The neurohormone tyramine stimulates the secretion of an Insulin-Like Peptide from the intestine to modulate the systemic stress response in C. elegans.

The DAF-2/insulin/insulin-like growth factor signaling (IIS) pathway plays an evolutionarily conserved role in regulating reproductive development, lifespan, and stress resistance. In C. elegans , DAF-2/IIS signaling is modulated by an extensive array of insulin-like peptides (ILPs) with diverse spatial and temporal expression patterns. However, the release dynamics and specific functions of these ILPs in adapting to different environmental conditions remain poorly understood. Here, we show that the ILP, INS-3, plays a crucial role in modulating the response to different types of stressors in C. elegans . ins-3 mutants display increased resistance to both heat and oxidative stress; however, under favorable conditions, this advantage is countered by slower reproductive development. ins-3 expression in both neurons and the intestine is downregulated in response to environmental stressors. Conversely, the neurohormone tyramine, which is released during the acute flight response, triggers an upregulation in ins-3 expression. Moreover, we found that tyramine negatively impacts environmental stress resistance by stimulating the release of INS-3 from the intestine. The subsequent release of INS-3 systemically activates the DAF-2 pathway, resulting in the inhibition of cytoprotective mechanisms mediated by DAF-16/FOXO and HSF-1. These studies offer mechanistic insights into the brain-gut communication pathway that weighs adaptive strategies to respond to acute and long-term stress scenarios.

Vitamin B12 produced by gut bacteria modulates cholinergic signalling.

A growing body of evidence indicates that gut microbiota influence brain function and behaviour. However, the molecular basis of how gut bacteria modulate host nervous system function is largely unknown. Here we show that vitamin B12-producing bacteria that colonize the intestine can modulate excitatory cholinergic signalling and behaviour in the host Caenorhabditis elegans. Here we demonstrate that vitamin B12 reduces cholinergic signalling in the nervous system through rewiring of the methionine (Met)/S-adenosylmethionine cycle in the intestine. We identify a conserved metabolic crosstalk between the methionine/S-adenosylmethionine cycle and the choline-oxidation pathway. In addition, we show that metabolic rewiring of these pathways by vitamin B12 reduces cholinergic signalling by limiting the availability of free choline required by neurons to synthesize acetylcholine. Our study reveals a gut-brain communication pathway by which enteric bacteria modulate host behaviour and may affect neurological health.

PARP knockdown promotes synapse reformation after axon injury.

Injured nervous systems are often incapable of self-repairing, resulting in permanent loss of function and disability. To restore function, a severed axon must not only regenerate, but must also reform synapses with target cells. Together, these processes beget functional axon regeneration. Progress has been made towards a mechanistic understanding of axon regeneration. However, the molecular mechanisms that determine whether and how synapses are formed by a regenerated motor axon are not well understood. Using a combination of in vivo laser axotomy, genetics, and high-resolution imaging, we find that poly (ADP-ribose) polymerases (PARPs) inhibit synapse reformation in regenerating axons. As a result, regenerated parp(-) axons regain more function than regenerated wild-type axons, even though both have reached their target cells. We find that PARPs regulate both axon regeneration and synapse reformation in coordination with proteolytic calpain CLP-4. These results indicate approaches to functionally repair the injured nervous system must specifically target synapse reformation, in addition to other components of the injury response.

Cannabinoids activate the insulin pathway to modulate mobilization of cholesterol in C. elegans.

The nematode Caenorhabditis elegans requires exogenous cholesterol to survive and its depletion leads to early developmental arrest. Thus, tight regulation of cholesterol storage and distribution within the organism is critical. Previously, we demonstrated that the endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) plays a key role in C. elegans since it modulates sterol mobilization. However, the mechanism remains unknown. Here we show that mutations in the ocr-2 and osm-9 genes, coding for transient receptors potential V (TRPV) ion channels, dramatically reduce the effect of 2-AG in cholesterol mobilization. Through genetic analysis in combination with the rescue of larval arrest induced by sterol starvation, we found that the insulin/IGF-1signaling (IIS) pathway and UNC-31/CAPS, a calcium-activated regulator of neural dense-core vesicles release, are essential for 2-AG-mediated stimulation of cholesterol mobilization. These findings indicate that 2-AG-dependent cholesterol trafficking requires the release of insulin peptides and signaling through the DAF-2 insulin receptor. These results suggest that 2-AG acts as an endogenous modulator of TRPV signal transduction to control intracellular sterol trafficking through modulation of the IGF-1 signaling pathway.

