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[
East Coast Worm Meeting,
1998]
We have extended recently described methods for in situ patch-clamp recording from C. elegans neurons (1, 2) to permit recording in adult animals. The main improvement is the use of a fixed-stage, upright microscope mounted on an x-y translation stage. In this way, worms can be visualized from above using a high N.A. water immersion objective (Zeiss 63X/0.9 or 100X/1.0). This arrangement gives superior optics compared to viewing worms on an inverted microscope and makes it possible to expose neurons in adult animals. In addition, methods for exposing neuronal cell bodies in the head were modified to expose neuronal cell bodies in the tail for in situ patch-clamp recording. With this apparatus, we plan to record the response of PLM cells to light touch. 1. Lockery, S. R. & Goodman, M. B. (1998) Tight-seal whole-cell patch clamping of C. elegans neurons. Methods in Enzymology (in press). 2. Goodman, M. B., Hall, D. H., Avery, L. & Lockery, S. R. (1998). Active currents regulate sensitivity and dynamic range in C. elegans neurons. Neuron (in press).
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[
International Worm Meeting,
2005]
AMP kinase is a highly conserved sensor of cellular and systemic energy status in eukaryotes. AMP kinase is activated by stressors that increase the cellular AMP:ATP ratio such as osmotic shock, hypoxia, oxidative damage, glucose deprivation, and exercise. AMP kinase can also be activated by the synthetic AMP analogue, 5 aminoimidazole-4-carboxamide riboside (AICAR). When activated, AMP kinase phosphorylates downstream signals that up-regulate ATP producing pathways and down-regulate ATP consuming pathways. The downstream effects of activated AMP kinase include both the regulation of cell-cycle progression and the maintenance of energy stores by increasing fatty acid oxidation and muscle glucose transport via distinct mechanisms. To understand the role of AMP kinase in C. elegans, we used AICAR to activate AMP kinase. We show that the addition of AICAR results in growth suppression and a rapid and reversible fat reduction. Using RNA inactivation we show that these effects are mediated through the catalytic subunit of AMP Kinase. Worms exposed to RNAi of the AMP kinase catalytic subunit are resistant to the effects of AICAR. Furthermore, our data confirm that the critical components of the AMP kinase signaling cascade to fatty acid oxidation are conserved in C. elegans. Using AICAR and RNA inactivation, we are currently exploring novel components of the AMP kinase signaling pathway.
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[
International Worm Meeting,
2015]
A pair of ASE chemosensory neurons, ASEL and ASER, are major salt sensors, and play critical roles in chemotaxis to NaCl. Calcium imaging has previously revealed that ASEL and ASER are activated by an increase and decrease in NaCl concentrations, respectively (Suzuki et al., 2008; Ortiz et al., 2009). These asymmetric responses by ASEL and ASER to changes of NaCl concentrations are crucial to efficient chemotaxis of C. elegans toward higher concentrations of NaCl. While Goodman et al. (1998) reported in situ whole-cell patch-clump recording of ASER, electrophysiological characterisation of ASE neurons is still required to understand how the neurons respond to the NaCl concentration changes.Toward the goal, we have investigated electrophysiological properties of ASE neurons in wild-type C. elegans by in vivo whole-cell patch-clamp recordings, and have found that both of ASE neurons showed resting membrane potentials of approximately -60 mV and membrane resistances of about 2 Gomega. In both of ASE neurons, voltage responses to current injections showed solitary action potentials. Depolarization of wild-type ASEL was observed when a puff of 150 mM NaCl was applied to the animal's nose in bath solution containing 50 mM NaCl. On the other hand, a puff of NaCl-free buffer induced ASER depolarization. These results are consistent with those of calcium imaging. To understand roles of the action potentials in ASE, we are currently trying to analyse electrophysiological properties of ASE neurons in various mutants.References1. Suzuki et al., Nature 454: 114-118 (2008)2. Ortiz et al., Current Biology 19: 996-1004 (2009)3. Goodman et al., Neuron 20: 763-772 (1998).
