Poorna Viswanathan et al. (2007) International Worm Meeting "Virulence characteristics of diverse B.cenocepacia isolates and the identification of virulence factors using the C.elegans model for pathogenicity."

Todd Ciche, Poorna Viswanathan, Martha Mulks, Luke Gray

[International Worm Meeting, 2007]

The C.elegans model for pathogenic host-bacteria interactions has been used extensively to study virulence many pathogens including Pseudomonas aeruginosa, an important opportunistic pathogen for cystic fibrosis patients. P.aeruginosa kills C.elegans by producing toxins on PGS media (fast killing) or by infecting nematodes on NGM media (slow killing). Burkholderia cenocepacia is a ubiquitous organism that is also an important opportunistic pathogen for CF patients. We used the nematode model to determine virulence characteristics of clinical, agricultural and environmental isolates and as an animal host to study virulence in select strains of B.cenocepacia. The virulence characteristics of some B.cenocepacia are similar to P.aeruginosa(e.g. Midwest clone PC184), however some strains cause rapid mortality on NGM (e.g. AU1054, HI2424, and MI onion field isolate 6RT-130) and PGS, while other strains kill slowly on PGS or do not kill at all. B.cenocepaciaAU1054 is a Mid-Atlantic CF-patient isolate, that is pathogenic to onions, virulent to worms and has anti-fungal properties. Because of this broad host range, we focused on this strain to understand its mechanism of C.elegans killing and to identify its virulence genes by transposon mutagenesis. We have used GFP-labeled B.cenocepaciaAU1054 in our experiments. B.cenocepaciaAU1054 appears to kill C. elegans by infections and by producing toxins. To identify the virulence genes, we used transposon mutagenesis and screened for mutants that did not kill C.elegans at 72 h on NGM plates. We have four mutants that appear to be avirulent to worms. Work is in progress to identify the gene(s) that have been interrupted by the transposon in these mutants, to determine if these genes encode general or specific virulence factors and to characterize the virulence of AU1054 to C.elegans. In summary, C.elegans has proved useful for assessing virulence characteristics of diverse B.cenocepacia strains and to identify virulence factors present in select strains.

Chalasani, Sreekanth H. et al. (2009) International Worm Meeting "AWC sensory neurons release two neurotransmitters to regulate exploratory behavior upon removal from food."

Albrecht, Dirk, Bargmann, Cornelia I., Chalasani, Sreekanth H., Kato, Saul

[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.

Chalasani S H et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Neuropeptide feedback modifies odor induced responses in Caenorhabditis elegans olfactory neurons"

Bargmann C I, Kato S, Chalasani S H, Albrecht D R

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Neural circuits transform sensory signals to generate behaviors on timescales from seconds to hours. In some C.elegans behaviors, sensory inputs lead to long lasting and complex behavioral outputs. Animals that have been removed from food spend about 15 minutes exploring a local area by interrupting long forward movements with reversals and turns (Wakabayashi et al., 2004, Gray et al 2005). AWC sensory neurons regulate this behavior by releasing two neurotransmitters, glutamate (promoting turns) and NLP-1 (inhibiting turns). AWC sensory neuron released glutamate activates AIB and inhibits AIY and AIA interneurons (Chalasani et al 2007). In contrast to glutamate, AWC neuron released NLP-1 acts on AIA interneurons to suppress reversals, indicating that turn frequencies are regulated by at least two opposing systems. AWC calcium responses are modulated in these neurotransmitter mutants suggesting that multiple pathways can influence AWC dependent behavior and neuronal activity. ReferencesChalasani, S. H., et. al. Dissecting a neural circuit for olfactory behaviour in Caenorhabditis elegans. Nature 450, 63-70 (2007).Gray, J.M., et. al. A circuit for navigation in Caenorhabditis elegans. Proc. Natl. Acad. Sci. 102, 3184-3191 (2005).Wakabayashi, T., et. al. Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111 (2004).

Sreekanth Chalasani et al. (2007) International Worm Meeting "Functional analysis of a neural circuit controlling turning behavior in Caenorhabditis elegans."

