Bowerman BA et al. (1994) Worm Breeder's Gazette "The Maternal Gene skn-4 and the Specification of Ventral Blastomere Fates in the Early C. elegans Embryo"

Bowerman BA, Shelton CA, Thorpe CJ, Martin PR

[Worm Breeder's Gazette, 1994]

THE MATERNAL GENE SKN-4 AND THE SPECIFICATION OF VENTRAL BLASTOMERE FATES IN THE EARLY C. ELEGANS EMBRYO Bruce Bowerman, Paula R. Martin, Christopher J. Thorpe, and Christopher A. Shelton. The Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

Wirak, Gregory et al. (2021) International Worm Meeting "Age-related decline of neuronal function is linked to a loss of inhibitory signaling in C. elegans"

Wirak, Gregory, Gabel, Christopher, Alkema, Mark, Connor, Christopher, Florman, Jeremy

[International Worm Meeting, 2021]

Neuronal function declines with age resulting in behavioral deficits. How this age-related decline is related to changes in neuronal connectivity and circuit function on the cellular level is largely unknown. Here we employ multi-neuronal fluorescence imaging in C. elegans to measure changes in neuronal activity and connectivity with age across the animal's nervous system. Employing transgenic expression of the fluorescent calcium indicator GCaMP6s in specific neurons, we identify changes in the activity of individual neurons and circuits linked to locomotory behaviors that are modulated by age. We further perform multi-neuronal imaging, employing light sheet microscopy to capture neuronal activity at single-cell resolution across the organism's entire head region. We identify substantial age-related alterations in the activity dynamics of neurons as well as changes in connectivity and functional organization. As the animals age, we observe a loss of system-wide organization and a corresponding shift in individual neuron activity toward higher frequencies. We also observe a specific loss of anti-correlative (i.e. inhibitory) signaling between neurons, resulting in an overall shift in the excitatory-inhibitory (E/I) balance of the system. In support of this, we find that application of the GABAA agonist muscimol diminishes certain aspects of nervous system decline in aged animals. Interestingly, age-related effects are recapitulated in young animals bearing a gain-of-function mutation within the unc-2 gene, encoding a pre-synaptic voltage-gated calcium channel. Conversely, aging effects are greatly diminished by loss-of-function mutations in either unc-2 or in ced-4. Ced-4 is a key mediator of the conserved apoptotic cell-death pathway and is known to play a role in synaptic elimination across multiple species. During development, UNC-2/CaV2 activity is known to trigger the removal of inhibitory GABAergic synapses in motor neurons through a CED-4 dependent pathway. Our results suggest that a similar process is occurring as the animals age, resulting in a loss of inhibitory signaling and disruption of the system dynamics. Through comprehensive multi-neuronal imaging, we are able to measure the progressive breakdown of neuronal activity and system dynamics with age and begin to identify the cellular processes and changes in synaptic signaling that contribute to this decline.

Chang, Andrew et al. (2021) International Worm Meeting "Imaging neuronal dynamics during recovery from anesthesia in C. elegans"

Wirak, Gregory, Gabel, Christopher, Awal, Mehraj, Chang, Andrew, Connor, Christopher

[International Worm Meeting, 2021]

The innovation of anesthesia with volatile anesthetic agents has made modern surgical practice possible. The anesthetized state is defined by behavioral criteria and is today largely monitored through physiological measurements such as hemodynamics and gas exchange. These measurements are sometimes supplemented by EEG derived metrics, but the relationship between these metrics and the neurophysiological mechanisms of anesthesia is entirely unknown. We have used cellular resolution multi-neuronal functional imaging in C. elegans as a powerful model for bridging the gap between single-neuron activity and whole nervous system dynamics under anesthesia. We previously found that the anesthetic state in C. elegans corresponds to a loss of synchrony in global neural dynamics as opposed to generalized depression of individual neuron activity. Here we extend this work by examining how the nervous system of C. elegans recovers from the anesthetic state of dissynchrony to normal function. Employing a light-sheet diSPIM microscope we can measure the activity of the majority of neurons in the C. elegans head as the animal emerges from anesthesia. We observe that correlation in activity between neuron pairs, which is significantly suppressed in the anesthetized state, recovers to near pre-exposure levels in a non-linear, cyclical manner over several hours. Notably, the time required for this recovery is significantly longer then the time needed to induce the neurological anesthetic state. This finding recapitulates the concept of "neural inertia", a proposed hysteresis in the processes of anesthesia induction and emergence well-characterized in humans by the observation that emergence from anesthesia tends to occur at lower concentrations of drug than are required for induction. Because C. elegans perform gas exchange through diffusion, the differences we observe in time needed for induction and emergence are consistent with the phenomenon characteristic of neural inertia in humans. This finding further demonstrates the utility of C. elegans as a model system for investigating the effects of anesthesia on neuronal function and the physiological basis of those effects.

