An imaging system for standardized quantitative analysis of C. elegans behavior

BACKGROUND: The nematode Caenorhabditis elegans is widely used for the genetic analysis of neuronal cell biology, development, and behavior. Because traditional methods for evaluating behavioral phenotypes are qualitative and imprecise, there is a need for tools that allow quantitation and standardization of C. elegans behavioral assays. RESULTS: Here we describe a tracking and imaging system for the automated analysis of C. elegans morphology and behavior. Using this system, it is possible to record the behavior of individual nematodes over long time periods and quantify 144 specific phenotypic parameters. CONCLUSIONS: These tools for phenotypic analysis will provide reliable, comprehensive scoring of a wide range of behavioral abnormalities, and will make it possible to standardize assays such that behavioral data from different labs can readily be compared. In addition, this system will facilitate high-throughput collection of phenotypic data that can ultimately be used to generate a comprehensive database of C. elegans phenotypic information. AVAILABILITY: The hardware configuration and software for the system are available from [email protected]

MAGI-1 Modulates AMPA Receptor Synaptic Localization and Behavioral Plasticity in Response to Prior Experience

It is well established that the efficacy of synaptic connections can be rapidly modified by neural activity, yet how the environment and prior experience modulate such synaptic and behavioral plasticity is only beginning to be understood. Here we show in C. elegans that the broadly conserved scaffolding molecule MAGI-1 is required for the plasticity observed in a glutamatergic circuit. This mechanosensory circuit mediates reversals in locomotion in response to touch stimulation, and the AMPA-type receptor (AMPAR) subunits GLR-1 and GLR-2, which are required for reversal behavior, are localized to ventral cord synapses in this circuit. We find that animals modulate GLR-1 and GLR-2 localization in response to prior mechanosensory stimulation; a specific isoform of MAGI-1 (MAGI-1L) is critical for this modulation. We show that MAGI-1L interacts with AMPARs through the intracellular domain of the GLR-2 subunit, which is required for the modulation of AMPAR synaptic localization by mechanical stimulation. In addition, mutations that prevent the ubiquitination of GLR-1 prevent the decrease in AMPAR localization observed in previously stimulated magi-1 mutants. Finally, we find that previously-stimulated animals later habituate to subsequent mechanostimulation more rapidly compared to animals initially reared without mechanical stimulation; MAGI-1L, GLR-1, and GLR-2 are required for this change in habituation kinetics. Our findings demonstrate that prior experience can cause long-term alterations in both behavioral plasticity and AMPAR localization at synapses in an intact animal, and indicate a new, direct role for MAGI/S-SCAM proteins in modulating AMPAR localization and function in the wake of variable sensory experience

Calcineurin and Protein kinase G regulate C. elegans behavioral quiescence during locomotion in liquid

<p>Abstract</p> <p>Background</p> <p>Most rhythmic motor behaviors in nature are episodic i.e. they alternate between different behavioral states, including quiescence. Electrophysiological studies in invertebrate behavioral switching, maintenance and quiescence have elucidated several neuronal mechanisms that generate a temporal pattern in behavior. However, the genetic bases of these processes are less well studied. We have previously uncovered a novel episodic behavior exhibited by <it>C. elegans </it>in liquid media where they alternate between distinct phases of rhythmic swimming and quiescence. Here, we have investigated the effect of several genes and their site of action on the behavioral quiescence exhibited in liquid by the nematode <it>C. elegans</it>.</p> <p>Results</p> <p>We have previously reported that high cholinergic signaling promotes quiescence and command interneurons are critical for timing the quiescence bout durations. We have found that in addition to command interneurons, sensory neurons are also critical for quiescence. We show that the protein phosphatase calcineurin homolog <it>tax-6 </it>promotes swimming whereas the protein kinase G homolog <it>egl-4 </it>promotes quiescence. <it>tax-6 </it>expression in the sensory neurons is sufficient to account for its effect. <it>egl-4 </it>also acts in multiple sensory neurons to mediate its effect on quiescence. In addition our data is consistent with regulation of quiescence by <it>egl-4 </it>acting functionally downstream of release of acetylcholine (ACh) by motor neurons.</p> <p>Conclusions</p> <p>Our study provides genetic evidence for mechanisms underlying the maintenance of a behavioral state operating at multiple neuronal levels through the activities of a kinase and a phosphatase. These results in a genetically tractable organism establish a framework for further dissection of the mechanism of quiescence during episodic behaviors.</p

