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[
International Worm Meeting,
2019]
Whole brain imaging of C. elegans holds incredible potential for investigating how nervous systems integrate sensory input and drive behavior. This is particularly true with the application of microfluidics to precisely manipulate worms' sensory environment. However, collecting high-quality volumetric images with sufficient speed to track individual neurons as a worm moves and deforms tests the limits of optical microscopy. Light sheet microscopy has proven to have several advantages for high-speed volumetric imaging, including low light exposure and high collection efficiency, which make it ideal for imaging small, transparent specimens. However, the geometry of conventional light sheet microscopes requires that the sample be suspended in the small volume between the separate illumination and detection objectives. This renders these microscopes incompatible with microfluidics, agarose pads, or any other planar sample mounting which would be physically blocked by the objectives. We present an open-top light sheet microscope capable of fast volumetric imaging that is compatible with planar microfluidics and demonstrate its utility for recording neural activity in C. elegans. This design uses remote focusing with an electrically tunable lens to rapidly reposition the image plane without physically moving the detection objective, enabling acquisition at camera-limited volume rates. We show that multi-channel fluorescence imaging at 10+ volumes/second with light sheet microscopy improves whole brain calcium imaging and reduces motion artifacts. We also demonstrate its ability to record fine anatomical features during mechanical stimulation through microfluidics.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Tracking development of individual animals can provide insights into dynamics of processes such as synaptogenesis, synaptic re-arrangement, and axonal growth cone development. However, such longitudinal studies are experimentally complicated and sometimes impossible to perform on the basis of individual worms; this is largely due to negative effects of anaesthetics on physiology and development and the difficulty of keeping track of individual worms. We developed a platform to i) repeatedly immobilize animals at physiological temperature and conditions, ii) perform high-magnification imaging while keeping track of individual specimen, and iii) culture animals between imaging cycles for proper development for days. The platform is based on the thermo-reversible sol-gel transition of a polymer solution (within 1 degree Celcius), which is used for the reversible immobilization of animals. A microfluidic system is used in conjunction for animal trapping, nutrient delivery, and fluid and precise temperature control. Worm embryos are trapped in individual chambers via connected embryo traps. Gentle flow of M9 with OP50 bacteria and cholesterol is delivered into each chamber, allowing animals to feed and develop while trapping them inside the chamber. Precise temperature modulation by integrated microelectrodes controls the sol-gel transition for immobilization; the devices are ease to set up, re-usable, and economical. During immobilization, the gel exerts uniform pressure along the animal body, and thus leads to negligible physical deformation. Image quality (e.g. amount of diffraction, photo-bleaching) when using the gel is comparable to that of standard methods with anaesthetics. We have verified the gel's biocompatibility with C. elegans; repeated and long-term exposure and immobilization show no effect on measured physiological traits such as pharyngeal pumping rate, number of progeny, and time to reach egg-laying. We tested the functionality of our system by monitoring the re-arrangement of synaptic connections of the six GABAergic motor neurons at the end of the L1 larval stage (Hallam and Jin, Nature, 1998). To our knowledge, our platform is the only system to date that facilitates longitudinal studies in the early stages of development on an individual-animal basis. It is also the first to enable live imaging of short-term (order of seconds) to long-term (hours and days) developmental events at arbitrary intervals on a single device.
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[
International Worm Meeting,
2013]
Imprinting, a special form of learning during an early developmental stage, can generate a long-lasting memory and change animal behavior. We discovered a novel behavior in which early exposure of larval C. elegans to pathogen elicits a long-term aversion that is maintained for days, which we call food choice imprinting. It has been previously reported that adult C. elegans can learn to avoid pathogen Pseudomonas aeruginosa (PA14) after a single 6-hour exposure, but will lose the memory after 12 hours and return to the same preference as naive worms (Zhang et al., 2005). We modified this learning assay by performing pathogen training of newly hatched C. elegans larvae. Remarkably, larval-trained (henceforth, imprinted) animals retain this food choice memory days later, showing aversion to pathogen even as aged adults. To dissect the neural circuits of food choice imprinting, we used an inhibitory chloride channel to disrupt neuronal activity at different developmental stages, asking whether candidate neurons are required for memory formation during training, memory consolidation, or memory retrieval. In initial experiments, we have found that inhibiting the AIB interneuron during larval training disrupts performance, whereas inhibiting AIB during adult training does not affect adult learning. This result suggests that AIB is required to form a memory of the imprinted food choice, but not required for avoidance learning in adults. Through a combination of genetic and functional methods, we hope to elucidate the molecular basis of food choice imprinting in C. elegans. Reference: Zhang, Y.; Lu, H.; Bargmann, C. I., Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature 2005, 438 (7065), 179-84.