Christopher J Cronin et al. (2005) International Worm Meeting "The Combined UC San Diego/Caltech Quantitative Behavior Analysis System"

William R Schafer, Zhaoyang Feng, Kuang-man Huang, Paul W Sternberg, Christopher J Cronin

[International Worm Meeting, 2005]

California Institute of Technology and University of California, San Diego have each developed practical, automated movement analysis systems to quantify aspects of worm behavior and morphology (see An imaging system for standardized quantitative analysis of C. elegans behavior, Zhaoyang Feng, et al, BMC Bioinformatics 2004, 5:115, An automated system for measuring parameters of nematode sinusoidal movement, Christopher J Cronin, et al, BMC Genetics 2005, 6:5, and previous meeting abstracts). We have consolidated our development efforts and merged our independent system designs to create a single, unified behavior analysis system. The combined system provides researchers with all of the quantification tools and database functionality of the individual UCSD and Caltech systems, but also establishes a common, open software and hardware foundation for continued development by Caltech, UCSD and the community. Current efforts include expanding system capabilities for the automated analysis of mating behavior with the challenge of tracking multiple moving individuals, both in contact with and separated from each other.

Pena-Gonzalez, I. et al. (2013) International Worm Meeting "Suppressors of TDP-1 toxicity in Caenorhadbitis elegans."

Link, CD., Pena-Gonzalez, I.

[International Worm Meeting, 2013]

Suppressors of TDP-1 toxicity in Caenorhadbitis elegans Ilana Pena-Gonzalez1 and Christopher D Link1, 2 1 Integrative Physiology, University of Colorado, Boulder, CO 80309, USA 2 Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA RNA binding protein TDP-43 forms damaging aggregates in multiple neurodegenerative diseases. We have found that the deletion of tdp-1, the C. elegans ortholog of TDP-43, leads to increased accumulation of double stranded RNA. TDP-1 is not believed to bind to dsRNA itself; however the marked increase in dsRNA accumulation in worms deleted for tdp-1 implies TDP-1 normally participates in limiting the stability and structure of dsRNA. Although worms deleted for TDP-1 do not have a severe phenotype, overexpressed nuclear TDP-1 is toxic in worms. One possible explanation for this is that too much TDP-1 leads to excessive disruption of structured RNAs needed for normal RNA metabolism. Making use of this overexpression model we have established a heat shock induction system to help identify components associated with TDP-1's prevention of dsRNA accumulation. Identifying mutations that suppress the neurotoxic effects of overexpressed TDP-1 could help identify others factors contributing to the complex mechanism of disassembling dsRNA. A mutagenesis screen has identified four mutant strains that partially suppress the toxicity of overexpressed TDP-1. We have also tested suppressors of other toxic proteins. Further identifying the pieces of the mechanism regulating dsRNA breakdown could help better explain the pathogenic pathway of TDP-1 and consequently TDP-43 in disease.

