Schwarz EM et al. (1996) East Coast Worm Meeting "Cellular and molecular analysis of thermosensation"

Chalfie M, Schwarz EM

[East Coast Worm Meeting, 1996]

While senses like sight, hearing, and mechanosensation are becoming well understood, thermosensation remains obscure. Work by Hedgecock (1) and Mori (2) has shown that C. elegans is a promising model system for metazoan thermosensation, but neither the cells nor the genes required for thermosensation in C. elegans are fully known. Previous work in this laboratory (3) has shown that at least three new deg mutations cause cells in the head to degenerate while partially crippling thermosensation; none of these are allelic to previously described ttx mutations . We have therefore begun to determine which cells are defective in these deg mutations. We have also begun genetic mapping experiments aimed at positional cloning of the wild-type deg loci. References: 1. Hedgecock, E.M. and Russell, R.L. (1975). Proc. Natl. Acad. Sci. U.S.A. 72, 4061-4065. 2. Mori, I. and Ohshima, Y. (1995). Nature 376, 344-348. 3. Treinin, M. and Chalfie, M. (1993). International C. elegans meeting abstracts, p. 446.

Witvliet, D. et al. (2017) International Worm Meeting "The C. elegans connectome consist of invariant, stochastic, and developmentally regulated synapses."

Berger, D.R., Wang, J., Cook, S.J., Holmyard, D., Lichtman, J.W., Neubauer, M., Laskova, V., Zhen, M., Emmons, S.W., Mulcahy, B., Samuel, A.D.T., Kersen, D., Chisholm, A.D., Schalek, R.L., Koh, W.X., Qian, J., Hall, D.H., Mitchell, J.K., Chang, M., Witvliet, D.

[International Worm Meeting, 2017]

When animals are born, neuronal networks are already in place to integrate sensory cues and effect appropriate behavioral or homeostatic responses. During the course of postnatal development, synapses and circuits undergo refinement and remodeling, updating or adapting sensorimotor behaviors. Rules for developmental remodeling are poorly characterized, because a circuit-level analyses at multiple time points, in multiple animals are missing. We use an isogenic C. elegans N2 population to examine how the neural circuit changes during development. At birth, C. elegans has 218 neurons (excluding CAN); 82 new neurons are incorporated into the nervous system before the end of larval development. These include sensory, motor, and interneurons that are located throughout the body, suggesting a system-wide modification of the juvenile circuit during post-embryonic development. Using serial-section electron microscopy, we mapped the synaptic connectivity in the head and tail ganglia for five animals, from hatching to late larval development. These datasets allow us to separate the connectome into core connections, synapses that are present throughout the lifetime, transient connections, synapses that are present only during early or late development, and variable connections, synapses that are not conserved between animals. We propose that the core connections may drive hard-wired behaviors, and developmentally regulated connections exert stage-specific roles, while variable connections may confer individual variability and/or be stochastic in nature. Our current data analyses lead to two notions: First, developmentally regulated connections are enriched for synapses between sensory neurons, and with neuromodulatory neurons, while connections between interneurons are remarkably stable. This implies that for C. elegans, core circuits for decision-making are already established at birth, but their modulation and multi-sensory integration are refined or shaped during development. Second, variable connections, comprising about half of the connection edges in the published adult dataset, are prominent. Stochastic or experience dependent variability in connections may contribute to variability of behaviors set by hard-wired core connections.

Gal Haspel et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Stretch Receptors and Pattern Generation in C. elegans Locomotion Circuit"