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[
International Worm Meeting,
2007]
Hydrogen sulfide (H<sub>2</sub>S), which is naturally produced in animal cells, has been shown to effect physiological changes that improve the capacity of mammals to survive environmental changes. We have investigated the physiological response of C. elegans to H<sub>2</sub>S to begin to elucidate the molecular mechanisms of H<sub>2</sub>S action. We show that nematodes exposed to H<sub>2</sub>S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. However, we observed that animals exposed to H<sub>2</sub>S had increased thermotolerance and lifespan and survived subsequent exposure to otherwise lethal concentrations of H<sub>2</sub>S. Increased thermotolerance and lifespan is not observed in the
sir-2.1(
ok434) deletion mutant exposed to H<sub>2</sub>S. However, mutants in the insulin signaling pathway (both
daf-2 and
daf-16), animals with mitochondrial dysfunction (
isp-1 and
clk-1) and a genetic model of caloric restriction (
eat-2) all exhibit H<sub>2</sub>S-induced increased thermotolerance. These data suggest that H<sub>2</sub>S activates a pathway including SIR-2.1 that is separate from dietary restriction and insulin signaling that results in increased lifespan. Moreover, these studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H<sub>2</sub>S increases thermotolerance and lifespan in nematodes are conserved, and that studies using C. elegans may help explain beneficial effects observed in mammals exposed to H<sub>2</sub>S.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
After experiencing an olfactory cue concurrent with harmful experience, animals can learn to avoid the odour. Studies in both vertebrates and invertebrates have implicated specific brain areas, neurons and neurotransmitters in olfactory learning. However, a systems-level analysis of how olfactory plasticity is encoded in a neural network has not yet been possible. Caenorhabditis elegans is able to learn to avoid the smell of pathogenic bacteria, a potential peril in their food source. Naive animals prefer smells of pathogenic bacteria but animals trained with pathogens lose this attraction (Zhang et al., 2005, Nature, 438:179). Here, we have mapped an olfactory neural network, from sensory neurons to motor outputs, that regulates this aversive olfactory learning ability. Using laser ablation of individual neurons and a novel single-animal learning assay, we mapped three interconnected neural circuits that generate olfactory preference in naive and learned animals. Two of the circuits are distinct, with one required for the nave preference and the other specifically for the trained preference; and both circuits are connected to the third circuit that transduces olfactory inputs to locomotor responses. Behavioral analyses and calcium imaging recordings revealed that the intrinsic properties of olfactory sensory neurons encode the nave preference while modulation of motor outputs alters the preference during learning. These findings reveal the organizational principles that allow a neural network for learning to encode both the nave response and experience-dependent plasticity and to produce the optimal behavior under specific conditions. Comparison of this neural network with those that regulate feeding-escaping choice in sea slug and fear extinction and renewal in mice suggests that it is a general principle that switch between alternative behavioral states is generated by the differential usage of anatomically distinct and connected neural circuits. References: Zhang, Y., Lu, H. and Bargmann, C. I. Pathogenic bacteria induce aversive olfactory learning in C. elegans. Nature 438, 179-184 (2005).