Cornelia Bargmann, Jesse Gray, Makoto Tsunozaki, Sreekanth Chalasani, Nikolas Chronis

[International Worm Meeting, 2007]

C. elegans increase its frequency of reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999) after removal of a food stimulus. 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 the sensory neuron AWC releases two neurotransmitters (glutamate and a neuropeptide, NLP-1) when the worm is removed from food. The released glutamate acts to activate AIB and inhibit AIY, promoting reversals. Strains with different reversal frequencies can be generated by manipulating the level of glutamate receptors on interneurons AIB and AIY. Decreasing receptor expression leads to fewer reversals, and increasing receptor expression results in more reversals than in wild-type. The AWC released neuropeptide NLP-1 serves to reduce reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. Consistent with behavioral responses, AWC and AIB respond (by increasing calcium concentration) to removal of stimulus. We plan to extend the imaging studies to other neurons in the circuit. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. References: 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. Pierce-Shimomura, J.T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci 19, 9557-9569. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Sreekanth H Chalasani et al. (2005) International Worm Meeting "Molecular analysis of a neural circuit for navigation in Caenorhabditis elegans."

Sreekanth H Chalasani, Nikolas Chronis, Jesse M Gray, Cornelia I Bargmann

[International Worm Meeting, 2005]

Navigation in C.elegans is achieved by sustained forward movement that is interrupted with reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999). We are interested in the neural circuit that controls the frequency of reversals and turns during exploratory behavior. After worms are taken off bacterial food, they exhibit an initial local search with a high frequency of pirouettes. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY interneurons inhibit pirouettes. (Tsalik and Hobert 2003, Wakabayashi et al 2004, Gray et al 2005). How is activity transmitted through this neuronal circuit? The neurotransmitters glutamate and dopamine regulate turning frequency (Hills et al 2004). We found that the vesicular glutamate transporter EAT-4 is essential for the generation of pirouettes after removal from food. Using cell-specific rescue of eat-4 mutants, we show that both AWC and ASK sensory neurons can release glutamate to stimulate pirouettes. The released glutamate appears to be sensed by a glutamate-gated chloride channel (GLC-3) that inhibits the AIY interneurons, and the glutamate-gated cation channel GLR-1, which stimulates the AIB interneurons. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. We are currently attempting to image neuronal activity in these neurons using genetically encoded calcium sensors. References: 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. Hills, T., Brockie, P.J., and Maricq, A.V. (2004). Dopamine and glutamate control area-restricted search behavior in Caenorhabditis elegans. J. Neurosci 24, 1217-1225. Pierce-Shimomura, T., Morse, T.M., and Lockery, S.R. (1999). The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis. J. Neurosci 19, 9557-9569. Wakabayashi, T., Kitagawa, I., and Shingai, R. (2004). Neurons regulating the duration of forward locomotion in Caenorhabditis elegans. Neurosci. Res. 50, 103-111.

Makoto Tsunozaki et al. (2005) International Worm Meeting "A mutation in ODR-11 reverses AWC-mediated butanone responses from attraction to avoidance"

Makoto Tsunozaki, Cori Bargmann

[International Worm Meeting, 2005]