Anne Hart, Gal Haspel

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

The anatomy of the nervous system of the nematode C. elegans has been comprehensively described compared to other animal species; the location and connectivity of each neuron are known. This affords us the opportunity to address the neural circuitry underlying various behaviors such as locomotion. Only five pairs of interneurons and six groups of motoneurons have been are central to locomotion based on ablation studies. Yet how, these neurons orchestrate their activity during locomotion is unclear. The sinusoid motion locomotive pattern might rise from a sensory feedback loop or involve a central pattern generator. Even in the latter case, sensory feedback might serve to adjust the pattern generator to environmental cues. Taking advantage of C. elegans optical transparency and the relative ease of genetic manipulation, we have used cameleon (a genetically encoded calcium indicator) to record the activity in specific motoneurons during fictive locomotion. Two initial findings stand out. First, mechanically moving the nematode activates motoneuron activity- suggesting the presence of stretch receptors. This supports a hypothesis first suggested by R.L. Russell based on motoneuron anatomy 30 years ago. Second, by presenting an aversive stimulus, we were able to trigger rhythmic activity in the motoneurons of immobilized animals, indicating the presence of a central pattern generator, active even in the absence of sensory feedback. These findings begin to unravel the characteristics of the nematode locomotion circuit. Understanding the orchestrated activity of neurons within this circuit will provide us with general insights into how neurons produce rhythmic movements in animals and into nematode behaviors that involve locomotion.

Stegeman, Gregory et al. (2011) International Worm Meeting "Variation in temperature-dependent behaviours among natural isolates of Caenorhabditis briggsae."

Shin, Jiwon, Ryu, William, Cutter, Asher, Bueno De Mesquita, Matthew, Stegeman, Gregory, Lin, Nan

[International Worm Meeting, 2011]

Natural genetic variation allows the discovery of new gene functions and novel alleles for genes already known to act in biologically important processes. We are applying this approach to temperature-dependent behaviours in nematode worms in order to better understand the genetics behind behaviour. We focus on Caenorhabditis briggsae because most wild caught individuals fall into two genetically distinct clades that correspond approximately with northern temperate or with tropical latitiudes. Interestingly, strains from the tropical clade have higher fecundity when reared at higher temperature than do the temperate strains, suggesting local adaptation to climate variables like temperature (Prasad et al. 2011). Movement through its thermal landscape is the main way for nematodes like C. briggsae to regulate body temperature, so we also expect to see heritable differences in temperature-dependent behaviours. Here we quantify for the first time classic thermal-response behaviours among several C. briggsae wild strains from different haplotype groups using assays like accumulation on a linear thermal gradient, isothermal tracking, and a new droplet based thermal gradient assay. We demonstrate that C. briggsae shows thermotaxis and isothermal tracking similar to C. elegans but with some differences. We also identify heritable differences among strains from wild genetic backgrounds within C. briggsae. We will continue to develop higher throughput assays for temperature-dependent behaviour in order to carry out a quantitative trait loci mapping project using recombinant inbred lines derived from tropical and temperate parental strains. Prasad, A., M. Croydon-Sugarman, R.L. Murray & A.D. Cutter. 2011. Temperature-dependent fecundity associates with latitude in Caenorhabditis briggsae. Evolution. 65: 52-63.

Witvliet, D. et al. (2019) International Worm Meeting "Structural plasticity of the C. elegans connectome across development and between individuals."

Neubauer, M., Witvliet, D., Mitchell, J.K., Maeng, S., Meirovitch, Y., Chisholm, A.D., Wang, M.D., Shavit, N., Wang, Z., Ho, C.Y., Samuel, A.D.T., Lichtman, J.W., Koh, W.X., Schalek, R.L., Rehaluk, C., Mulcahy, B., Holmyard, D., Berger, D.R., Zhen, M., Cook, S.J.

[International Worm Meeting, 2019]

Nervous system development is widely thought to be optimized for precise wiring and efficient networks. Maintaining plasticity, however, is necessary for animals to adapt, learn, and evolve. This is especially evident during postembryonic development when newly born neurons are integrated into functioning neurocircuits. Rules for circuit plasticity and variability during and after development are poorly because circuit-level analyses across multiple animals and developmental time points are missing. Using electron microscopy, we mapped the connectome of the central nervous system for eight isogenic C. elegans animals spanning from birth to adulthood. These datasets reveal a highly dynamic circuit architecture, with a five-fold increase in synapse number across postembryonic development. Synapses are built and removed for multiple reasons: (1) to strengthen a remarkably stable core circuitry for decision-making, (2) to fine-tune multi-sensory integration and motor responses by building new unique cell-cell connections, (3) to streamline circuit computations, and (4) to fill connectivity gaps as a result of evolutionary pressures to hatch before initial wiring is complete. This remodeling is driven independently of the contact area between neurons and instead is dependent on neuron class and centrality. Remodeling is overrepresented between connections from sensory neurons to motor and interneurons, while connections between interneurons are remarkably stable. Well-connected, central neurons (typically interneurons), are more heavily regulated than other neurons, but surprisingly this regulation does not originate from other central neurons. Thus, the driving force for synaptic growth is not uniform, and may depend on neuron identity, suggesting an underpinning by genetic or functional identity. Together, our findings show that even in one of the most deterministic animals, nervous system wiring retains a high degree of plasticity. The connectome should be separated into core connections, that are present throughout life, developmentally changing connections, that depend on the stage of development, and variable connections, that are not conserved among individuals. Core connections are the strongest class of connections, but they make up less than half of all connections. The prevalence of wiring plasticity implicates relevance to nervous system adaptation and variability. Our data will be available on nemanode.org before the end of the summer.

