgensen, WCWM 1996). The phenotype of unc-47 worms and the expression pattern of unc-47 suggest that it might be regulated by unc-30. We crossed the unc-47-GFP array (gift of Kim Schuske and Erik Jorgensen) into an unc-30 null mutant. In the unc-30 mutant background, unc-47 expression was abolished in the D neurons whereas its expression in other GABAergic neurons was unaffected, suggesting that unc-30 regulates transcription of unc-47 in the D neurons. Experiments to identify the unc-47 promoter elements regulated by unc-30 are underway. We will present this analysis at the meeting.

Adam W Tearle et al. (2005) International Worm Meeting "Investigating nicotinic acetylcholine receptors in vulval muscles by calcium imaging."

William R Schafer, Adam W Tearle

[International Worm Meeting, 2005]

We are studying the properties of acetylcholine receptors in C. elegans vulval muscles using the genetically expressed calcium indicator cameleon. The worm is dissected using a technique adapted from Richmond and Jorgensen (1999). Animals are glued down in a perfusion chamber, a small incision made on the ventral side opposite the vulva and the viscera removed. This exposes the vulval muscles and allows the direct application of drugs to these muscles at physiological concentrations. Dissected vulval muscles continue to contract and give corresponding calcium transients. Wild type vulval muscles show a large increase in intracellular calcium in response to 500M nicotine or 100M levamisole. Levamisole receptor mutants, unc-29 and lev-1 do not respond to levamisole but show a smaller response to nicotine than wild type. Further experiments will be performed to determine the response to acetylcholine and the effect of different pharmacological blockers of nicotinic receptors. The effects of adaptation to repeated nicotine stimulation, or long term exposure to nicotine, will also be described. Richmond, JE. and Jorgensen, EM., Nat Neurosci. 1999 (9):791-7

Edward Yeh et al. (2003) International Worm Meeting "A screen to identify factors involved in active zone formation."

Mei ZHEN, Edward YEH

[International Worm Meeting, 2003]

The active zone is a specialized presynaptic structure where vesicles containing neurotransmitters fuse with the plasma membrane, releasing the neurotransmitters to the post-synaptic membrane. At the EM level, electron dense active zones are surrounded by clusters of vesicles. A few components of the active zone have been identified such as UNC-10 (Koushika et al., 2001), UNC-13 (Richmond et al., 1999; Aravamudan et al., 1999) and SYD-2 (Zhen and Jin, 1999) but even fewer have been implicated in active zone establishment, differentiation or maintainance.syd-2 encodes a homolog of liprin- and has been shown to be a component of the active zone. Mutations in syd-2 give rise to larger active zones. At a previous meeting, we reported a SYD-2::GFP marker that could be used to visualize active zones in live animals. The SYD-2::GFP protein marker was placed under the promoter of unc-25 which expresses in GABAergic neurons. We have performed a SYD-2::GFP screen to identify a dditional factors involved in the formation of the active zone. An EMS mutagenesis screen was performed looking for mutations affecting the formation of the active zone. Approximately 2500 haploid genomes were screened and we are currently characterizing 5 isolated mutant alleles. hp77 and hp121 represent two loci on chromosome X, hp102 and hp198 represent two loci on chromosome IV and hp129 is on chromosome V. hp121 and hp102 have behavioural unc phenotypes, hp198 are slightly dumpy while hp77 and hp129 do not appear to have any behaviour or physical phenotypes. Initial characterization and phenotypic analysis will be presented on these mutants. It is our hope that the molecular characterization of these mutants will provide information on factors involved in the establishment, differentiation, and maintenance of active zones. Ultimately, this information will provide insight into the genetic pathways used to establish neural synapses in general.References: Aravamudan B, Fergestad T, Davis WS, Rodesch CK, and Broadie K. (1999) Nature Neuroscience. 2:965; Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM and Nonet M. (2001) Nature Neuroscience. 4:997-1005; Richmond JE, Davis WS and Jorgensen EM. (1999) Nature Neuroscience. 2:959-64; Zhen M and Jin Y. (1999) Nature. 401:371-5

Allikian MJ et al. (1991) International C. elegans Meeting "GENOMIC SEQUENCE ANALYSIS OF THE UNC-104 GENE"