When wild-type C. elegans is exposed to an odor, the identity of the cell that senses the odor specifies its behavioral response. The AWA and AWC neurons mediate attraction, and AWB mediates an avoidance response. Ectopic expression, in AWB, of a receptor for a normally attractive odor can reprogram the animals response to the ligand from attraction to avoidance (Troemel et al., 1997). Here we describe a mutant, odr-11(ky713), in which AWC mediates an avoidance response to a normally attractive odor, butanone. odr-11 animals are defective in chemotaxis to AWC-sensed odors but have normal responses to AWA- and AWB-sensed odors. Mutant animals avoid butanone and show reduced chemotaxis to benzaldehyde, isoamylalcohol, and 2,3-pentanedione. A ceh-36 mutation eliminates AWC neurons, and butanone avoidance is absent in odr-11;ceh-36 mutants. Mutations in odr-1, a transmembrane guanylyl cyclase, and tax-4, a cyclic nucleotide-gated channel subunit, also suppress butanone avoidance in odr-11. These and other double mutant phenotypes suggest that butanone avoidance in odr-11 mutants requires cGMP signaling in the AWC neurons. We are also using an odor flow assay to quantitatively analyze the behavior of wild-type and odr-11 animals in response to temporal changes in olfactory stimuli. Wild-type animals respond to a step increase in butanone concentration by suppressing reversals, while they respond to a step decrease by increasing the frequency of reversals. These observations are consistent with the biased random-walk model for chemotaxis (Pierce-Shimomura et al., 1999). We are currently examining odr-11 responses. The removal-from-food assay measures AWC-regulated behavioral responses (Gray et al., 2005). Wild-type animals have a high frequency of reversals immediately after removal from food, and gradually suppress reversals over 30 minutes. odr-11 animals show a higher frequency, relative to wild type, of reversals in the earlier times off of food, and the subsequent decline in reversal frequency is faster. We are testing the idea that ODR-11 regulates the time course of behavioral state changes in response to sensory stimuli. References: Troemel et al. (1997) Cell 91:161. Pierce-Shimomura et al. (1999) J. Neurosci. 19:9557. Gray et al. (2005) PNAS 102:3184.

Stephen C Pak et al. (2005) International Worm Meeting "Protein aggregate formation and disruption of post-embryonic development in worms expressing SRP-2::GFP"

Clifford J Luke, Stephen C Pak, Gary A Silverman

[International Worm Meeting, 2005]

Serpins are high molecular weight serine proteinase inhibitors that irreversibly inactivate target proteinases using a suicide substrate-like mechanism. Although in vitro biochemical characterizations have provided some clues as to the identity of the target proteinases, the pathways in which serpins play a role are currently unknown. To determine whether serpins plays a role in C. elegans development, growth and longevity, we generated mutants that lack or over-express serpin genes. In general, with the exception of srp-6 (see poster by Luke et al.,), null mutants displayed no overt phenotypes. In contrast, transgenic animals over-expressing serpins displayed a variety of developmental and growth defects. In particular, over-expression of SRP-2::GFP resulted in distruption of post-embryonic development characterized by early larval arrest, slow growth and molting defects. Over-expression of SRP-2 mutants harboring amino-acid substitutions that diminish the serpins ability to inhibit proteinases failed to suppress the phenotype. This result suggests that the phenotype is independent of the proteinase-inhibitory function of SRP-2. Preliminary structure/function mapping results suggest that a short, N-terminal portion of SRP-2 is sufficient for the phenotype. Interestingly, we observed large, insoluble SRP-2::GFP aggregates in the cytosol of anterior hypodermal cells. The relationship between aggregate formation and the developmental defects is currently unclear. We are currently conducting a genetic screen to identify modifiers of the SRP-2 developmental and aggregation phenotypes. We hope to present the results of the genetic screen at the conference.

Jordan Boyle et al. (2008) C.elegans Neuronal Development Meeting "Agar: Not So Groovy After All"

Stefano Berri, Netta Cohen, Ian Hope, Jordan Boyle

[C.elegans Neuronal Development Meeting, 2008]

