Bruce A Bamber et al. (2005) International Worm Meeting "Homeostatic regulation of the UNC-49 GABAA receptor"

Janet E Richmond, Aaron M Rowland, Alice Benham, Bruce A Bamber

[International Worm Meeting, 2005]

Inhibitory synaptic strength depends on the density of postsynaptic GABAA receptors. Plasticity of postsynaptic GABAA receptor density contributes to homeostatic regulation in the nervous system, and is associated with disease states such as epilepsy and anxiety. To understand the mechanisms that control GABAA receptor density at synapses, we are studying the inhibitory neuromuscular junction in C. elegans. When exposed to the GABA agonist muscimol, worms initially become paralyzed, but then adapt. Adapted worms have a shrinker Unc phenotype characteristic of worms that lack GABA neurotransmission. We hypothesized that adaptation is the result of downregulation of GABAA receptors at the neuromuscular junction, which are encoded by the unc-49 gene. We tested this hypothesis by comparing GABA currents in naïve and muscimol-adapted worms. GABA currents were six-fold reduced in amplitude in adapted animals. Using quantitative immunofluorescence, we demonstrated that UNC-49 abundance at the synapse was reduced nine-fold. A transcriptional mechanism does not appear to be involved: quantitative RT-PCR analysis demonstrated that unc-49 mRNA levels were reduced, but only transiently. unc-49 mRNA returned to its pre-exposure level before the majority of the protein was lost. Using the immunofluorescence assay, we are characterizing protein trafficking and cell signaling mutants to understand the mechanism of this homeostatic regulation of the UNC-49 GABAA receptor.

Bruce A Bamber et al. (2005) International Worm Meeting "Identification of residues that confer neurosteroid sensitivity on the UNC-49 GABAA receptor"

Bryan Wardell, Bruce A Bamber, Purba S Marik, Erik M Jorgensen

[International Worm Meeting, 2005]

We are using the C. elegans GABAA receptor, encoded by unc-49, to study the structural basis of neurosteroid action. Neurosteroids are endogenous steroid molecules that control many aspects of mammalian nervous system function, principally by modulating GABAA receptors. Attempts to identify residues involved in neurosteroid modulation using standard domain swap approaches have been unsuccessful to date because there is little variation in neurosteroid sensitivity among mammalian GABAA receptors. UNC-49 provides a solution to this problem. The unc-49 gene encodes two subunits, UNC-49B and UNC-49C, by alternative splicing. Normally these subunits coassemble to form a heteromultimer, which is highly sensitive to the inhibitory neurosteroid pregnenolone sulfate. However UNC-49B also forms a homomultimer which is insensitive to pregnenolone sulfate. By swapping segments of UNC-49C in to UNC-49B, followed by site-directed mutagenesis, we have identified seven important residues. When mutated together, these residues produce a 58-fold increase in pregnenolone sulfate sensitivity. These mutations have little effect on other allosteric regulators, suggesting they may be specific for neurosteroids. The residues cluster in regions important for channel gating, suggesting that pregnenolone sulfate binding changes how key gating residues are positioned, or move during GABA activation, resulting in decreased channel function.

Debnath, Arunima et al. (2021) International Worm Meeting "Neuromodulation and the relationship between Ca2+ transients and neuronal states"

Bamber, Bruce, Debnath, Arunima

[International Worm Meeting, 2021]

Neuromodulators (monoamines and neuropeptides) shape nervous system function by regulating neuronal depolarization and synaptic strengths. We are studying neuromodulation in the context of nociception, to better understand pain perception and pain treatment strategies. The ASH neuron is a major nociceptor in C. elegans. ASH senses 1-octanol and drives an aversive response, modulated by the monoamines serotonin (5-HT, potentiating) and octopamine (OA, inhibitory). To better understand neuromodulation, we are focusing on 1-octanol stimulated Ca2+ dynamics in ASH, and the quantitative relationship between Ca2+ signals and depolarization amplitudes. We showed that 5-HT potentiates ASH depolarization, but surprisingly, inhibits ASH Ca2+ transient amplitudes. These effects, like the 5-HT stimulation of behavior, depend on the SER-5 receptor in ASH. This paradoxical finding is explained by existence of a Ca2+-dependent inhibitory feedback loop: Ca2+ inhibits ASH depolarization through SLO-1 IKCa channels, and 5-HT inhibits EGL-19 L-type Ca2+ channels in ASH (via SER-5), thus disinhibiting the neuron. We are currently investigating modulation of 1-octanol responses by OA. OA inhibits 1-octanol behavioral responses, and antagonizes 5-HT potentiation, dependent on the OCTR-1 receptor in ASH. OA also inhibits ASH Ca2+ transients, representing another paradox: how can OA and 5-HT have opposite effects on ASH-dependent aversive behaviors, but the same effect on ASH Ca2+ transients? Our results show that 5-HT and OA utilize distinct signaling pathways in ASH (Galphaq for 5-HT and Galphao for OA), and that OA does not inhibit EGL-19. We are currently testing the hypothesis that OA hyperpolarizes ASH through Galphao-dependent activation of IRK K+ channels. These results further emphasize that neuronal Ca2+ transients, as key reporters of neuronal depolarization, are also critical signaling intermediates in and of themselves, with multiple upstream inputs and downstream consequences.