Age-associated changes to neuronal dynamics involve a disruption of excitatory/inhibitory balance in C. elegans.

In the aging brain, many of the alterations underlying cognitive and behavioral decline remain opaque. Caenorhabditis elegans offers a powerful model for aging research, with a simple, well-studied nervous system to further our understanding of the cellular modifications and functional alterations accompanying senescence. We perform multi-neuronal functional imaging across the aged C. elegans nervous system, measuring an age-associated breakdown in system-wide functional organization. At single-cell resolution, we detect shifts in activity dynamics toward higher frequencies. In addition, we measure a specific loss of inhibitory signaling that occurs early in the aging process and alters the systems' critical excitatory/inhibitory balance. These effects are recapitulated with mutation of the calcium channel subunit UNC-2/CaV2α. We find that manipulation of inhibitory GABA signaling can partially ameliorate or accelerate the effects of aging. The effects of aging are also partially mitigated by disruption of the insulin signaling pathway, known to increase longevity, or by a reduction of caspase activation. Data from mammals are consistent with our findings, suggesting a conserved shift in the balance of excitatory/inhibitory signaling with age that leads to breakdown in global neuronal dynamics and functional decline.

Co-transmission of neuropeptides and monoamines choreograph the C. elegans escape response.

Co-localization and co-transmission of neurotransmitters and neuropeptides is a core property of neural signaling across species. While co-transmission can increase the flexibility of cellular communication, understanding the functional impact on neural dynamics and behavior remains a major challenge. Here we examine the role of neuropeptide/monoamine co-transmission in the orchestration of the C. elegans escape response. The tyraminergic RIM neurons, which coordinate distinct motor programs of the escape response, also co-express the neuropeptide encoding gene flp-18. We find that in response to a mechanical stimulus, flp-18 mutants have defects in locomotory arousal and head bending that facilitate the omega turn. We show that the induction of the escape response leads to the release of FLP-18 neuropeptides. FLP-18 modulates the escape response through the activation of the G-protein coupled receptor NPR-5. FLP-18 increases intracellular calcium levels in neck and body wall muscles to promote body bending. Our results show that FLP-18 and tyramine act in different tissues in both a complementary and antagonistic manner to control distinct motor programs during different phases of the C. elegans flight response. Our study reveals basic principles by which co-transmission of monoamines and neuropeptides orchestrate in arousal and behavior in response to stress.

A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior.

Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.

Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone.

Dioecious species are a hallmark of the animal kingdom, with opposing sexes responding differently to identical sensory cues. Here, we study the response of C. elegans to the small-molecule pheromone, ascr#8, which elicits opposing behavioral valences in each sex. We identify a novel neuropeptide-neuropeptide receptor (NP/NPR) module that is active in males, but not in hermaphrodites. Using a novel paradigm of neuropeptide rescue that we established, we leverage bacterial expression of individual peptides to rescue the sex-specific response to ascr#8. Concurrent biochemical studies confirmed individual FLP-3 peptides differentially activate two divergent receptors, NPR-10 and FRPR-16. Interestingly, the two of the peptides that rescued behavior in our feeding paradigm are related through a conserved threonine, suggesting that a specific NP/NPR combination sets a male state, driving the correct behavioral valence of the ascr#8 response. Receptor expression within pre-motor neurons reveals novel coordination of male-specific and core locomotory circuitries.

The transcription factor LAG-1/CSL plays a Notch-independent role in controlling terminal differentiation, fate maintenance, and plasticity of serotonergic chemosensory neurons.

During development, signal-regulated transcription factors (TFs) act as basal repressors and upon signalling through morphogens or cell-to-cell signalling shift to activators, mediating precise and transient responses. Conversely, at the final steps of neuron specification, terminal selector TFs directly initiate and maintain neuron-type specific gene expression through enduring functions as activators. C. elegans contains 3 types of serotonin synthesising neurons that share the expression of the serotonin biosynthesis pathway genes but not of other effector genes. Here, we find an unconventional role for LAG-1, the signal-regulated TF mediator of the Notch pathway, as terminal selector for the ADF serotonergic chemosensory neuron, but not for other serotonergic neuron types. Regulatory regions of ADF effector genes contain functional LAG-1 binding sites that mediate activation but not basal repression. lag-1 mutants show broad defects in ADF effector genes activation, and LAG-1 is required to maintain ADF cell fate and functions throughout life. Unexpectedly, contrary to reported basal repression state for LAG-1 prior to Notch receptor activation, gene expression activation in the ADF neuron by LAG-1 does not require Notch signalling, demonstrating a default activator state for LAG-1 independent of Notch. We hypothesise that the enduring activity of terminal selectors on target genes required uncoupling LAG-1 activating role from receiving the transient Notch signalling.