Rezsohazy R et al. (1995) International C. elegans Meeting "Towards Tc1- and Tc3-mediated enhancer detection assays in Caenorhabditis elegans"

Rezsohazy R, Plasterk RHA

[International C. elegans Meeting, 1995]

We aim to set up an assay to detect tissue- or stage-specific regulatory sequences. A screen of gene expression patterns has been developed by Hope, using promoter-reporter fusions generated by shotgun cloning (1). Our assay will rely on the insertion of the transposable elements Tc1 and Tc3 carrying an appropriate reporter gene. As a preliminary step we developed a selection that allows detection of Tc1, or Tc3 jumping events from an extrachromosomal array to any chromosomal locus in the germ-line. To detect such events the transposable elements have been marked by inserting the pha-1 gene (2) which complements the thermosensitive embryonic lethal allele pha- 1(e2123). The ability of marked elements to jump in somatic cells was confirmed by PCR detection. Transgenes carrying both the rol-6(su1006) gene and the Tc1-pha-1 or Tc3-pha-1 construct have been established in mutator strains (3) that are a homozygous for the pha-1(e2123) allele. This strain is now an obligatory roller at the non-permissive temperature. Among the progeny we look for non-rolling worms able to develop at non- permissive temperature, in which presumably the transposable module has inserted into new chromosomal locations. For use as an enhancer-trapping technique, large scale detection of transposition events will be needed. Therefore it will be necessary to select rather than detect the loss of the transgene. For this purpose the cha-1 gene could be used as a counter-selection marker in homozygous cha- 1(p1152) mutants resistant to acetylcholine esterase inhibitors (4). (1) Hope, I.A. (1991); Development, 113, 399-408. (2) Granato, M., et al. (1994); Nucl Acids Res, 22, 1762-1763. (3) Collins, J., et al. (1987); Nature, 328, 726-728. (4) Rand, J.B. and Russell, R.L. (1984); Genetics, 106, 227-248.

Byerly L et al. (1975) Worm Breeder's Gazette "Monitoring Growth and Development with a Nematode Counter"

Cassada RC, Russell RL, Scherer S, Byerly L

[Worm Breeder's Gazette, 1975]

We have been using the sizing nematode counter (Byerly, L., Cassada, R.C., and Russell, R.L., Rev. Sci. Inst. 46, 517-521, 1975) to study the growth and reproduction of the wild type and some mutants of C. elegans. For the wild type we have obtained growth curves and egg- laying curves at 16 C, 20 C, and 25 C; growth rate shows an exponential temperature dependence with a Q10 of 2.1, but egg-laying rate does not, dropping off relative to expectation at 25 C. Egg yield is also reduced by about one-third at 25 C. As a restrictive temperature for ts mutants, 25 C seems on the borderline of acceptability, and certainly should not be exceeded. To simplify the analysis of growth and reproduction in mutants, we have been using a '3-egg' method. Cultures started from three synchronously laid eggs are allowed to develop to the point where the second-generation progeny of the original 3 animals are beginning to hatch. The resulting size distribution is then measured and a computerized fitting procedure is used to fit this distribution as closely as possible, using either wild type growth parameters or simple modifications thereof. The goodness of fit indicates how well a given mutant conforms to the wild type growth and egg-laying pattern, and the modifications indicate quantitative differences from the wild type in growth rate, egg yield, and/or size. We find this to be a very efficient way to screen mutants for growth and/or reproductive differences, and it will probably come as no surprise to learn that almost all mutants so far examined have such changes. We would be happy to examine mutants which anyone would like to send, and if anyone is interested in building a counter of his/her own ( total materials cost ~ $500; needs someone with electronics experience to construct) we would be happy to provide plans and instructions.