Zahratka, Jeffrey et al. (2013) International Worm Meeting "Multiple independent calcium pools in a nociceptive neuron in C. elegans."

Bamber, Bruce, Komuniecki, Richard, Zahratka, Jeffrey, Williams, Paul

[International Worm Meeting, 2013]

To navigate constantly changing environments, animals integrate sensory inputs and internal state information to formulate proper locomotory commands. Monoamines and neuropeptides encode internal state information, modulating sensorimotor circuits at several levels, including the sensory neurons themselves. We study aversive behaviors in C. elegans to better understand how neuromodulators affect circuit function, in terms of the physiological states of single identifiable neurons. C. elegans reacts to the noxious odorant 1-octanol by reversing locomotory direction, to move away from the odor source. Worms off food respond to diluted 1-octanol in 10s, but in the presence of food, response time shortens to 5s. Endogenous 5-HT and neuropeptides are key signaling intermediates in this food-dependent modulation, relying in part on a 5-HT receptor (SER-5) in the ASHs (the pair of nociceptive neurons that sense 1-octanol). Using the GCaMP3 Ca++ indicator, we show that a saturating concentration of 1-octanol dissolved in buffer (~2 mM) elicits robust excitation of ASHs when applied to the nose. At least 3 distinct Ca++ pools exist in ASHs: distal dendrite, proximal dendrite/soma, and axon. Proximal dendrite/soma Ca++ responses are sensitive to Nemadipine-A, a blocker of L-type Ca++ channels. Distal dendrite responses, and more importantly, axonal responses are resistant to Nemadipine-A. This finding suggests that somal Ca++ influx is unnecessary for signal propagation from one end of the cell to the other, consistent with isopotentiality within C. elegans sensory neurons. What then is the role of somal Ca++ influx? Rather than amplifying or relaying chemosensory signals to axons, we hypothesize it is intermediate in sensory modulation, and in support, monoamine signals regulating aversive behavior also regulate ASH somal Ca++ signals. We are currently testing whether the different Ca++ pools within ASHs are the result of regionalized expression of Ca++ channels, characterizing the monoamine and neuropeptide signaling cascades affecting ASH somal Ca++, and elucidating the roles for ASH somal Ca++ by direct measurement of ASH synaptic release onto postsynaptic targets and behavioral assays.

Summers, Philip et al. (2015) International Worm Meeting "Modulation of glutamatergic signaling in C. elegans."

Komuniecki, Richard, Summers, Philip, Ortega, Amanda, Bamber, Bruce

[International Worm Meeting, 2015]

The glutamatergic ASH sensory neurons are essential for the initiation of aversive responses to dilute 1-octanol. To identify and localize the glutamate receptors involved, we screened animals with null alleles for each of the previously identified or predicted glutamate-gated cation and anion channel subunits. Not surprisingly, given the array of ASH downstream synaptic partners, a number of the glutamate receptor mutants yielded phenotypes in various aspects of the modulated ASH mediated aversive responses. For example, the initiation of aversive responses in animals with null alleles for either avr-14 or glc-4, predicted to encode glutamate-gated Cl- channel subunits, was not stimulated by food or 5-HT, compared to wild type animals. In contrast, animals overexpressing either avr-14 or glc-4 initiated aversive response more rapidly than wild type animals off food. AVR-14 has been characterized extensively and encodes a homomeric glutamate-gated Cl- channel. In contrast, GLC-4 is the most distantly related of the predicted glutamate-gated Cl- channel subunits and has not been characterized. Therefore, GLC-4 was expressed heterologously in Xenopus oocytes to determine ligand-specificity and over the 100 ligands were examined. None activated a Cl- current in the oocytes. In contrast, GLC-4 expression with AVR-14 or AVR-15, well characterized glutamate-gated Cl- channel subunits, reduced peak Cl- currents and glutamate affinity, suggesting the GLC-4 interacted with these subunit to alter channel properties. This result appeared to be specific for glutamate-gated Cl- channels, as GLC-4 co-expression with MOD-1 that forms a 5-HT dependent Cl- channel had no effect on MOD-1 signaling. These studies are continuing to localize AVR-14 and GLC-4 in the ASH-mediated locomotory circuit at both the cellular and subcellular levels to determine their roles in ASH signaling and, more importantly, to determine if they associate in vivo.