S. Luedtke et al. (2006) European Worm Meeting "The Physiological Role of Peptidergic Transmission in the Pharyngeal Nervous System"

V. O'Connor, N.A. Hopper, R.J. Walker, S. Luedtke, L. Holden-Dye

[European Worm Meeting, 2006]

S. Luedtke, R.J. Walker, N.A. Hopper, V. O''Connor and L. Holden-Dye. We are interested in the peptidergic regulation of neuronal function. As the pharyngeal muscle potently responds to a number of flp neuropeptides, we are determining whether peptidergic signalling is involved in the physiological regulation of pumping activity. In a first approach we wish to identify genes that are involved in pharyngeal pumping activity and peptidergic signalling. To achieve this we have described the pumping activity on and off food in wild-type and mutant animals. Here we show data for several different mutants with a dysfunctional feeding phenotype and how this might help in understanding the interactions in the neuronal circuit underlying pharyngeal pumping.. In another approach, we are developing an optical assay for peptidergic signalling. The neuropeptide we are focusing on initially is FLP-13, as this is expressed in pharyngeal neurones and has been shown to have a potent inhibitory effect on pharyngeal pumping. We have generated a flp-13::dsRed2 fusion construct in which the optical marker is inserted within the coding sequence of one of the FLP-13 peptides. This construct has been injected into animals using the pha-1 protocol as a selection for transgenic animals. Stable lines that were rescued for pha-1 were recovered, PCR confirmed they had the flp-13::dsRed transgene and RT-PCR confirmed that this construct was expressed. However, none of the animals exhibited fluorescence. Currently we are experimenting with other fluorophores that may be more suitable for use with the dense core secretory pathway. Funded by the Gerald Kerkut Charitable Trust. We are grateful to the C. elegans Genetics Center for the provision of strains.

Bauer CC et al. (1998) West Coast Worm Meeting "a, b AND g-FILAGENIN - NEW PROTEINS IN THE CORE'S OF THICK FILAMENTS"

Schmid MF, Ortiz I, Epstein HF, Liu FZ, Bauer CC

[West Coast Worm Meeting, 1998]

The cores of muscle thick filaments are composed of an outer layer of paramyosin surrounding an inner tubular region containing additional core proteins. In order to study the structure of the thick filament core and the mechanisms and molecules involved in the formation of this length-regulated, multi-protein structural assemblage, we have isolated and characterized three novel proteins: a, b and g-filagenin. We obtained partial peptide sequences from each of the filagenin proteins and determined their predicted protein sequences from the C. elegans Genome Project's database and correlated this information with cDNA sequence obtained from RT-PCR or phage libraries. All three filagenins are novel proteins with no significant homologies to any known proteins (although homologues in parasitic nematodes have been found in the databases). All filagenins also possess the distinction of having calculated isoelectric points of over pH 10 and having a higher than average number of aromatic amino acids. Both b and g-filagenin are predicted to have secondary structures containing only beta strands and loops, whereas a-filagenin is composed of both alpha helices and beta strands. Structural studies of the core indicate that there are seven paramyosin subfilaments crosslinked by additional internal proteins. We tested our structural model with Western blotting using an affinity-purified antibody and have showed that a, b and g-filagenins are associated with the cores. All three filagenins were localized by immunofluorescence microscopy to the A bands of body wall muscles, but there was no detectable reaction with the pharynx, with the exception of a slight reaction with anti-a-filagenin antibody. b-Filagenin has been shown by immunoelectron microscopy to be assembled with paramyosin into the tubular cores of wild-type nematodes at the same periodicity of 72-nm as observed with paramyosin. In CB1214 paramyosin null mutants, b-filagenin assembles with myosin to form abnormal tubular filaments with a periodicity identical to that of wild type. These results verify that the filagenins are core proteins that co-assemble with either myosin or paramyosin in C. elegans to form tubular filaments. Further studies on nematode muscle thick filaments are intended to elucidate the mechanisms behind the formation and regulation of these stable assemblies of myosin and associated proteins. *Supported by grants from the Muscular Dystrophy Association, the National Science Foundation and a NRSA postdoctoral fellowship for Christopher Bauer.