Corollary discharge promotes a sustained motor state in a neural circuit for navigation.

Animals exhibit behavioral and neural responses that persist on longer timescales than transient or fluctuating stimulus inputs. Here, we report that Caenorhabditis elegans uses feedback from the motor circuit to a sensory processing interneuron to sustain its motor state during thermotactic navigation. By imaging circuit activity in behaving animals, we show that a principal postsynaptic partner of the AFD thermosensory neuron, the AIY interneuron, encodes both temperature and motor state information. By optogenetic and genetic manipulation of this circuit, we demonstrate that the motor state representation in AIY is a corollary discharge signal. RIM, an interneuron that is connected with premotor interneurons, is required for this corollary discharge. Ablation of RIM eliminates the motor representation in AIY, allows thermosensory representations to reach downstream premotor interneurons, and reduces the animal's ability to sustain forward movements during thermotaxis. We propose that feedback from the motor circuit to the sensory processing circuit underlies a positive feedback mechanism to generate persistent neural activity and sustained behavioral patterns in a sensorimotor transformation.

Flexible motor sequence generation during stereotyped escape responses.

Complex animal behaviors arise from a flexible combination of stereotyped motor primitives. Here we use the escape responses of the nematode Caenorhabditis elegans to study how a nervous system dynamically explores the action space. The initiation of the escape responses is predictable: the animal moves away from a potential threat, a mechanical or thermal stimulus. But the motor sequence and the timing that follow are variable. We report that a feedforward excitation between neurons encoding distinct motor states underlies robust motor sequence generation, while mutual inhibition between these neurons controls the flexibility of timing in a motor sequence. Electrical synapses contribute to feedforward coupling whereas glutamatergic synapses contribute to inhibition. We conclude that C. elegans generates robust and flexible motor sequences by combining an excitatory coupling and a winner-take-all operation via mutual inhibition between motor modules.

HIF-1 Has a Central Role in Caenorhabditis elegans Organismal Response to Selenium.

Selenium is a trace element for most organisms; its deficiency and excess are detrimental. Selenium beneficial effects are mainly due to the role of the 21st genetically encoded amino acid selenocysteine (Sec). Selenium also exerts Sec-independent beneficial effects. Its harmful effects are thought to be mainly due to non-specific incorporation in protein synthesis. Yet the selenium response in animals is poorly understood. In Caenorhabditis elegans, Sec is genetically incorporated into a single selenoprotein. Similar to mammals, a 20-fold excess of the optimal selenium requirement is harmful. Sodium selenite (Na2SeO3) excess causes development retardation, impaired growth, and neurodegeneration of motor neurons. To study the organismal response to selenium we performed a genetic screen for C. elegans mutants that are resistant to selenite. We isolated non-sense and missense egl-9/EGLN mutants that confer robust resistance to selenium. In contrast, hif-1/HIF null mutant was highly sensitive to selenium, establishing a role for this transcription factor in the selenium response. We showed that EGL-9 regulates HIF-1 activity through VHL-1, and identified CYSL-1 as a key sensor that transduces the selenium signal. Finally, we showed that the key enzymes involved in sulfide and sulfite stress (sulfide quinone oxidoreductase and sulfite oxidase) are not required for selenium resistance. In contrast, knockout strains in the persulfide dioxygenase ETHE-1 and the sulfurtransferase MPST-7 affect the organismal response to selenium. In sum, our results identified a transcriptional pathway as well as enzymes possibly involved in the organismal selenium response.

The flight response impairs cytoprotective mechanisms by activating the insulin pathway.

