An Image-Free Opto-Mechanical System for Creating Virtual Environments and Imaging Neuronal Activity in Freely Moving Caenorhabditis elegans

Non-invasive recording in untethered animals is arguably the ultimate step in the analysis of neuronal function, but such recordings remain elusive. To address this problem, we devised a system that tracks neuron-sized fluorescent targets in real time. The system can be used to create virtual environments by optogenetic activation of sensory neurons, or to image activity in identified neurons at high magnification. By recording activity in neurons of freely moving C. elegans, we tested the long-standing hypothesis that forward and reverse locomotion are generated by distinct neuronal circuits. Surprisingly, we found motor neurons that are active during both types of locomotion, suggesting a new model of locomotion control in C. elegans. These results emphasize the importance of recording neuronal activity in freely moving animals and significantly expand the potential of imaging techniques by providing a mean to stabilize fluorescent targets

Dopamine Signaling Is Essential for Precise Rates of Locomotion by C. elegans

Dopamine is an important neuromodulator in both vertebrates and invertebrates. We have found that reduced dopamine signaling can cause a distinct abnormality in the behavior of the nematode C. elegans, which has only eight dopaminergic neurons. Using an automated particle-tracking system for the analysis of C. elegans locomotion, we observed that individual wild-type animals made small adjustments to their speed to maintain constant rates of locomotion. By contrast, individual mutant animals defective in the synthesis of dopamine made larger adjustments to their speeds, resulting in large fluctuations in their rates of locomotion. Mutants defective in dopamine signaling also frequently exhibited both abnormally high and abnormally low average speeds. The ability to make small adjustments to speed was restored to these mutants by treatment with dopamine. These behaviors depended on the D2-like dopamine receptor DOP-3 and the G-protein subunit GOA-1. We suggest that C. elegans and other animals, including humans, might share mechanisms by which dopamine restricts motor activity levels and coordinates movement

The Retrograde IFT Machinery of C. elegans Cilia: Two IFT Dynein Complexes?

We analyzed the relatively poorly understood IFT-dynein (class DYNC2)-driven retrograde IFT pathway in C. elegans cilia, which yielded results that are surprising in the context of current models of IFT. Assays of C. elegans dynein gene expression and intraflagellar transport (IFT) suggest that conventional IFT-dynein contains essential heavy (CHE-3), light-intermediate (XBX-1), plus three light polypeptide chains that participate in IFT, but no “essential” intermediate chain. IFT assays of XBX-1::YFP suggest that IFT-dynein is transported as cargo to the distal tip of the cilium by kinesin-2 motors, but independent of the IFT-particle/BBSome complexes. Finally, we were surprised to find that the subset of cilia present on the OLQ (outer labial quadrant) neurons assemble independently of conventional “CHE-3” IFT-dynein, implying that there is a second IFT-dynein acting in these cilia. We have found a novel gene encoding a dynein heavy chain, DHC-3, and two light chains, in OLQ neurons, which could constitute an IFT-dynein complex in OLQ neuronal cilia. Our results underscore several surprising features of retrograde IFT that require clarification

Inositol 1,4,5-Trisphosphate Signalling Regulates the Avoidance Response to Nose Touch in Caenorhabditis elegans

When Caenorhabditis elegans encounters an unfavourable stimulus at its anterior, it responds by initiating an avoidance response, namely reversal of locomotion. The amphid neurons, ASHL and ASHR, are polymodal in function, with roles in the avoidance responses to high osmolarity, nose touch, and both volatile and non-volatile repellents. The mechanisms that underlie the ability of the ASH neurons to respond to such a wide range of stimuli are still unclear. We demonstrate that the inositol 1,4,5-trisphosphate receptor (IP3R), encoded by itr-1, functions in the reversal responses to nose touch and benzaldehyde, but not in other known ASH-mediated responses. We show that phospholipase Cβ (EGL-8) and phospholipase Cγ (PLC-3), which catalyse the production of IP3, both function upstream of ITR-1 in the response to nose touch. We use neuron-specific gene rescue and neuron-specific disruption of protein function to show that the site of ITR-1 function is the ASH neurons. By rescuing plc-3 and egl-8 in a neuron-specific manner, we show that both are acting in ASH. Imaging of nose touch–induced Ca2+ transients in ASH confirms these conclusions. In contrast, the response to benzaldehyde is independent of PLC function. Thus, we have identified distinct roles for the IP3R in two specific responses mediated by ASH