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[
International Worm Meeting,
2019]
Animals change their locomotion or gaits in response to environmental condition. In vertebrates, gait transition has been shown to be mediated by monoamines, which is conserved across many species including the nematode Caenorhabditis elegans (Vidal-Gadea et al., 2011). However, molecular mechanisms of gait transition are still unclear. C. elegans exhibits two gaits, swimming in liquids and crawling on dense gels. C. elegans genome contains evolutionarily conserved 28 DEG/ENaC channels, of which functions may be involved in mechanosensory transduction and locomotion (Goodman and Schwarz, 2003). We first hypothesized that mechanosensitive channels could act as a gait transition initiator and examined crawl-to-swim transition phenotype in DEG/ENaC mutants. We found that while
acd-5 mutants show normal crawling, transition from crawling to swimming upon liquid exposure is defective, suggesting roles of ACD-5 in gait transition. We are currently generating
acd-5 rescue lines and examining expression pattern of
acd-5.
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[
International Worm Meeting,
2007]
The stomatin-related protein MEC-2 and the paraoxonase-related protein MEC-6 are required for mechanical activation of native force detection channels in C. elegans touch receptor neurons [1]. Prior work indicates that such channels contain at least two pore-forming degenerin subunits, MEC-4 and MEC-10. Both MEC-4 and MEC-10 belong to the DEG/ENaC superfamily of amiloride-sensitive Na+ channel subunits. Wild-type copies of all four proteins are needed for touch sensation in vivo. Co-expressing MEC-2 and MEC-6 with constitutively-active isoforms of MEC-4 and MEC-10 in Xenopus oocytes leads to a dramatic and synergistic increase in amiloride-sensitive whole-cell Na+ current. This effect is not explained by a MEC-2- or MEC-6-dependent increase in the amount of MEC-4 or MEC-10 protein in the plasma membrane [2, 3], suggesting that MEC-2 and MEC-6 alter the activity of individual channels in the plasma membrane. To test this, we analyzed single, amiloride-sensitive Na+ channels in membrane patches drawn from oocytes expressing MEC-4 and MEC-10 in the presence or absence of MEC-2, MEC-6 or both MEC-2 and MEC-6. We found that while MEC-2 coexpression leads to a modest, but significant increase in single channel conductance, <font face=symbol>g</font>, the effect is too small to explain the MEC-2-dependent increase in whole-cell current. Otherwise, <font face=symbol>g</font> and steady-state open probability (P<sub>o</sub>) are not significantly altered by the presence or absence of MEC-2 and MEC-6. We conclude that, in the absence of MEC-2 and MEC-6, the vast majority of channels occupy a non-conducting state despite correct expression at the plasma membrane. 1.O''Hagan, R., M. Chalfie, and M.B. Goodman, Nat Neurosci, 2005. 8(1):43 2.Chelur, D.S., et al., Nature, 2002. 420(6916):669 3.Goodman, M.B., et al., Nature, 2002. 415(6875):1039.
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[
East Coast Worm Meeting,
1998]
We are testing a number of biocontrol bacteria (eg. Burkholderia, Pseudomonas and Stenotrophomonas) for toxicity to bacterial feeding nematodes including C. elegans N2 and
pgp-3. Serratia marcescens in particular had a measurable effect on number of eggs over 24 hours and relative motility in microtiter wells. Serratia marcescens strains with increasing amounts of red pigmentation (BF1-5@ (albino), D1, N4-5, NIMA) were tested for relative motility to C. elegans N2,
pgp3, Oscheius myriophila DF5020, Panagrellus redivivus PS 1163, Mesorhabditis sp. PS1170 and Zeldia punctata PS 1153. Toxicity directly increases with decreasing amount of pigment in Caenorhabditis, Oscheius and Panagrellus. The order of relative toxicity is reversed for the 2 highly pigmented strains and the 2 least pigmented strains (2143 vs. 1234) in Mesorhabditis and Zeldia. These nematodes are phylogenetically distant from C. elegans. Bacterial strains were provided by Phyllis Martin and Dan Roberts, USDA.
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[
International Worm Meeting,
2013]
It is time-consuming to manually quantify C. elegans phenotypes in lifespan, locomotion, body size, and egg laying We developed an automated image acquisition and analysis system to quantify multiple C. elegans phenotypes. The imaging system is composed of a microscope equipped with a digital camera, a motorized stage, and image analysis software. The image analysis software package contains Lifespan Assay, Locomotion Assay, WormLength Assay, and Egg Counter software. The Lifespan Assay software counts the number of moving worms using two time-lapse images. Inspired by the Parallel Worm Tracker developed by the Goodman lab, the Locomotion Assay software conducts fully automated video analysis and computes the velocity of moving worms. The WormLength Assay and Egg counter software have been developed for body size measurement and automated egg counting, respectively. This software suite can run on any operating system since they were written in Java. We evaluate the performance of the software by various benchmarks. We also demonstrate the application of the software in a pilot chemical screen.
