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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.

In the aging brain, many of the alterations underlying cognitive and behavioral decline remain opaque. C. 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.

SUMMARYNeuromodulators 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 C. 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.Impact statementInvestigation of neuromodulatory control of ethologically conserved area-restricted food search behavior shows that NLP-12 stimulation of the head motor circuit promotes food searching through the previously uncharacterized CKR-1 GPCR.

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.

Editor’s Perspective 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.

Complex animal behaviors arise from flexible combinations of stereotyped motor primitives. How do nervous systems generate dynamics to purposefully explore the action space? Here we study escape responses of the nematode C. elegans, in which the animal predictably moves away from a potential threat. However, the motor sequences and the timing that constitute the whole behavior are variable. We report that a rapid feedforward pathway embedded in a recurrent attractor network underlies robust motor sequence generation, and neurons in different functional modules exploit synaptic inhibitions to flexibly control the timing of motor sequences. We reveal that electrical synapses contribute to feedforward coupling while glutamatergic synaptic transmission contributes to selective inhibitions between motor programs. The rates of motor state transitions are consistent with a stochastic neural dynamic model with short term synaptic depression. Our findings identify a neural computation for robust and flexible motor sequence generation in a compact nervous system.

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 processes1,2,3. 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. Fil: de Rosa, Maria Jose. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Veuthey, Tania Vanesa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina Fil: Florman, Jeremy. University of Massachussets; Estados Unidos Fil: Grant, Jeff. University of Massachussets; Estados Unidos Fil: Blanco, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Andersen, Natalia Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Donnelly, Jamie. University of Massachussets; Estados Unidos Fil: Rayes, Diego Hernán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina. University of Massachussets; Estados Unidos Fil: Alkema, Mark. University of Massachussets; Estados Unidos

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., eLife digest Nervous systems are complex networks of interconnected cells called neurons. These networks vary in size from a few hundred cells in worms, to tens of billions in the human brain. Within these networks, each individual neuron forms connections – called synapses – with many others. But these partner neurons are not necessarily alike. In fact, they may be different cell types. How neurons form distinct connections with different partner cells remains unclear. Part of the answer may lie in specialized proteins called cell adhesion molecules. These proteins occur on the cell surface and enable neurons to recognize one another. This helps ensure that the cells form appropriate connections via synapses. Cell adhesion molecules are therefore also known as synaptic organizers. Philbrook et al. have now examined the role of synaptic organizers in wiring up the nervous system of the nematode worm and model organism Caenorhabditis elegans. Motor neurons form connections with two types of partner cell: muscle cells and neurons. Philbrook et al. screened C. elegans that have mutations in genes encoding various synaptic organizers. This revealed that a protein called neurexin must be present for motor neurons to form synapses with other neurons. By contrast, neurexin is not required for the same neurons to establish synapses with muscles. Philbrook et al. found that neuron-to-neuron synapses arise at specialized finger-like projections. These resemble the dendritic spines at which synapses form in the brains of mammals, and had not been previously identified in C. elegans. In worms that lack neurexin, these spine-like structures do not form correctly, disrupting the formation of neuron-to-neuron connections. Previous work has implicated neurexin in synapse formation in the mammalian brain. But this is the first study to reveal a role for neurexin in establishing partner-specific synaptic connections. Mutations in synaptic organizers, including neurexin, contribute to disorders of brain development. These include schizophrenia and autism spectrum disorders. Learning more about how neurexin helps establish specific synaptic connections may help us understand how these disorders arise.

Persistent activation of the “fight-or-flight” response accelerates aging and increases the susceptibility to disease. We show that repeated induction of the C. elegans flight response inhibits conserved cytoprotective mechanisms. This acute-stress response activates neurons that release tyramine, the invertebrate analog of adrenaline/noradrenaline. Tyramine stimulates the DAF-2/Insulin/IGF-1 pathway and precludes the nuclear translocation of the DAF-16/FOXO transcription factor through the activation of an adrenergic-like receptor TYRA-3 in the intestine. In contrast, environmental long-term stressors, such as heat or oxidative stress, reduce tyramine release allowing the induction of FOXO-dependent cytoprotective genes. These findings demonstrate how a neural stress-hormone signaling provides a state-dependent neural switch between acute and long-term stress responses, and provide mechanistic insights how acute stress impairs cellular defensive systems.One Sentence Summary: The “fight-or-flight” response reduces resistance to environmental challenges.