Cook, Steven et al. (2019) International Worm Meeting "Olfactory circuit structure is reconfigured between divergent nematode species."

Sommer, Ralf, Riebesell, Metta, Carstensen, Heather, Bumbarger, Dan, Sarpolaki, Tahmineh, Hobert, Oliver, Moreno, Eduardo, Siebriebriennikov, Bogdan, Castrejon, Jessica, Hong, Ray, Cook, Steven, Cochella, Luisa

[International Worm Meeting, 2019]

The ability of an organism to process sensory information is dependent upon the structure and connectivity of its neurons. The nematodes P. pacificus and C. elegans have a repertoire of different behaviors and sensory preferences, yet the neural substrate for these differences is unknown. To understand structural differences of the pharyngeal nervous system related to predatory feeding behavior, serial section EM series were generated of P. pacificus. Through a volumetric reconstruction and subsequent analysis, it was shown that an identical set of pharyngeal neurons with similar morphology generated many different synaptic patterns (Bumbarger et al 2013). Toward the goal of better understanding differences in olfactory behavior between P. pacificus and C. elegans we further analyzed these EMs and identified homologous amphid sensory and interneurons between P. pacificus and C. elegans based upon cell body position, dendritic morphology, dye staining, and reporter gene expression (see abstract by R.L. Hong et al). We determined the set of chemical and gap junction connections for the amphid circuit and compared the resultant network to C. elegans. We observed that neurons with the most similar morphology (AFD and AUA) have the most similar connectivity across species, while neurons with different dendritic structure (AWA, AWB, AWC) show a greater number of species-specific connections. Overall, we found that 60% of strong chemical connections and 40% of gap junctions are conserved across species. We next asked whether differences in connectivity are due to changes in neuronal neighborhoods, or synaptic partner choice. We found that in each instance of a discrepant synapse, the pair of neurons in questions are adjacent in both species. We are actively extending our analysis to determine whether sensory neurons are more divergent in their structure and connectivity compared to downstream inter- and motorneurons. Our results suggest evolutionary pressure has kept the overall nervous system plan of nematodes very similar, but has allowed for different synaptic configurations to permit species-specific behavioral differences.

H A Zariwala et al. (2001) International C. elegans Meeting "The trampoline assay: A new method for measuring the step response of the thermotaxis mechanism in C. elegans."

H A Zariwala, S R Lockery, S Faumont

[International C. elegans Meeting, 2001]

Worms in a thermal gradient migrate to the temperature at which they were cultivated (thermotaxis). Qualitative analysis of the effects of mutations and neuronal ablations on thermosensory behavior[1,2] suggests that thermotaxis may involve a mixture of two strategies: isothermal tracking, in which the head is aligned orthogonal to the thermal gradient, and pirouetting, in which course correction is achieved by a cluster of sharp turns, as in chemotaxis[3]. As a direct test of whether pirouettes play a role in thermotaxis, we devised a new procedure--the "trampoline assay"--in which worms were placed on a thin (~100 m m) agarose film suspended over a chamber filled with buffer at the cultivation temperature (21 o C). After a 5 min. adaptation period, we quickly replaced the first chamber with a second containing buffer at a lower temperature (18 o C), and observed the worm for an additional 5 min. Temperature measurements on the surface of the film indicated that the time constant of the temperature shift was ~4 sec. When we shifted wild type worms (n = 14) to the lower temperature (down-shifts), we observed a transient increase in the probability of initiating sharp turns (time to peak = ~30 sec; decay time constant = ~50 sec). This result is consistent with a role for pirouettes in thermotaxis because, in a spatial temperature gradient, a pirouette induced by a temperature drop tends to return the worm to its cultivation temperature. The increase in sharp turn probability is almost certainly not a mechanical artifact because we saw no increase in turning when the second chamber was at the cultivation temperature (n = 8). Conversely, when we down-shifted the cryophilic mutant ttx-3 (ks5) (n = 15), we observed a sustained decrease in sharp-turn probability. In a spatial temperature gradient, a decrease in turning induced by a temperature drop tends to move the worm toward lower temperatures, consistent with the cryophilic phenotype of ttx-3 . We conclude that pirouettes are likely to be important for thermotaxis, at least for excursions below the cultivation temperature. Experiments are in progress to determine if the same is true for excursions above cultivation temperature. 1. Hedgecock, E.M. & Russel, R.L. PNAS 72, 4061-4065 (1975). 2. Mori, I. & Ohshima, Y. Nature 376, 344-348 (1995). 3. Pierce-Shimomura, J.T. & Lockery, S.R. J. Neurosci. 19, 9557-9569(1999).