Isolation and Culture of Larval Cells from C. elegans

Cell culture is an essential tool to study cell function. In C. elegans the ability to isolate and culture cells has been limited to embryonically derived cells. However, cells or blastomeres isolated from mixed stage embryos terminally differentiate within 24 hours of culture, thus precluding post-embryonic stage cell culture. We have developed an efficient and technically simple method for large-scale isolation and primary culture of larval-stage cells. We have optimized the treatment to maximize cell number and minimize cell death for each of the four larval stages. We obtained up to 7.8×104 cells per microliter of packed larvae, and up to 97% of adherent cells isolated by this method were viable for at least 16 hours. Cultured larval cells showed stage-specific increases in both cell size and multinuclearity and expressed lineage- and cell type-specific reporters. The majority (81%) of larval cells isolated by our method were muscle cells that exhibited stage-specific phenotypes. L1 muscle cells developed 1 to 2 wide cytoplasmic processes, while L4 muscle cells developed 4 to 14 processes of various thicknesses. L4 muscle cells developed bands of myosin heavy chain A thick filaments at the cell center and spontaneously contracted ex vivo. Neurons constituted less than 10% of the isolated cells and the majority of neurons developed one or more long, microtubule-rich protrusions that terminated in actin-rich growth cones. In addition to cells such as muscle and neuron that are high abundance in vivo, we were also able to isolate M-lineage cells that constitute less than 0.2% of cells in vivo. Our novel method of cell isolation extends C. elegans cell culture to larval developmental stages, and allows use of the wealth of cell culture tools, such as cell sorting, electrophysiology, co-culture, and high-resolution imaging of subcellular dynamics, in investigation of post-embryonic development and physiology

A TRPV Channel Modulates C. elegans Neurosecretion, Larval Starvation Survival, and Adult Lifespan

For most organisms, food is only intermittently available; therefore, molecular mechanisms that couple sensation of nutrient availability to growth and development are critical for survival. These mechanisms, however, remain poorly defined. In the absence of nutrients, newly hatched first larval (L1) stage Caenorhabditis elegans halt development and survive in this state for several weeks. We isolated mutations in unc-31, encoding a calcium-activated regulator of neural dense-core vesicle release, which conferred enhanced starvation survival. This extended survival was reminiscent of that seen in daf-2 insulin-signaling deficient mutants and was ultimately dependent on daf-16, which encodes a FOXO transcription factor whose activity is inhibited by insulin signaling. While insulin signaling modulates metabolism, adult lifespan, and dauer formation, insulin-independent mechanisms that also regulate these processes did not promote starvation survival, indicating that regulation of starvation survival is a distinct program. Cell-specific rescue experiments identified a small subset of primary sensory neurons where unc-31 reconstitution modulated starvation survival, suggesting that these neurons mediate perception of food availability. We found that OCR-2, a transient receptor potential vanilloid (TRPV) channel that localizes to the cilia of this subset of neurons, regulates peptide-hormone secretion and L1 starvation survival. Moreover, inactivation of ocr-2 caused a significant extension in adult lifespan. These findings indicate that TRPV channels, which mediate sensation of diverse noxious, thermal, osmotic, and mechanical stimuli, couple nutrient availability to larval starvation survival and adult lifespan through modulation of neural dense-core vesicle secretion

A gene expression fingerprint of C. elegans embryonic motor neurons

BACKGROUND: Differential gene expression specifies the highly diverse cell types that constitute the nervous system. With its sequenced genome and simple, well-defined neuroanatomy, the nematode C. elegans is a useful model system in which to correlate gene expression with neuron identity. The UNC-4 transcription factor is expressed in thirteen embryonic motor neurons where it specifies axonal morphology and synaptic function. These cells can be marked with an unc-4::GFP reporter transgene. Here we describe a powerful strategy, Micro-Array Profiling of C. elegans cells (MAPCeL), and confirm that this approach provides a comprehensive gene expression profile of unc-4::GFP motor neurons in vivo. RESULTS: Fluorescence Activated Cell Sorting (FACS) was used to isolate unc-4::GFP neurons from primary cultures of C. elegans embryonic cells. Microarray experiments detected 6,217 unique transcripts of which ~1,000 are enriched in unc-4::GFP neurons relative to the average nematode embryonic cell. The reliability of these data was validated by the detection of known cell-specific transcripts and by expression in UNC-4 motor neurons of GFP reporters derived from the enriched data set. In addition to genes involved in neurotransmitter packaging and release, the microarray data include transcripts for receptors to a remarkably wide variety of signaling molecules. The added presence of a robust array of G-protein pathway components is indicative of complex and highly integrated mechanisms for modulating motor neuron activity. Over half of the enriched genes (537) have human homologs, a finding that could reflect substantial overlap with the gene expression repertoire of mammalian motor neurons. CONCLUSION: We have described a microarray-based method, MAPCeL, for profiling gene expression in specific C. elegans motor neurons and provide evidence that this approach can reveal candidate genes for key roles in the differentiation and function of these cells. These methods can now be applied to generate a gene expression map of the C. elegans nervous system

A wake-active locomotion circuit depolarizes a sleep-active neuron to switch on sleep