Sujkowski, Alyson L. et al. (2009) International Worm Meeting "Homeostatic regulation of GABA neuromuscular synapses."

Moraitis, Stavros, Mohammed, Nizar, Sujkowski, Alyson L., Bamber, Bruce A., Davis, Kathleen M., Sturt, Brianne L.

[International Worm Meeting, 2009]

Modulation of GABA synapse function affects the excitation-inhibition balance in neural circuits, and plays a significant role in human brain function. The GABA neuromuscular synapse in C. elegans is a simple, genetically accessible model to study this modulation. The GABA receptor in muscle is encoded by UNC-49, a ligand-gated chloride channel homologous to the mammalian GABAA receptor. Postsynaptically, GABA neuromuscular junctions exhibit two forms of homeostatic regulation. First, exposure to the GABA agonist muscimol causes flaccid paralysis. Worms eventually adapt to muscimol, adopting the shrinker phenotype typical of GABA-defective mutants. Adaptation is due to a 6-10 fold reduction of GABA receptor abundance, and a proportional reduction in muscle GABA sensitivity, caused by increased GABA receptor trafficking to the lysosome. Levamisole responses are unaffected by muscimol exposure, suggesting that this plasticity is specific for GABA receptors. Second, GABA receptor levels are reduced in twk-18(cn110) mutants. TWK-18 is a twin-pore potassium channel expressed in body wall muscles that exhibits steep temperature dependence of activity. cn110 is a gain-of-function allele with proportionally higher K+ currents across the full temperature range. twk-18(cn110) mutants move normally at 20 deg C, but show flaccid paralysis at 30 deg C, presumably because the elevated K+ currents interfere with muscle contraction. GABA receptor levels are reduced 2-3 fold compared to wild type in twk-18(cn110) mutants raised at 20 deg C and 25 deg C, but not at 15 deg C, possibly as compensation for elevated muscle K+ conductance at the higher temperatures. These two forms of plasticity suggest that receptor activation, chloride influx, and membrane excitation state can influence postsynaptic GABA receptor expression to establish and maintain appropriate cellular excitability. We are using pharmacological and genetic approaches to independently manipulate these parameters to better understand how GABA receptor abundance is controlled. We are also using microarrays and RNA interference screening to identify the relevant signaling pathways. Analysis of muscimol-treated worms also suggests that presynaptic GABA release may be modulated as well. Upon removal of muscimol, locomotion recovers several hours before muscle GABA responsiveness recovers, consistent with suppression and relatively fast recovery of presynaptic GABA release. We are presently characterizing the structure and function of presynaptic GABA neurons in muscimol-treated and twk-18(cn110) worms, to test the extent to which presynaptic and postsynaptic aspects of GABA synapse function are coordinately regulated.

Sabrina G C Shore et al. (2002) West Coast Worm Meeting "The temperature sensitive lethal mutation or566 may define a new gene that functions in early morphogenesis"

Andrew D Chisholm, Sarah L Moseley, Sabrina G C Shore

[West Coast Worm Meeting, 2002]

Mutations in the EPH receptor tyrosine kinase VAB1 and in its ephrin ligands cause incompletely penetrant defects in gastrulation cleft closure and epidermal enclosure. Fully penetrant defects in cleft closure are seen when multiple pathways are blocked (e.g. vab-1 and ptp-3 (Harrington et al., 2002, Development 129: 2141). To find genes that may have essential functions in gastrulation cleft closure we have begun to examine conditional lethal mutants for possible defects in this process. Bruce Bowermans lab identified several new temperature-sensitive (ts)-lethal mutations affecting morphogenesis in their large scale ts-lethal screens, and very kindly provided some of them to us.