Otsuka AJ, Allikian MJ, Whiteaker K

[International C. elegans Meeting, 1991]

When mutant, the unc-104 gene in C. elegans yields a slow and uncoordinated phenotype. It has been shown that the protein product of this gene is a kinesin-related protein. Kinesin is thought to be involved in anterograde axonal transport in the neuron, consistent with the phenotypic effect of an unc-104 mutant. The unc-104 gene was cloned by transposon tagging of the unc-104 (e2184) mutant. However, rhlO16 and rhlO17 are two additional alleles which yielded 1.6 Kb DNA insertions. With these three alleles and physical markers, the unc-104 gene was cloned (Otsuka et al., Neuron, 6:113-122, 1991). From the cDNA sequence, an open reading frame was found which was able to encode a protein with a motor domain sequence similar to that of a kinesin heavy chain. ItS relatedness to kinesin was noted at a protein primary and secondary structural level. While the majority of the unc- 104 gene has been sequenced, the S' end and promoter region has yet to be determined. A 10.7 Kb segment of the unc-104 genomic DNA containing the S' end and promoter region has been ligated into a PTZl9 vector. Sequencing of the 5 Kb to the left of the known genomic sequence is currently underway using Eco Rl and Sst I subclones of the cosmid ZK323.

Christian Abraham et al. (2006) European Worm Meeting "Evolutionary Conservation of Synaptic Tetraspan Vesicle Membrane Proteins but Lack of Defects in Mutant C. elegans Strains"

Robby Weimer, Gabriele Spatkowski, Harald Hutter, Erik Jorgensen, Reinhard Windoffer, Christian Abraham, Lin Bai, Rudolf E. Leube, Mark Palfreyman

[European Worm Meeting, 2006]

1Christian Abraham, 1Lin Bai, 2Harald Hutter, 3Mark Palfreyman, 1Gabriele Spatkowski, 4Robby Weimer, 1Reinhard Windoffer, 3Erik Jorgensen and 1Rudolf E. Leube. Tetraspan vesicle membrane proteins (TVPs) comprise a major portion of synaptic vesicle proteins, yet their contribution to the synaptic vesicle cycle is poorly understood. TVPs are grouped in three mammalian gene families, the physins, gyrins and secretory carrier associated membrane proteins (SCAMPs). Remarkably, each family is represented by only a single polypeptide in C. elegans and all nematode TVPs co-localize to the same vesicular compartment when expressed in mammalian cells. To examine their function, C. elegans null-mutants were isolated for each gene. Triple mutants were generated by combination of all three mutant alleles. Careful analyses of these worms revealed no morphological or behavioral phenotypic defects. Nervous system architecture and synaptic contacts appear normal in the triple mutants. Mutant worms moved normally and released normal levels of neurotransmitter as assayed pharmacologically and electrophysiologically. Worms also responded normally to chemical or mechanosensory stimuli. We therefore conclude that TVPs are not needed for the basic neuronal machinery. Current transrciptome analyses identify potential regulatory components whose expression is altered in the mutant strains.

Kaplan JM et al. (2000) West Coast Worm Meeting "Electrophysiological analysis of serotonin modulation of body wall neuromuscular physiology."

Madison JM, Kaplan JM

[West Coast Worm Meeting, 2000]