Sleep-active neurons depolarize during sleep to suppress wakefulness circuits. Wake-active wake-promoting neurons in turn shut down sleep-active neurons, thus forming a bipartite flip-flop switch. However, how sleep is switched on is unclear because it is not known how wakefulness is translated into sleep-active neuron depolarization when the system is set to sleep. Using optogenetics in Caenorhabditis elegans, we solved the presynaptic circuit for depolarization of the sleep-active RIS neuron during developmentally regulated sleep, also known as lethargus. Surprisingly, we found that RIS activation requires neurons that have known roles in wakefulness and locomotion behavior. The RIM interneurons-which are active during and can induce reverse locomotion-play a complex role and can act as inhibitors of RIS when they are strongly depolarized and as activators of RIS when they are modestly depolarized. The PVC command interneurons, which are known to promote forward locomotion during wakefulness, act as major activators of RIS. The properties of these locomotion neurons are modulated during lethargus. The RIMs become less excitable. The PVCs become resistant to inhibition and have an increased capacity to activate RIS. Separate activation of neither the PVCs nor the RIMs appears to be sufficient for sleep induction; instead, our data suggest that they act in concert to activate RIS. Forward and reverse circuit activity is normally mutually exclusive. Our data suggest that RIS may be activated at the transition between forward and reverse locomotion states, perhaps when both forward (PVC) and reverse (including RIM) circuit activity overlap. While RIS is not strongly activated outside of lethargus, altered activity of the locomotion interneurons during lethargus favors strong RIS activation and thus sleep. The control of sleep-active neurons by locomotion circuits suggests that sleep control may have evolved from locomotion control. The flip-flop sleep switch in C. elegans thus requires an additional component, wake-active sleep-promoting neurons that translate wakefulness into the depolarization of a sleep-active neuron when the worm is sleepy. Wake-active sleep-promoting circuits may also be required for sleep state switching in other animals, including in mammals

Using C. elegans to decipher the cellular and molecular mechanisms underlying neurodevelopmental disorders

Prova tipográfica (uncorrected proof)Neurodevelopmental disorders such as epilepsy, intellectual disability (ID), and autism spectrum disorders (ASDs) occur in over 2 % of the population, as the result of genetic mutations, environmental factors, or combination of both. In the last years, use of large-scale genomic techniques allowed important advances in the identification of genes/loci associated with these disorders. Nevertheless, following association of novel genes with a given disease, interpretation of findings is often difficult due to lack of information on gene function and effect of a given mutation in the corresponding protein. This brings the need to validate genetic associations from a functional perspective in model systems in a relatively fast but effective manner. In this context, the small nematode, Caenorhabditis elegans, presents a good compromise between the simplicity of cell models and the complexity of rodent nervous systems. In this article, we review the features that make C. elegans a good model for the study of neurodevelopmental diseases. We discuss its nervous system architecture and function as well as the molecular basis of behaviors that seem important in the context of different neurodevelopmental disorders. We review methodologies used to assess memory, learning, and social behavior as well as susceptibility to seizures in this organism. We will also discuss technological progresses applied in C. elegans neurobiology research, such as use of microfluidics and optogenetic tools. Finally, we will present some interesting examples of the functional analysis of genes associated with human neurodevelopmental disorders and how we can move from genes to therapies using this simple model organism.The authors would like to acknowledge Fundação para a Ciência e Tecnologia (FCT) (PTDC/SAU-GMG/112577/2009). AJR and CB are recipients of FCT fellowships: SFRH/BPD/33611/2009 and SFRH/BPD/74452/2010, respectively

Cross-Modulation of Homeostatic Responses to Temperature, Oxygen and Carbon Dioxide inC. elegans

Different interoceptive systems must be integrated to ensure that multiple homeostatic insults evoke appropriate behavioral and physiological responses. Little is known about how this is achieved. Using C. elegans, we dissect cross-modulation between systems that monitor temperature, O₂ and CO₂. CO₂ is less aversive to animals acclimated to 15°C than those grown at 22°C. This difference requires the AFD neurons, which respond to both temperature and CO₂ changes. CO₂ evokes distinct AFD Ca²⁺ responses in animals acclimated at 15°C or 22°C. Mutants defective in synaptic transmission can reprogram AFD CO₂ responses according to temperature experience, suggesting reprogramming occurs cell autonomously. AFD is exquisitely sensitive to CO₂. Surprisingly, gradients of 0.01% CO₂/second evoke very different Ca²⁺ responses from gradients of 0.04% CO₂/second. Ambient O₂ provides further contextual modulation of CO₂ avoidance. At 21% O₂ tonic signalling from the O₂-sensing neuron URX inhibits CO₂ avoidance. This inhibition can be graded according to O₂ levels. In a natural wild isolate, a switch from 21% to 19% O₂ is sufficient to convert CO₂ from a neutral to an aversive cue. This sharp tuning is conferred partly by the neuroglobin GLB-5. The modulatory effects of O₂ on CO₂ avoidance involve the RIA interneurons, which are post-synaptic to URX and exhibit CO₂-evoked Ca²⁺ responses. Ambient O₂ and acclimation temperature act combinatorially to modulate CO₂ responsiveness. Our work highlights the integrated architecture of homeostatic responses in C. elegans