Johnstone DB et al. (1996) West Coast Worm Meeting "SCREENING FOR ADAPTATION MUTANTS AT AN IONOTROPIC RECEPTOR"

Thomas JH, Johnstone DB

[West Coast Worm Meeting, 1996]

Adaptation limits the efficacy of most clinical drug therapies and is also a form of synaptic plasticity, which is important in learning and memory.[1] Adaptation has been studied extensively with electrophysiology and biochemistry, but rarely with genetics. A genetic screen using C. elegans was recently described for defects in adaptation to serotonin and dopamine[2], which may occur at metabotropic (G-protein coupled) receptors. Many mutants were isolated from this screen, and the identity and biology of two mutants, unc-2 and unc-36, was described. While metabotropic receptors and ionotropic receptors (ion channels) are thought to share some mechanisms of adaptation, there are also important differences.[3] A genetic approach to adaptation at ionotropic receptors has not yet been reported. We have initiated a screen for mutants that fail to undergo adaptation to muscimol, an agonist of the ionotropic GABA-A receptor. Muscimol inhibits both body wall and pharyngeal muscle[4]. We used the rate of pharyngeal pumping as a quantitative measure of the degree of inhibition by muscimol. When incorporated into NG plates muscimol causes a dose- dependent decrease in the rate of pharyngeal pumping, reaching complete inhibition of pumping at 6mM. With wild type, pumping slowly increases after 1 hour, and reaches 80-90 percent of untreated after 4 hours. By visually screening mutagenized animals we have isolated 5 mutations that result in a significant reduction in pumping rate after a 3 hour muscimol exposure. The reduction is statistically significant but subtle, approximately 3-fold for all mutants. To rule out the possibility that the mutants are simply hypersensitive to muscimol, we examined dose-response curves of the mutants and wild type. None of the mutants is hypersensitive. Therefore, all five mutations presumably cause defects in adaptation at the ionotropic GABA-A receptor. Defects in adaptation to other neurotransmitters have not yet been examined. In parallel, we examined the previously characterized adaptation mutants unc-2 and unc-36 for possible effects on adaptation to muscimol. Both mutants regain their normal rates of pumping significantly more slowly than wild type on 6mM muscimol. This effect is particularly evident with unc-36, which shows an approximately 15-fold reduction in adaptation to 6mM muscimol. However, the effects of unc-2 on the presumed adaptation defect at the GABA-A receptor may be attributed to muscimol hypersensitivity. Low doses of muscimol that inhibit wild type pumping only 2 to 5 percent inhibit pumping in unc-2 by about 45 percent. It is interesting that unc-36, which encodes a Ca++ channel subunit, is defective in adaptation to several different neurotransmitter agonists. We speculate that calcium influx may be a shared first step in transmitter adaptation pathways. I. Huaginir, R.L. and Greengard, P. (1990) Neuron 5:555-567. 2. Schafer, W.R and Kenyon, C.J. (1995) Nature 375:73-78. 3. Lohse, M.J. (1993) Biochem et Biophys Acta 1179:171-188. Leidenheimer, N.J. et al. (1991), TIPS 12:84-87. Raymond, L.A. et al. (1993) TINS 16:147-153. 4. McIntire, S.L., Jorgensen, E. and Horvitz, R.H. (1993) Nature 364:334-337.