The ability of neurons to alter their synaptic function underlies our ability to control behavior. We are interested in understanding how neurotransmitters like serotonin (5-HT) cause changes in neuron and synapse function to effect behavioral changes. In C. elegans, 5-HT modulates the rate of locomotion. Recent analysis has shown that 5-HT acts presynaptically on motor neurons through a G-protein Gao subunit, GOA-1, to reduce acetylcholine (ACh) release at body wall neuromuscular junctions (NMJs) [1]. Using a recently developed dissection technique and whole cell voltage clamp recordings [2], we have recorded the effects of 5-HT on adult body wall NMJ physiology. We have recorded from body wall muscle both miniature excitatory post-synaptic currents (mEPSCs) and muscle responses to ACh application in the presence of 5-HT. We see two effects of 5-HT: a reduction in EPSC frequency and a reduction in ACh-activated current amplitude. The amplitude of ACh-activated current is reduced by 40% in the presence of 5-HT. Previous physiological analysis by Richmond and Jorgensen has shown there to be two classes of ACh receptors at the body wall NMJ; one ACh receptor class is sensitive to the cholinergic agonist levamisole and one receptor is insensitive [2]. Recordings of levamisole-activated current show that 5-HT reduces this current up to 90%. These data suggest that the levamisole activated ACh receptors may be specifically modulated by 5-HT. To resolve the contribution of post-synaptic modulation by 5-HT to presynaptic changes in mEPSC frequency and to further understand the mechanism of the post-synaptic response, we have recorded from mutants that might eliminate the post-synaptic ACh receptor modulation. Since 5-HT's downstream effects are mediated by G-protein coupled pathways, we have tested a number of candidate signal transduction mutants expressed in muscle for defects in ACh receptor modulation. We have tested the G-protein Ga subunit mutants, gpa-14 and gpa-7, and an adenylate cyclase, acy-1, and all are normal for ACh receptor modulation by 5-HT. Future physiologic and genetic experiments will help further our understanding of 5-HT's modulation of synaptic function and behavior. 1. Nurrish et al., Neuron 24: 231-242 1999. 2. Richmond and Jorgensen, Nat Neurosci 2: 791-797 1999.

Lucinda Carnell et al. (2001) International C. elegans Meeting "Measuring neurotransmitter levels in isolated C.elegans vesicles by HPLC."

Jennifer Hsu, Steven McIntire, Lucinda Carnell

[International C. elegans Meeting, 2001]

We are in the process of developing an assay to measure transmitter content in synaptic vesicles from C.elegans in order to study the function of various vesicular neurotransmitter transporters. We circumvented the problem of obtaining large amounts of tissue sample by measuring transmitter levels using HPLC. Starting with about half a gram of worms, we can by differential centrifugation isolate a crude vesicle fraction. We can demonstrate enrichment of synaptic vesicles in this fraction by the detection of full length unc-47 ::GFP (the GABA vesicular transporter) using Western blot analysis. The presence of unc-47 ::GFP in this fraction suggests it contains synaptic vesicles because this construct is localized to synaptic vesicles and can rescue the unc-47 mutant phenotype (McIntire et al. (1997), Nature 389: p 870-879). We measured amino acid neurotransmitter levels in these fractions by HPLC which is sensitive enough to allow small concentrations (ng/ml) to be reliably quantified. Although we used this procedure to primarily examine the neurotransmitter GABA, this method could also be used to measure quantities of other neurotransmitters such as dopamine, serotonin, octopamine and acetylcholine. In vesicle preparations from N2 animals we demonstrated that GABA is contained in this fraction. As a control for the purity of the synaptic vesicle preparation, we measured GABA levels in unc-47 mutants which should contain no GABA in their vesicles and observed a 3-4 fold reduction compared to N2 animals. We also examined GABA levels in synaptic vesicles of unc-46 mutant animals. unc-46 encodes a novel protein and unc-46 mutants have a similar phenotype to unc-47 animals (K. Schuske and E. Jorgensen, personal communication). Since overexpression of UNC-47 is capable of rescuing the unc-46 mutant phenotype, UNC-46 may function together with UNC-47 to regulate vesicular transport of GABA (K. Schuske and E. Jorgensen, personal communication). We found GABA levels were also 3-4 fold lower in these mutants further supporting UNC-46's role in regulating transport of GABA into synaptic vesicles. This technique can also be used to quantitate neurotransmitter levels in whole worm extracts. For example, unc-25 animals, which are missing the enzyme glutamic acid decarboxylase, have significantly decreased GABA levels in their extracts compared to N2 animals. We currently are focusing our attention on obtaining a purer vesicle preparation to reduce the levels of GABA we see in the unc-47 mutants. Once established, this technique can be used to characterize and identify genes involved in the function and regulation of vesicular transporters in C.elegans .