An animal's stress response requires different adaptive strategies depending on the nature and duration of the stressor. Whereas acute stressors, such as predation, induce a rapid and energy-demanding fight-or-flight response, long-term environmental stressors induce the gradual and long-lasting activation of highly conserved cytoprotective processes1-3. In animals across the evolutionary spectrum, continued activation of the fight-or-flight response weakens the animal's resistance to environmental challenges4,5. However, the molecular and cellular mechanisms that regulate the trade-off between the flight response and long-term stressors are poorly understood. Here we show that repeated induction of the flight response in Caenorhabditis elegans shortens lifespan and inhibits conserved cytoprotective mechanisms. The flight response activates neurons that release tyramine, an invertebrate analogue of adrenaline and noradrenaline. Tyramine stimulates the insulin-IGF-1 signalling (IIS) pathway and precludes the induction of stress response genes by activating an adrenergic-like receptor in the intestine. By contrast, long-term environmental stressors, such as heat or oxidative stress, reduce tyramine release and thereby allow the induction of cytoprotective genes. These findings demonstrate that a neural stress hormone supplies a state-dependent neural switch between acute flight and long-term environmental stress responses and provides mechanistic insights into how the flight response impairs cellular defence systems and accelerates ageing.

Gain-of-function mutations in the UNC-2/CaV2α channel lead to excitation-dominant synaptic transmission in Caenorhabditis elegans.

Mutations in pre-synaptic voltage-gated calcium channels can lead to familial hemiplegic migraine type 1 (FHM1). While mammalian studies indicate that the migraine brain is hyperexcitable due to enhanced excitation or reduced inhibition, the molecular and cellular mechanisms underlying this excitatory/inhibitory (E/I) imbalance are poorly understood. We identified a gain-of-function (gf) mutation in the Caenorhabditis elegans CaV2 channel α1 subunit, UNC-2, which leads to increased calcium currents. unc-2(zf35gf) mutants exhibit hyperactivity and seizure-like motor behaviors. Expression of the unc-2 gene with FHM1 substitutions R192Q and S218L leads to hyperactivity similar to that of unc-2(zf35gf) mutants. unc-2(zf35gf) mutants display increased cholinergic and decreased GABAergic transmission. Moreover, increased cholinergic transmission in unc-2(zf35gf) mutants leads to an increase of cholinergic synapses and a TAX-6/calcineurin-dependent reduction of GABA synapses. Our studies reveal mechanisms through which CaV2 gain-of-function mutations disrupt excitation-inhibition balance in the nervous system.

Co-option of neurotransmitter signaling for inter-organismal communication in C. elegans.

Biogenic amine neurotransmitters play a central role in metazoan biology, and both their chemical structures and cognate receptors are evolutionarily conserved. Their primary roles are in cell-to-cell signaling, as biogenic amines are not normally recruited for communication between separate individuals. Here, we show that in the nematode C. elegans, a neurotransmitter-sensing G protein-coupled receptor, TYRA-2, is required for avoidance responses to osas#9, an ascaroside pheromone that incorporates the neurotransmitter, octopamine. Neuronal ablation, cell-specific genetic rescue, and calcium imaging show that tyra-2 expression in the nociceptive neuron, ASH, is necessary and sufficient to induce osas#9 avoidance. Ectopic expression in the AWA neuron, which is generally associated with attractive responses, reverses the response to osas#9, resulting in attraction instead of avoidance behavior, confirming that TYRA-2 partakes in the sensing of osas#9. The TYRA-2/osas#9 signaling system represents an inter-organismal communication channel that evolved via co-option of a neurotransmitter and its cognate receptor.

Diapause induces functional axonal regeneration after necrotic insult in C. elegans.

Many neurons are unable to regenerate after damage. The ability to regenerate after an insult depends on life stage, neuronal subtype, intrinsic and extrinsic factors. C. elegans is a powerful model to test the genetic and environmental factors that affect axonal regeneration after damage, since its axons can regenerate after neuronal insult. Here we demonstrate that diapause promotes the complete morphological regeneration of truncated touch receptor neuron (TRN) axons expressing a neurotoxic MEC-4(d) DEG/ENaC channel. Truncated axons of different lengths were repaired during diapause and we observed potent axonal regrowth from somas alone. Complete morphological regeneration depends on DLK-1 but neuronal sprouting and outgrowth is DLK-1 independent. We show that TRN regeneration is fully functional since animals regain their ability to respond to mechanical stimulation. Thus, diapause induced regeneration provides a simple model of complete axonal regeneration which will greatly facilitate the study of environmental and genetic factors affecting the rate at which neurons die.