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[
International Worm Meeting,
2019]
Screens in C. elegans can be a challenging task when analyzing behavioral response to chemical stimulation for large libraries or genetic screens. Major challenges reside in designing an efficient method to screen large number of chemicals, recovering animals on demand, and minimizing reagent consumption. Here we present a platform for screening C. elegans behavior in a high-throughput manner using droplet microfluidics. We have integrated on the same device several modules that enable precise manipulation of single nematodes. First, an encapsulation module allows for trapping animals in microdroplets, which provides a robust way to transport animals through the microfluidic network. Second, we have designed two microfluidic systems allowing for precise control of the animal's chemical environment. One system merges droplets, allowing for creating mixtures, while the other one exchanges the content of a droplet with another, allowing for full control over the worm's chemical environment. Finally, using on-chip valves, real-time image processing, and a custom LabVIEW program, the operation of the platform is fully automated. We have demonstrated the operation of the platform with a throughput of 100 worms per hour when monitoring behavioral response for 30 s and, as a proof-of-concept, we have studied male response to pheromones. In addition, active control over droplets allows for recovery of animals on demand. This platform can handle adult animals and can be adapted as well for more challenging situations, such as handling first developmental stage larvae. Finally, we demonstrate ultra-low reagent consumption with 100 nL of reagent per animal, which will facilitate screening libraries of expensive or rare compounds.
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[
International Worm Meeting,
2021]
The ability to discriminate between nutritious and harmful food is essential to survival. As a result, learned avoidance to harmful food sources is conserved from invertebrates to humans. The mechanisms enabling the nervous system to associate sensory cues from a food source with an internal state of sickness to trigger aversive memory formation remain elusive. After prolonged exposure to pathogenic food, C. elegans can learn to avoid the pathogen upon subsequent encounter1. This learned aversion requires infection; non virulent forms of bacteria are not sufficient for memory formation. In response to exposure to pathogenic food, serotonin is induced in a pair of sensory neurons called ADF and remodels downstream circuits2. Learned aversion to pathogen requires serotonin signaling from ADF, suggesting that ADF serves as a site of integration for detecting bacterial cues and internal sickness caused by the pathogen. We seek to understand how internal state changes the coupling between sensory activation and serotonin release in ADF neurons. As a first step, we are using calcium imaging to examine ADF responses to bacterial cues in both naive and pathogen-exposed animals. ADF responds robustly to conditioned media from both pathogenic and non-pathogenic bacteria in a dose-dependent fashion, and ADF activity can be modulated by previous odor history. We are screening mutants using these quantitative parameters to assess ADF responses to direct chemosensory stimuli, indirect signaling from other sensory neurons, and signaling from non-neuronal tissues indicating bacterial infection. Our goal is to uncover cell-biological mechanisms through which ADF neurons mediate learned pathogenic behavior in C. elegans. 1. Zhang, Y., Lu, H., & Bargmann, C.I. (2005). Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans. Nature, 438(7065), 179. 2. Morud, J., Hardege, I., Liu, H., Wu, T., Basu, S., Zhang, Y., & Schafer, W. (2020). Deoprhanisation of novel biogenic amine-gated ion channels identifies a new serotonin receptor for learning. bioRxiv. doi: https://doi.org/10.1101/2020.09.17.301382.
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[
International Worm Meeting,
2003]
Class A and class B synthetic Multivulva (synMuv) genes function in genetically redundant pathways to inhibit vulval fate specification. While the exact mechanism of inhibition in C. elegans is unknown, many synMuv genes encode proteins that have been implicated in transcriptional repression in other systems, such as the class B synMuv gene
lin-35 Rb (Lu and Horvitz, 1998). The cellular focus of synMuv genes is currently unclear. Originally, synMuv genes were proposed to act non-autonomously to repress vulval fates from within the
hyp7 (Herman and Hedgecock, 1990; Hedgecock and Herman, 1995), but were subsequently proposed to act autonomously within the vulval precursor cells (VPCs) (Lu and Horvitz, 1998; Thomas and Horvitz, 1999). It is also conceivable that synMuv genes, which in general are broadly expressed, may have more than one cellular focus. Determining the cellular focus of synMuv gene activity is critical to understanding their roles during vulval development and the signalling events that lead to the invariant pattern of VPC fates, as models for the role of synMuv genes depend on inferences about their cellular focus. For example, the existence of an inhibitory signal was inferred based on the interpretation that
hyp7 is the focus of
lin-15 (Herman and Hedgecock, 1990); additionally, the view that LIN-35 Rb and transcription factors activated by the Ras-MAP kinase cascade compete with target gene promoters to antagonize vulval induction (Lu and Horvitz, 1998) requires that the focus of
lin-35 activity be the VPCs. Utilizing both mosaic analysis and tissue-specific promoters for rescue experiments, we are currently trying to determine the focus of class B synMuv gene
lin-35. We will report on our progress at the meeting. Hedgecock E.M., and Herman R.K. (1995). Genetics 141, 989-1006; Herman R.K., and Hedgecock E.M. (1990). Nature 348, 169-171; Lu X., and Horvitz, H.R. (1998). Cell 95, 981-991; Thomas, J.H., and Horvitz, H.R. (1999). Development 126, 3449-3459.