We present an imaging system for pan-neuronal recording in crawling Caenorhabditis elegans. A spinning disk confocal microscope, modified for automated tracking of the C. elegans head ganglia, simultaneously records the activity and position of ∼80 neurons that coexpress cytoplasmic calcium indicator GCaMP6s and nuclear localized red fluorescent protein at 10 volumes per second. We developed a behavioral analysis algorithm that maps the movements of the head ganglia to the animal's posture and locomotion. Image registration and analysis software automatically assigns an index to each nucleus and calculates the corresponding calcium signal. Neurons with highly stereotyped positions can be associated with unique indexes and subsequently identified using an atlas of the worm nervous system. To test our system, we analyzed the brainwide activity patterns of moving worms subjected to thermosensory inputs. We demonstrate that our setup is able to uncover representations of sensory input and motor output of individual neurons from brainwide dynamics. Our imaging setup and analysis pipeline should facilitate mapping circuits for sensory to motor transformation in transparent behaving animals such as C. elegans and Drosophila larva.

Chronic and acute nicotine exposure causes pregnancy and fertility problems as well as increased blood pressure and heart rate, vasoconstriction and cardiovascular disease. These physiological processes are regulated by the neuropeptides arginine vasopressin and oxytocin, which are synthesized and released from magnocellular neurons (MCNs) in the hypothalamic-neurohypophyseal system (HNS).Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels which bind nicotine as well as the endogenous neurotransmitter acetylcholine and are widely distributed throughout the central nervous system. However, little is currently known about the distribution and function of nAChRs in the HNS. Using RT-PCR in conjunction with immunolabeling, Ca imaging and release studies, we provide a comprehensive description of the types, localization and role of nAChRs in the HNS. RT-PCR identified the presence of alpha7, alpha4, alpha2, beta4 and beta2 subunits in tissue obtained from the Supraoptic and Paraventricular nuclei; with alpha7 and beta2 being predominately expressed. Immunolabeling supports this finding and was used to quantify subunit expression and determine the anatomical location of subunits within these neurons. Immunolabeling identified alpha7, alpha4, alpha2, beta4 and beta 2 subunits in MCN terminals. The presence of alpha7 and alpha4 nAChRs was also confirmed using pharmacological and electrophysiological measurements from isolated terminals. Furthermore, neuropeptide release in the presence of nicotine and/or anatoxin was enhanced in isolated terminals and whole glands. Finally, we confirmed the presence of the vesicular acetylcholine transporter in MCN terminals indicating a possible cholinergic autocrine modulation of peptide release in the HNS. (Supported by UMass Grant P60037094900000 to SOM)

Each fall, eastern North American monarch butterflies in their northern range undergo a long-distance migration south to their overwintering grounds in Mexico. Migrants use a time-compensated sun compass to determine directionality during the migration. This compass system uses information extracted from sun-derived skylight cues that is compensated for time of day and ultimately transformed into the appropriate motor commands. The central complex (CX) is likely the site of the actual sun compass, because neurons in this brain region are tuned to specific skylight cues. To help illuminate the neural basis of sun compass navigation, we examined the neuronal composition of the CX and its associated brain regions. We generated a standardized version of the sun compass neuropils, providing reference volumes, as well as a common frame of reference for the registration of neuron morphologies. Volumetric comparisons between migratory and nonmigratory monarchs substantiated the proposed involvement of the CX and related brain areas in migratory behavior. Through registration of more than 55 neurons of 34 cell types, we were able to delineate the major input pathways to the CX, output pathways, and intrinsic neurons. Comparison of these neural elements with those of other species, especially the desert locust, revealed a surprising degree of conservation. From these interspecies data, we have established key components of a conserved core network of the CX, likely complemented by species-specific neurons, which together may comprise the neural substrates underlying the computations performed by the CX. J. Comp. Neurol. 521:267298, 2013. (c) 2012 Wiley Periodicals, Inc. (Less)