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

[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.

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

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

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

Jesse Gray, Cornelia Bargmann, Nikolas Chronis, Sreekanth Chalasani

[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.

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

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

[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.

Lau, Hiu et al. (2015) International Worm Meeting "Acute food deprivation activates neuropeptide signaling in diverse tissues to modify avoidance behavior."

Chalasani, Sreekanth, Lau, Hiu

[International Worm Meeting, 2015]

Animals detect relevant cues in the environment and modify their behaviors to maximize survival and fitness. Underlying neural circuits integrate information about external stimuli with those from internal states to generate appropriate behaviors. In particular, food status signals are crucial internal state signals with profound effects on an animal's behavior. The underlying neural and molecular machinery for communicating internal state remain incompletely understood.We used acute food deprivation to probe the effects of internal states on behavior. In a sensory integration assay, C. elegans cross a copper barrier and chemotax toward a spot of attractive diacetyl odor. We find that two hours of food deprivation experience is sufficient to elicit robust behavior modification. Animals deprived of food cross the copper barrier more readily compared to animals fed E. coli. Food deprivation acts on different tissues including neurons, intestine and body wall muscle, which process and release peptides. All of these tissues use the AEX-5 peptide processing enzyme and UNC-31 calcium-dependent activator protein for secretion, respectively. Downstream, the insulin receptor, DAF-2, receives these tissue-released peptide signals and modifies neural circuits generating behavioral plasticity. Our analyses of signaling machinery reveal that conserved components and pathways relay the internal status of the animal to neurons to elicit robust modification in behavioral responses. Together, this research provides important insight into the complex signaling required for animals to respond to changes in food availability.

Lau, Hiu et al. (2013) International Worm Meeting "Food signals modulate sensory integration behavior."

Chalasani, Sreekanth, Lau, Hiu

[International Worm Meeting, 2013]

Neural circuits integrate information to generate behavior outputs. One challenge in neuroscience is to understand how neural circuits generate flexible behaviors. We use a sensory integration assay1 to ask how exposure to various food signals influences behavior. Here, C. elegans crosses a repellent barrier (copper) and chemotax towards a spot of an attractant (diacetyl)1. After C. elegans are exposed to the bacteria P. aeruginosa for 3 hours, we observe a two-fold increase in the number of animals that reach the attractant compared to those fed E. coli. We find that exposure to multiple strains of bacteria (including non-pathogenic ones) causes a similar behavioral change. To test the persistence of this behavior modulation, we transferred animals back to a diet of E. coli after P. aeruginosa exposure. We find that behavioral modulation by P. aeruginosa persists for two hours. Together these results present a form of neural circuit flexibility, where food signals modify behavioral outputs. From a pilot screen of signaling molecules, we found that knocking down neuropeptides impairs integration behavior. Neuropeptides are cleaved by specific proprotein convertase enzymes to form mature neuropeptides. To evaluate the role of individual subsets of neuropeptides, we tested proprotein convertase mutants bli-4, kpc-1, aex-5 and egl-3 2. We found that aex-5 mutants do not change behavioral response even after exposure to P. aeruginosa. This result suggests that neuropeptide(s) processed by AEX-5 is required for behavior modulation in response to food changes. We are in the process of identifying peptides involved in modulation of sensory integration behavior. Using this model, we aim to reveal the mechanisms regulating the dynamics of neural circuit functions in response to changes in prior food experience. 1. Ishihara T et al. Cell, 109: 639-649 (2002). 2. Li, C. and Kim, K. Neuropeptides (September 25, 2008), WormBook, ed. The C. elegans Research Community, WormBook, doi/10.1895/wormbook.1.142.1, http://www.wormbook.org.

Leinwand, Sarah et al. (2013) International Worm Meeting "Neuropeptide signaling remodels chemosensory circuit composition."

Chalasani, Sreekanth, Leinwand, Sarah

[International Worm Meeting, 2013]

Sensory neural circuits detect dynamic changes in the environment and drive behavior. In particular, chemosensory neural circuits have the challenge of representing a potentially limitless set of novel smell and taste stimuli. Neural circuits may achieve this by switching their path of information flow between alternative circuit configurations1,2; however, the mechanisms underlying circuit remodeling are poorly understood. We combine genetics, in vivo calcium imaging, and behavioral analysis in C. elegans to understand how a neural circuit driven by the ASE sensory neuron represents specific changes in salt stimuli to drive appropriate behavioral responses. We define a novel, sensory context-dependent and neuropeptide-regulated switch in the composition of a C. elegans salt sensory circuit. The primary salt detectors, ASE sensory neurons, use a specific peptide-processing enzyme (the proprotein convertase, BLI-4), which previously had no known function in the nervous system, to release insulin neuropeptides in response to large but not small changes in external stimuli. The insulin neuropeptides signal through the tyrosine kinase receptor, daf-2, and PI3-Kinase, age-1, to functionally transform the AWC olfactory sensory neuron into an interneuron in the salt neural circuit. Consistent with these results, animals with disrupted ASE-AWC neuropeptide signaling show a specific deficit in high, but not low, salt driven behaviors, suggesting that peptidergic signaling potentiates the normal response to high salt. This novel, peptide-regulated high salt circuit configuration may be critical in reinforcing salt appetite to maintain ion homeostasis. Our results show that sensory context and peptidergic signaling select the active routes of information flow from alternative neural circuit configurations, which may be a general mechanism for encoding dynamic environments and driving appropriate behaviors. 1Anderson, C. & Van Essen, D. PNAS (1987). 2Weimann, J.M. & Marder, E. Curr Biol (1994).