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[
International Worm Meeting,
2009]
Animals increase their pirouette frequency in response to removal from food stimulus for a period of 15 min. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY and AIA interneurons inhibit pirouettes (Wakabayashi et al 2004, Gray et al 2005). We have found that AWC sensory neurons become active in response to removal of stimulus, releasing two neurotransmitters (glutamate and a neuropeptide NLP-1). The released glutamate acts to activate AIB and inhibit AIY interneurons, promoting reversals (Chalasani et al 2007). In contrast to glutamate, AWC-released NLP-1 acts on AIA interneurons to suppress reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. AWC calcium responses are modulated in these neurotransmitter mutants, suggesting that feedback pathways affect AWC neuronal activity. References: Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., and Bargmann, C. I. (2007). Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70. Gray, J.M., Hill, J.J., and Bargmann, C.I. (2005). A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.
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Yen, Jessica, Tan, Trudy, Jin, Suying, Zadoorian, Arbi, Arisaka, Katsushi, Huang, Rebecca, Narain, Shreya, D'Orazio, Etta, Yamada, Mandi, Mai, Phat, Park, Jane, Yang, Karen, Carmona, Javier, Kim, Ted, Liu, Junliang, Mendoza, Steve, Watson, Sonya
[
International Worm Meeting,
2017]
Caenorhabditis elegans exhibit a well-characterized host of thermosensory behaviors necessary for efficient navigation, foraging, and survival in the natural environment. Many of the neural structures responsible for temperature sensing have evolved to exceptional sensitivity over time, giving rise to cognitively complex, deterministic behaviors such as bias orientation and 90 degree turning. In order to better comprehend the underlying neural circuitry responsible for such behaviors, multiple PID-controlled hardware systems have been constructed to generate and control various thermal gradient conditions within 0.05 degrees C, over durations of ~ 30 minutes. Additionally, an IR laser-based thermosensory system has been fabricated, to provide spatiotemporally controlled thermal stimulus at a highly localized region of the worm's body, enabling the establishment of a virtual thermal environment on which the worm can behave. Using these tools, we have investigated several intricacies of C. elegans' response to temperature, including the mechanism governing 90 degree turns and biased orientation during negative thermotaxis. Making use of line confocal calcium imaging microscopy methods, the dynamics of the AFD sensory neuron and the AIY interneuron were observed, yielding multiple noteworthy datasets. Custom-written MatLab image processing tools based on Goodman and NEMO were utilized to conduct a systematic motional analysis in a variety of experimental thermal conditions. This poster aims to outline the some of these recent advancements in thermotaxis investigation in the Elegant Mind Club, along with preliminary supporting datasets, on a case-by-case basis.
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[
West Coast Worm Meeting,
2000]
In C. elegans, chemotaxis to the attractant NaCl is largely controlled by a pair of left-right homologous chemosensory neurons ASER and ASEL. Although the two neurons are bilaterally symetric in their morphology and pattern of connectivity , recent work argues for functional asymetry between these neurons in chemotaxis (Pierce-Shinomura et al., abstr. WCWM). Laser ablation of ASER greatly reduces chemotaxis to Cl- but not Na+; conversely, ablation of ASEL greatly reduces chemotaxis to Na+ but not Cl-. To determine if this functional asymetry reflects a difference in the ionic currents expressed in the two neurons, we made whole-cell voltage clamp recordings fron ASEL (n=20) for comparison with previous recording from ASER (Goodman et al., Neuron 20: 763-72, 1998). We found that ASEL is similar to ASER in three main respects. First, ASEL exhibits an outward current activated by depolarisation (-30 to 100 mV) and an inward current activated by hyperpolarisation below -70 mV. Little or no current is activated in the region between -70 and -30 mV. Second, the outward current comprises inactivating and sustained components which are probably carried by K+ ions, because both components are eliminated by substitution of N-methyl-glutamine (NMG) for K+ in the recording pipette. Third, outward current inactivation is voltage-dependent, because prepulses more positive than -70mV decreases peak amplitude of the outward current. These results suggest that the functional asymetry reflect differences in the response to chemical stimuli rather than differences in voltage dependent ionic current. Support: NIMH 51383 and NSF IBN-9458102.