Crowell T et al. (1995) International C. elegans Meeting "SYNAPTOTAGMIN-DEFICIENT (SNT-1) MUTANTS OF C. ELEGANS: MOLECULAR AND PHENOTYPIC ANALYSIS"

Crowell T, Rand JB, McManus JR

[International C. elegans Meeting, 1995]

Synaptotagmin is a major synaptic vesicle protein; it contains 2 apparent calcium-binding (PKC-CII) domains and seems to be involved in both exocytosis and endocytosis. The cloning of snt-1 and the isolation of mutants were reported by Nonet et al. (1993). We have now analyzed 17 independent spontaneous snt-1 mutations isolated in our laboratory, as well as alleles isolated by Leon Avery and Erik Jorgensen. Molecular analysis indicates that 12 of the alleles are associated with DNA rearrangements (3 Tc1 insertions, 1 non- Tc1 insertion and 8 deletions), with the remaining alleles due to point mutations. Some of the deletions provide guaranteed molecular null alleles; null mutants are quite uncoordinated, but viable. Phenotypic analysis of snt-1 mutants included quantitative behavioral studies (pharyngeal pumping and head thrashing) and immunoblots. Several of the mutants clearly retain some gene function, and it was possible to establish an alleleic series of phenotypic severity. Partial gene function is retained not only by some of the point mutants, but also by a 66-bp (inframe) deletion which eliminates most of the first CII domain, and by a 9-bp deletion which disrupts the second CII domain. Supported by a grant from MDA

Edwige Martin et al. (2003) International Worm Meeting "Analysis of Mos insertions as a preliminary step for a genome-wide approach"

Edwige Martin, Laure Granger, Laurent Segalat

[International Worm Meeting, 2003]

Tc transposons have been used extensively for both forward and reverse genetics purposes and the mechanisms of transposition have been well characterized. However, the use of endogenous Tc elements to generate large-scale libraries of mutants has intrinsic limitations which can be overcomed by using non-C. elegans transposons. We are studying the feasibility of using the Mos transposon to generate a comprehensive library of transposon-tagged genes. As a pilot experiment, we generated approximately 1000 random insertions using the Mos system developped by the Bessereau and Jorgensen labs. Since Mos is not naturally present in the genome of C. elegans, its detection is much easier than that of Tc novel insertions. Localization of insertions is determined by amplying the transposon flanking region by inverse PCR on lysed worms. Sequencing of the PCR product and comparison with the genome sequence then reveals the exact location of the insertion. We will present the analysis of our pilot study and particularly address the questions of chromosome distribution, exon/intron/intergenic distribution, potential bias due to hot spots, and mutagenic properties of the insertions. The insertions are available to the scientific community. Requests for strains should be addressed to segalat@cgmc.univ-lyon1.fr

Meghan Jobson et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Characterization of an Excitatory GABA Receptor"

Erik Jorgensen, Meghan Jobson, Asim Beg

[Neuronal Development, Synaptic Function, and Behavior Meeting, 2006]

Ionotropic GABA (gamma;-aminobutyric acid) receptors are GABA-gated chloride channels. The binding of GABA to these receptor results in a chloride current that usually hyperpolarizes the postsynaptic cell. In C. elegans the GABA-gated chloride current mediates behaviors such as locomotion and foraging. More recently it has been found C. elegans also contains a GABA-gated cation channel encoded by exp-1 (Beg and Jorgensen, 2001). EXP-1 is expressed in the enteric muscles and the EXP-1 GABA-gated cation current mediates enteric muscle contraction during defecation. We identified Y46G5A.26 as a possible relative of EXP-1 based on sequence homology. Expression of Y46G5A.26 in Xenopus oocytes results in a GABA-gated cation current, demonstrating that it is an excitatory GABA receptor. Surprisingly, expression of a GFP reporter construct indicates that Y46G5A.26 is expressed in the GABA neurons and not in the targets of GABA neurons as expected. A knockout allele was obtained from the Mitani lab. No gross defects in locomotion, defecation, or foraging were noted. Based on expression in the GABA neurons and lack of a severe phenotype we propose that Y46G5A.26 is acting as an autocrine receptor to upregulate GABA neuron function in C. elegans. We are currently testing this hypothesis by evaluating locomotion and defecation behavior at a closer level using video tracking and high-speed video analysis.