James Dillon et al. (2006) European Worm Meeting "The Molecular, Cellular and Functional Organization of the C. elegans Metabotropic Glutamate Receptor, MGL-1"

Lindy Holden-Dye, James Dillon, Vincent O'Connor, Neil A. Hopper

[European Worm Meeting, 2006]

James Dillon, Neil A. Hopper, Lindy Holden-Dye and Vincent O''Connor. Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors that are responsible for the fine-tuning of glutamatergic signalling within neural networks that subserve complex brain functions and behaviours. The C. elegans mGluR, mgl-1, is selectively expressed in the nervous system, where it is distributed in neurons of the tail, the nerve ring and the pharynx. We have defined a role for MGL-1 in the modulation of pharyngeal function and feeding behaviour. In the presence of the mammalian mGluR agonists, trans-ACPD (500?M) and L-CCG-I (EC50: 3?M) pharyngeal pumping is inhibited. The basal pharyngeal pumping frequency of the mgl-1 mutant strain mgl-1(tm1811) is not significantly different to wild-type levels in the absence of drug and is not inhibited by drug application (trans-ACPD 500?M and L-CCG-I in the range 1-12.5?M). The inhibition by L-CCG-I was rescued in mgl-1(tm1811) by the re-introduction of the mgl-1 gene, suggesting MGL-1 is responsible for the agonist-mediated regulation of pharyngeal function. The intracellular C-terminals of the eight mammalian mGluR subtypes have been shown to be responsible for directing protein-protein interactions with accessory proteins that scaffold the receptors function. Scaffolding entails the targeting of the receptor to specific compartments of the neuron, anchoring within specialized subcellular domains and the organised assembly of the receptors intracellular signalling pathway. A yeast-2-hybrid screen was performed with the MGL-1 C-terminal to identify proteins that scaffold MGL-1 function. The multi-PDZ domain protein MPZ-1a was identified as a strong interactor in yeast. MPZ-1a is encoded by the gene mpz-1, which we have identified as being co-expressed with mgl-1 in neurons of the pharyngeal nervous system and the nerve ring. The inhibition of pharyngeal pumping by MGL-1 in response to the agonist L-CCG-I was used as a behavioural assay to assess the functional significance of the interaction between MGL-1 and MPZ-1 in this neural network. Pharyngeal pumping was recorded from the available mpz-1 mutant animals mpz-1(tm1136), mpz-1(gk273/+) and mpz-1(tm1136/gk273) trans-heterozygotes. In each case a wild-type response was recorded in the presence of L-CCG-I, suggesting the MGL-1 signalling complex is intact. Although our study provides the first evidence for the function of MGL-1 in worm behaviour the significance of the PDZ-domain dependent interaction of MGL-1 and MPZ-1a requires further investigation.

P. Mitchell et al. (2006) European Worm Meeting "Evidence that Ethanol Equilibriates Rapidly Across C. elegans Cuticle"

L. Holden-Dye, N.A. Hopper, K. Bull, V. O'Connor, P. Mitchell, S. Glautier

[European Worm Meeting, 2006]

P. Mitchell, K. Bull, N.A. Hopper, S. Glautier1, L. Holden-Dye, V. O''Connor Ethanol has concentration-dependent effects on neural function. At concentrations in the low mM range, equivalent to the drink-driving limit, it has an intoxicating action, which is often associated with a transient excitability, followed by inhibition. At higher concentrations, greater than 100 mM, it has an anesthetic action and elicits muscle paralysis. C. elegans has been used to define the genetic basis for behavioural responses to ethanol exposure. In these studies intact animals have been exposed to 500 mM ethanol with the prediction that the internal concentration is more than ten-fold lower and therefore equivalent to an intoxicating, rather than an anesthetic, dose (Davies and McIntire, 2004). In order to underpin further studies, we have analysed the relationship between the external and internal ethanol concentration of C. elegans. Immersion of C. elegans in a sealed chamber containing ethanol (range 100 mM to 500 mM) elicits an inhibition of thrashing rate. Inhibition reaches a steady-state value within 5 min. This effect is concentration-dependent (half-maximal effect at 300 mM). At all concentrations of ethanol a steady-state inhibition is observed within 5 min. The animals rapidly recovered when removed from ethanol. Notably, this recovery was complete within 2.5 min. This suggests that ethanol may rapidly equilibriate across the cuticle and that the internal concentration that is required to inhibit motility approximates to the external concentration. In order to directly assay the effects of ethanol on muscle we used the pharyngeal muscle preparation. In this assay the muscle is exposed and therefore the concentration of ethanol is known. This muscle is inhibited by ethanol at concentrations in excess of 100 mM. Although it could be argued that the pharyngeal muscle behaves in a different fashion to body wall muscle in terms of ethanol sensitivity, the results support the contention that concentrations in excess of 100 mM are required to inhibit muscle activity. Internal ethanol concentrations in C. elegans following exposure to ethanol have been estimated using a biochemical assay kit. However, this procedure requires that the animals are briefly washed in cold buffer prior to the measurement. Our observation that animals exposed to ethanol fully recover from the inhibition of motility within 2.5 min suggests that a significant amount of ethanol may be lost from the inside of the animals during the protocol. We tested this by determining the influence of the wash volume on the apparent estimate in internal ethanol concentration. This shows that the estimate of internal ethanol concentration increases as the volume of the wash buffer decreases. This suggests that the measurement of internal ethanol concentration using this method underestimates the true internal concentration.. Davies, A.G. and McIntire, S.L. 2004, Biol. Proced. Online 6, 113-119.. Funded by the BBSRC, UK