Mendel, Jane et al. (2015) International Worm Meeting "WormBook News."

Harris, Todd, Mendel, Jane, Chalfie, Martin, Sternberg, Paul, Wang, Qinghua, Hobert, Oliver

[International Worm Meeting, 2015]

WormBook (http://www.wormbook.org/) is a comprehensive, open-access collection of original peer-reviewed chapters covering the biology of C. elegans and other nematodes. WormBook also includes WormMethods, which contains protocols ranging from the basics of C. elegans culture to advanced imaging techniques, WormHistory, which contains personal recollections of the early days of nematode research, and the Worm Breeder's Gazette, an informal newsletter. We are continually updating the older chapters in WormBook and WormMethods, as well as adding chapters on new topics. For example, we will shortly add several WormMethods chapters describing aspects and functions of WormBase.We are very happy to announce the addition of an Introduction to WormBook: "A Transparent Window into Biology: A Primer on Caenorhabditis elegans", by Ann Corsi, Bruce Wightman, and WormBook editor-in-chief, Marty Chalfie, is scheduled to be copublished by Genetics and WormBook in June.Additional developments include listing WormBook chapters in PubMed Central and upgrading the search feature of WormBook, WormMethods, and The Worm Breeder's Gazette.We welcome your feedback and comments on how we can improve WormBook, WormMethods, and The Worm Breeder's Gazette to make them more useful to you, the worm community.

Topper, Stephen M. et al. (2013) International Worm Meeting "Ethanol induces state-dependent behavioral transition."

Vidal-Gadea, Andres, Pierce-Shimomura, Jonathan, Topper, Stephen M., Aguilar, Sara, Young, Layla

[International Worm Meeting, 2013]

Alcohol, the most widely abused drug in the world, has a wide range of effect on individuals and there are many factors that predispose one to addiction. Chief among them are stress, depression, and anxiety, all of which include altered monoamine signaling. It remains unclear how behavioral states differentially influence responses to ethanol. C. elegans displays distinct behavioral states associated with locomotion (crawling and swimming) that are mediated by dopamine and serotonin respectively. We found that ethanol-induced responses in C. elegans depended on crawl or swim behavioral state. Whereas alcohol non-specifically impaired locomotion and feeding in crawling worms on land, alcohol induced a specific transition from swim to crawl state in worms submerged in ethanol. This response was dependent on dopamine. Loss of the D1-like dopamine receptor DOP-4 impaired the EtOH-induced transition to crawl, while optogenetic inhibition of DOP-4 receptor-expressing neurons via the Arch chloride pump recovered the swim state. These results suggest that alterations of dopamine-dependent behavioral states can drastically influence EtOH-induced behaviors with implications for prevention of addiction. Acknowledgements: CGC and the Knockout Consortium for strains, Bruce/Jones Award for Alcohol and Addiction Research, Karl Deisseroff for reagents.

RV Aroian et al. (1999) International C. elegans Meeting "Using C. elegans to Teach Embryonic Development to Undergraduates"

G Wienhausen, RV Aroian, D Johnson

[International C. elegans Meeting, 1999]

We have begun to use C. elegans as a model system in our undergraduate embryology laboratory class at the University of California, San Diego. In the past, this 10 week class has used Sea Urchin, Xenopus, Chick, and other non-genetic systems to demonstrate to students embryonic development. In this poster we will outline the 6 new weeks of C. elegans modules. These modules allow the students to use advanced techniques to explore widely applicable principals of embryonic development. These techniques include wild-type lineage analysis, mutant lineage analysis, use of specific inhibitors to alter embryonic development, immunofuorescence analysis of wild type and mutants using antibodies to various cell biology and developmental markers, genome searching, and knocking out genes using RNA-mediated interference. Our goal is to one day publish an article on our results in the Worm Breeder's Gazette. The embryonic development of more advanced model organisms such as Xenopus and chick are later used to compare and contrast to C. elegans. This work is greatly enhanced by the cooperativity of the C. elegans community in the sharing of antibodies, mutants, and other reagents such as GFP strains. Last year we used protocols, stains, and antibodies from to Geraldine Seydoux, Craig Mello, Pete Okkema, Michel Labouesse, Bruce Bowerman and the CGC. Thanks to them and others that have generously shared their reagents.