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Cook, S., Sun, G., Lu, H., Han, H., Otopalik, A., Hobert, O., Majeed, M., Berghoff, E., Zhang, K.
[
International Worm Meeting,
2019]
The National Science Foundation funds a program called Next Generation Networks for Neuroscience (NeuroNex), which aims to aid the research community to study the brain. In the context of this NeuroNex initiative, we plan to develop and disseminate tools that will empower the C. elegans neuroscience community to study the connectome of C. elegans. In the first phase, our so-called technology hub will develop two sets of tools: The Hobert lab will use fluorescent-based reporter technology (
rab-3::gfp,
cla-1::gfp, GRASP) to generate a large number of transgenic C. elegans strains in which the main "edges" of the entire wiring diagram (i.e. pairwise combinations of neurons) are visualized. These strains will eventually be distributed throughout the C. elegans community to enable labs with long-standing interest in various aspects of neuronal development and function and with a focus on specific neuronal circuits and behaviors to use these synaptic labels to examine variability, development and plasticity of these connections. In parallel, the Lu lab will develop microfluidic-based and automated image analysis technologies to precisely quantify the structure of the connectome and to enable high-throughput screening of worm population for defects in synaptic wiring. Computer vision and machine learning will be used to automatically score disruptions of synaptic wiring to remove human bias and detect subtle and therefore potentially changes previously unseen.
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[
Neuronal Development, Synaptic Function and Behavior, Madison, WI,
2010]
Neural plasticity is a remarkable feature of the nervous system because it enables organisms to modify their behavior by learning. One way to study neural plasticity is to identify and characterize conserved molecular and cellular mechanisms underlying learning and memory. To this end, we exploit Caenorhabditis elegans, because its nervous system is unusually simple and well-characterized and because it is accessible to genetic, molecular and imaging methods. Our previous work has shown that C. elegans learns to avoid the smell of bacteria that are pathogenic, such as Pseudomonas aeruginosa PA14, while remaining attracted to the smell of the ones that serve as food source, such as Escherichia coli OP50 (Zhang et al, 2005, Nature, 438:179). Our study on the functional assembly of the neuronal network for olfactory learning has identified a circuit that is specifically required for the learning to occur (Meeting abstract #18717). This learning circuit consists of a pair of serotonergic neurons and their downstream interneurons and motor neurons. Laser ablation of these neurons specifically compromised the ability of animals to learn to avoid pathogens without affecting their ability to recognize and distinguish different bacteria strains. We have conducted a genetic screen for novel molecular regulators that function in this learning circuit to generate aversive learning by screening for mutants whose phenotypes mimic the effects of killing the neurons in this circuit. We used EMS as the mutagen and screened about 1,000 haploid genomes with an automated learning assay. This screen has allowed us to identify 3 candidate mutants with different defects in their learning ability. One of the learning mutants appears to be learning defective while two other mutants showed increased learning ability. We are currently conducting backcrossing and plan to identify the molecular nature of the mutations with genome sequencing. Based on the efficiency of EMS and the size of the C. elegans genome, our screen is far from saturation. One immediate extension of the work is to continue the forward genetic screen to isolate additional mutants with learning defects. References:Zhang, Y., Lu, H. and Bargmann, C. I. Pathogenic bacteria induce aversive olfactory learning in C. elegans. Nature 438, 179-184 (2005).