C. elegans locomotion is typically studied on agar, with locomotion models generally being restricted to modeling the worm''s crawling behavior. One key question regards the relative contributions of active forces (due to the worm''s muscle actuation) versus external forces (due to resistance from the groove) in shaping the locomotion waveform. Models of C. elegans crawling fall into two classes: (i) those that generate a muscle activation pattern that determines body shape in the absence of any environmental forces (BC, Bryden and Cohen, 2004, 2008), and (ii) models in which the agar groove constrains the body to follow the head, with body muscles generating forward thrust along the groove (NE, Niebur and Erdos, 1991). We set out to determine the importance of the groove in shaping the undulation waveform of the worm. We compared locomotion on agar to that of worms on a flat, solid substrate, precluding the formation of a groove, and found that the worm was able to generate and propagate a crawling waveform. However, the worm failed to make significant forwards progress, confirming the absence of a groove. Next we developed a physics simulator of worm locomotion which models the environment as a viscoelastic fluid (following Gray and Hancock, 1955; Gray and Lissman, 1964), and validated the model against experimental crawling data on agar. We then estimated the groove strength (or viscoelastic properties of agar) by feeding worm skeletons extracted from recorded movies to this simulator. To resolve the respective roles of the groove and active muscle forces during crawling, we adapted the NE model and successfully replicated their results in a strong groove environment (Boyle et al., 2007). However, for sufficiently weak grooves the mechanism breaks down. This occurs with a groove strength significantly greater than that estimated on agar, indicating that the groove alone is not sufficient to determine the body shape. We conclude that the worm''s crawling waveform is fully determined by the pattern of muscle activation. This finding should have important implications for locomotion models in general and for predictions about the distribution of postulated stretch receptors along ventral cord motoneurons, in particular. Finally, we suggest that the use of agar as a medium for conducting locomotion assays could mask certain defects, and propose assays using other environments that could offer complementary insight. This work was funded by the EPRSC grants EP/C011953 and EP/C011961.

Graves J et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Fast and Customizable Video Analysis of C. Elegans Locomotion"

Mailler R, Graves J

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Currently, various software solutions exist to analyze and extract features from videos of the worm C. Elegans. However, these software packages have not been widely accepted by the community because they implement a single process for converting video into data, are implemented using Matlab's outdated, undocumented image processing algorithms, tend to be slow, and are poorly documented. The software we have developed rectifies these deficiencies by providing a versatile toolkit of video analysis techniques where the end user can customize the analysis pipeline. Our software, written in Java, not only provides versions of the image analysis algorithms found in current software packages, it also provides alternate and improved techniques to allow the user to mix and match processing steps to accommodate their specific needs. These algorithms include gray-scale conversion, histogram analysis, noise reduction, contrast adjustment, binarization, thinning, edge detection, straight line and spline fitting, as well as polygonalization. To facilitate the pipeline creation and management process, our program provides an intuitive drag and drop interface that allows a user to select processing algorithms, set their parameters, and connect them together. Users can then test and refine their pipeline by viewing the video at each stage of processing. The output of the process is a standardized file format that contains the key characteristics of the worm's motion in a format that is compatible with statistical analysis software.

Sreekanth Chalasani et al. (2006) Neuronal Development, Synaptic Function, and Behavior Meeting "Molecular analysis of a neural circuit for navigation in Caenorhabditis elegans"

Sreekanth Chalasani, Cornelia Bargmann, Jesse Gray, Nikolas Chronis

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

Navigation in C. elegans is achieved by sustained forward movement that is interrupted with reversals and turns (jointly termed pirouettes, Pierce-Shimomura et al 1999). We are interested in the neural circuit that controls the frequency of reversals and turns during exploratory behavior. After worms are taken off bacterial food, they exhibit an initial local search with a high frequency of pirouettes. The AWC and ASK sensory neurons and the AIB interneurons stimulate pirouettes immediately after removal from food, while the AIY interneurons inhibit pirouettes. (Tsalik and Hobert 2003, Wakabayashi et al 2004, Gray et al 2005). How is activity transmitted through this neuronal circuit? The neurotransmitters glutamate and dopamine regulate turning frequency (Hills et al 2004). We have found that the sensory neuron AWC releases two neurotransmitters (glutamate and a neuropeptide, NLP-1) when the worm is removed from food. The released glutamate acts to activate AIB and inhibit AIY, promoting reversals. By contrast, the neuropeptide NLP-1 serves to reduce reversals, suggesting that reversal frequencies are regulated by at least two opposing signaling systems. Strains with different reversal frequencies can be generated by manipulating the level of glutamate receptors on interneurons AIB and AIY. These results provide a plausible molecular explanation that links neurotransmitters, their receptors, and neuronal circuitry to generate behavior. We are currently using genetically encoded calcium sensors to image neuronal activity in these neurons.