Tong, Ada et al. (2013) International Worm Meeting "Exploring the role of rapsyn-1 in regulating C. elegans behavior."

Tong, Ada, Chalasani, Sreekanth

[International Worm Meeting, 2013]

Acetylcholine (ACh) signaling is a well-studied pathway in the neuromuscular junction (NMJ), but its role in the nervous system is poorly characterized. Rapsyn is a 43-kD protein that clusters and anchors acetylcholine receptors (AChR) in the postsynaptic membrane of the NMJ. Mutations in rapsyn have been associated with neuromuscular diseases in humans. For example, the N88K mutation in the RAPSN gene causes congenital myasthenic syndrome (1). In mice, targeted disruption of the RAPSN gene causes death within a few hours of birth (2). In C. elegans, however, rapsyn-1 (rpy-1) mutants are viable and appear wildtype in their locomotory ability. We plan to combine genetics and behavioral analysis to understand the role of rpy-1 in the worm. We find that rpy-1 mutants display a gain-of-function behavioral phenotype in the local search assay. They make twice as many turns in the first 15 minutes off food when compared to N2 wildtype animals. In order to test where rpy-1 functions, we performed knockdown experiments by expressing sense and antisense rpy-1 transcripts in muscles or in neurons. Surprisingly, neuronal knockdowns of rpy-1 are similar to rpy-1 mutants, while muscle knockdowns appear wildtype. This suggests that rpy-1 is required in the neurons rather than in the muscle. We will use transgenic rescue experiments to identify the cellular sites of rpy-1 action. We also plan on using candidate gene mutants and biochemical methods to identify components of the rpy-1 signaling pathway. These studies will provide insights into ACh signaling in the nervous system. (1) Dunne, V., Maselli, R. A., Hum. Genet. 49: 366-369 (2004) (2) Gautam, M., et al., Nature 377: 232-236 (1995).

Calhoun, Adam et al. (2013) International Worm Meeting "A circuit for working memory in C. elegans."

Sharpee, Tatyana, Calhoun, Adam, Chalasani, Sreekanth

[International Worm Meeting, 2013]

How do organisms use neural circuits to learn about their environment and then use that knowledge to guide behavior? The nervous system of C. elegans contains 302 neurons whose connectivity is fully mapped, simplifying the process of investigating neural circuits. These animals are faced with the common problem of how to best find new sources of food when they are transferred to a food-free plate. Their strategy is to produce many turns to stay in a small area when it believes food is nearby and to suppress those turns when it cannot find food. We have found that C. elegans' off-food search strategy is dependent on the size of the food patch it has most recently been exploring suggesting that it uses its prior experience to guide search behavior. Using a dimensional reduction technique, we have identified the relevant information in the on-food behavior and in the structure of the bacterial patch that predicts off-food search. The information is decoded by two sensory neurons, ASI and ASK, whose responses to bacteria match the relevant on-food statistics. Downstream of these sensory neurons, we identify several interneurons required for learning: removal of any individual neuron prevents the animal from learning the patch size. We additionally identify a downstream dopaminergic neuron, CEP, which is crucial for learning. Dopamine interacts with the broader circuit via two D1-like receptors, dop-1 and dop-4 on sensory neurons and postsynaptic interneurons respectively. We also find that the CREB homologue, crh-1, is required in the same interneurons as dop-4. The neurons required for learning overlap with the neurons required to perform a search suggesting that the same network that generates behavior also contains within itself the ability to modify it.

Haghani, Nadia et al. (2021) International Worm Meeting "A closer look at cuticle-resident microbes and their impact on host physiology"

Matty, Molly, Chalasani, Sreekanth, Haghani, Nadia

[International Worm Meeting, 2021]

All animals are in contact with communities of microbes, termed a microbiome. Microbiomes play a large role in the determination of host physiology, health, and behavior. While the gut microbiome takes the spotlight of current research, the skin microbiome is an unrecognized area of study despite harboring a diverse community of microorganisms. This is the case for the model nematode, Caenorhabditis elegans; despite subtle yet convincing implications of surface-adherent bacteria, microbial interactions with its cuticle (skin) remain understudied and a "skin microbiome" unacknowledged in literature. C. elegans is constantly surrounded by microorganisms in their natural habitat, proven by their common isolation from rotting plants filled with microbes. These microbes inevitably come into contact with the worm cuticle and so the adherence of these microbes is a likely reality. Existence of these surface-adherent microbes is further supported by the adoption of extensive washing protocols which aim to rid the cuticle surface of all residual microbes. We seek to identify, characterize, and define a role for these cuticle-resident microbes in C. elegans using a natural model microbiota, CeMbio. Significantly, we characterize a discrepancy between the sheer number of bacteria between surface-bleached and unbleached animals via Colony Forming Unit (CFU) counts. We demonstrate that a large number of cutaneous bacteria reside on the C. elegans skin. Furthermore, our preliminary results suggest that bacterial isolates within CeMbio can be primarily gut- or skin-dominating based on the relative bacterial abundances from 16S rRNA sequencing. To understand how skin-dominating bacteria affects host physiology, we use Hoechst 34580 uptake to assess how CeMbio variably affects cuticle integrity in mutant animals. From our results, we hypothesize that CeMbio bacteria interact with the cuticle structures of C. elegans to impact worm integrity both positively and negatively. These studies can provide a deeper understanding of how environmental microbes elicit changes in host physiology and explain the role of natural microbes in an animal's primary defense-the skin.