Mitchell PH et al. (2008) European Worm Meeting "Modelling alcohol dependence in Caenorhabditis elegans"

James C, Mitchell PH, Mould RN, Hopper NA, Glautier SP, Dillon J, Oconnor VM, Holden-Dye LM,, Adrianakis I

[European Worm Meeting, 2008]

Ethanol is one of the most widely used and socially acceptable drugs in the. world. However its chronic use can lead to serious problems due to the. development of dependence. Ethanol has been shown have wide-ranging but. selective effects on many different neurotransmitter circuits including;. glutamatergic, GABAergic, serotonergic, dopaminergic and opioid peptidergic. systems. The basis for alcohol tolerance and withdrawal involves. counteradaptive neurochemical changes within these systems to adapt to the. prolonged presence of ethanol in the brain. Here we are using C. elegans to. enable an analysis at all levels of organization from gene, molecule, and. neurone through to neuronal circuit and behaviour. We want to use C.. elegans to investigate the development of neuroadaptation to ethanol. In. order to achieve this we have first provided evidence that ethanol rapidly. equilibriates across the cuticle of C. elegans (Mitchell et al. 2007) thus. enabling experiments in which the concentration-dependent effects of. ethanol on behaviour can be defined. In line with previous studies (Davies. et al. 2003) we have established that external concentrations of ethanol. >100mM are required to overtly affect the visually observable measures of. behaviour. This is equivalent to concentrations that are supra-intoxicating. in mammals. In order to determine whether ethanol elicits any effects on. C. elegans at concentrations equivalent to those that are intoxicating in. mammals we have performed more discreet analyses of behaviours. Pharyngeal. recordings have revealed that concentrations as low as 10mM ethanol have. significant effects on the activity of neural networks, thus providing an. opportunity to investigate the molecular determinants of acute ethanol. effects. We are also in the process of refining the analysis of locomotory. behaviour with the aim of determining whether ethanol elicits effects at. low mM concentrations. As part of this effort we have developed a paradigm. in which we can demonstrate withdrawal from ethanol, and relief from. withdrawal due to low concentrations of acute ethanol. We have used this to. study the onset and recovery rates of this neuroadaptation and the. concentration dependence of this effect in order to understand the. mechanisms involved. This has been extended to investigate if genes already. implicated in acute effects of ethanol in C. elegans are involved in. controlling the withdrawal and tolerance behaviours. These studies are. aimed at better defining how well C. elegans can model aspects of. alcoholism.. Funded by the BBSRC, UK.. References. Davies, A. G., Pierce-Shimomura, J. T., Kim, H., VanHoven, M. K., Thiele,. T. R., Bonci, A., Bargmann, C. I., & McIntire, S. L. 2003, "A central role. of the BK potassium channel in behavioral responses to ethanol in C.. elegans", Cell, vol. 115, no. 6, pp. 655-666.. Mitchell, P. H., Bull, K., Glautier, S., Hopper, N. A., Holden-Dye, L., &. O''connor, V. 2007, "The concentration-dependent effects of ethanol on. Caenorhabditis elegans behaviour", Pharmacogenomics.J., vol. 7, no. 6, pp.. 411-417.