Breakdown of Neural Function under Isoflurane Anesthesia: In Vivo, Multineuronal Imaging in Caenorhabditis elegans.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Previous work on the action of volatile anesthetics has focused at either the molecular level or bulk neuronal measurement such as electroencephalography or functional magnetic resonance imaging. There is a distinct gulf in resolution at the level of cellular signaling within neuronal systems. The authors hypothesize that anesthesia is caused by induced dyssynchrony in cellular signaling rather than suppression of individual neuron activity. METHODS: Employing confocal microscopy and Caenorhabditis elegans expressing the calcium-sensitive fluorophore GCaMP6s in specific command neurons, the authors measure neuronal activity noninvasively and in parallel within the behavioral circuit controlling forward and reverse crawling. The authors compare neuronal dynamics and coordination in a total of 31 animals under atmospheres of 0, 4, and 8% isoflurane. RESULTS: When not anesthetized, the interneurons controlling forward or reverse crawling occupy two possible states, with the activity of the "reversal" neurons AVA, AVD, AVE, and RIM strongly intercorrelated, and the "forward" neuron AVB anticorrelated. With exposure to 4% isoflurane and onset of physical quiescence, neuron activity wanders rapidly and erratically through indeterminate states. Neuron dynamics shift toward higher frequencies, and neuron pair correlations within the system are reduced. At 8% isoflurane, physical quiescence continues as neuronal signals show diminished amplitude with little correlation between neurons. Neuronal activity was further studied using statistical tools from information theory to quantify the type of disruption caused by isoflurane. Neuronal signals become noisier and more disordered, as measured by an increase in the randomness of their activity (Shannon entropy). The coordination of the system, measured by whether information exhibited in one neuron is also exhibited in other neurons (multiinformation), decreases significantly at 4% isoflurane (P = 0.00015) and 8% isoflurane (P = 0.0028). CONCLUSIONS: The onset of anesthesia corresponds with high-frequency randomization of individual neuron activity coupled with induced dyssynchrony and loss of coordination between neurons that disrupts functional signaling.

Neurexin directs partner-specific synaptic connectivity in C. elegans.

In neural circuits, individual neurons often make projections onto multiple postsynaptic partners. Here, we investigate molecular mechanisms by which these divergent connections are generated, using dyadic synapses in C. elegans as a model. We report that C. elegans nrx-1/neurexin directs divergent connectivity through differential actions at synapses with partnering neurons and muscles. We show that cholinergic outputs onto neurons are, unexpectedly, located at previously undefined spine-like protrusions from GABAergic dendrites. Both these spine-like features and cholinergic receptor clustering are strikingly disrupted in the absence of nrx-1. Excitatory transmission onto GABAergic neurons, but not neuromuscular transmission, is also disrupted. Our data indicate that NRX-1 located at presynaptic sites specifically directs postsynaptic development in GABAergic neurons. Our findings provide evidence that individual neurons can direct differential patterns of connectivity with their post-synaptic partners through partner-specific utilization of synaptic organizers, offering a novel view into molecular control of divergent connectivity.

Excitatory motor neurons are local oscillators for backward locomotion.

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron's oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron's intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

Selenoprotein T is required for pathogenic bacteria avoidance in Caenorhabditis elegans.

Selenoprotein T (SELENOT) is an endoplasmatic reticulum (ER)-associated redoxin that contains the amino acid selenocysteine (Sec, U) within a CXXU motif within a thioredoxin-like fold. Its precise function in multicellular organisms is not completely understood although it has been shown in mammals to be involved in Ca2+ homeostasis, antioxidant and neuroendocrine functions. Here, we use the model organism C. elegans to address SELENOT function in a whole organism throughout its life cycle. C. elegans possess two genes encoding SELENOT protein orthologues (SELT-1.1 and SELT-1.2), which lack Sec and contain the CXXC redox motif instead. Our results show that a Sec→Cys replacement and a gene duplication were two major evolutionary events that occurred in the nematode lineage. We find that worm SELT-1.1 localizes to the ER and is expressed in different cell types, including the nervous system. In contrast, SELT-1.2 exclusively localizes in the cytoplasm of the AWB neurons. We find that selt-1.1 and selt-1.2 single mutants as well as the double mutant are viable, but the selt-1.1 mutant is compromised under rotenone-induced oxidative stress. We demonstrate that selt-1.1, but not selt-1.2, is required for avoidance to the bacterial pathogens Serratia marcescens and Pseudomonas aeruginosa. Aversion to the noxious signal 2-nonanone is also significantly impaired in selt-1.1, but not in selt-1.2 mutant animals. Our results suggest that selt-1.1 would be a redox transducer required for nociception and optimal organismal fitness. The results highlight C. elegans as a valuable model organism to study SELENOT-dependent processes.