Functional Phenotypic Rescue of Caenorhabditis elegans Neuroligin-Deficient Mutants by the Human and Rat NLGN1 Genes

Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C) and worm NLG-1 (R437C) proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X) and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA), both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1) or pan-muscular (myo-3) specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders

The Caenorhabditis elegans Mucin-Like Protein OSM-8 Negatively Regulates Osmosensitive Physiology Via the Transmembrane Protein PTR-23

The molecular mechanisms of animal cell osmoregulation are poorly understood. Genetic studies of osmoregulation in yeast have identified mucin-like proteins as critical regulators of osmosensitive signaling and gene expression. Whether mucins play similar roles in higher organisms is not known. Here, we show that mutations in the Caenorhabditis elegans mucin-like gene osm-8 specifically disrupt osmoregulatory physiological processes. In osm-8 mutants, normal physiological responses to hypertonic stress, such as the accumulation of organic osmolytes and activation of osmoresponsive gene expression, are constitutively activated. As a result, osm-8 mutants exhibit resistance to normally lethal levels of hypertonic stress and have an osmotic stress resistance (Osr) phenotype. To identify genes required for Osm-8 phenotypes, we performed a genome-wide RNAi osm-8 suppressor screen. After screening ∼18,000 gene knockdowns, we identified 27 suppressors that specifically affect the constitutive osmosensitive gene expression and Osr phenotypes of osm-8 mutants. We found that one suppressor, the transmembrane protein PTR-23, is co-expressed with osm-8 in the hypodermis and strongly suppresses several Osm-8 phenotypes, including the transcriptional activation of many osmosensitive mRNAs, constitutive glycerol accumulation, and osmotic stress resistance. Our studies are the first to show that an extracellular mucin-like protein plays an important role in animal osmoregulation in a manner that requires the activity of a novel transmembrane protein. Given that mucins and transmembrane proteins play similar roles in yeast osmoregulation, our findings suggest a possible evolutionarily conserved role for the mucin-plasma membrane interface in eukaryotic osmoregulation

A Soluble Acetylcholinesterase Provides Chemical Defense against Xenobiotics in the Pinewood Nematode

The pinewood nematode genome encodes at least three distinct acetylcholinesterases (AChEs). To understand physiological roles of the three pinewood nematode AChEs (BxACE-1, BxACE-2, and BxACE-3), BxACE-3 in particular, their tissue distribution and inhibition profiles were investigated. Immunohistochemistry revealed that BxACE-1 and BxACE-2 were distributed in neuronal tissues. In contrast, BxACE-3 was detected from some specific tissues and extracted without the aid of detergent, suggesting its soluble nature unlike BxACE-1 and BxACE-2. When present together, BxAChE3 significantly reduced the inhibition of BxACE-1 and BxACE-2 by cholinesterase inhibitors. Knockdown of BxACE-3 by RNA interference significantly increased the toxicity of three nematicidal compounds, supporting the protective role of BxACE-3 against chemicals. In summary, BxACE-3 appears to have a non-neuronal function of chemical defense whereas both BxACE-1 and BxACE-2 have classical neuronal function of synaptic transmission

Neogenin May Functionally Substitute for Dcc in Chicken

Dcc is the key receptor that mediates attractive responses of axonal growth cones to netrins, a family of axon guidance cues used throughout evolution. However, a Dcc homolog has not yet been identified in the chicken genome, raising the possibility that Dcc is not present in avians. Here we show that the closely related family member neogenin may functionally substitute for Dcc in the developing chicken spinal cord. The expression pattern of chicken neogenin in the developing spinal cord is a composite of the distribution patterns of both rodent Dcc and neogenin. Moreover, whereas the loss of mouse neogenin has no effect on the trajectory of commissural axons, removing chicken neogenin by RNA interference results in a phenotype similar to the functional inactivation of Dcc in mouse. Taken together, these data suggest that the chick neogenin is functionally equivalent to rodent Dcc

Pheromone-sensing neurons regulate peripheral lipid metabolism in <i>Caenorhabditis elegans</i>

It is now established that the central nervous system plays an important role in regulating whole body metabolism and energy balance. However, the extent to which sensory systems relay environmental information to modulate metabolic events in peripheral tissues has remained poorly understood. In addition, it has been challenging to map the molecular mechanisms underlying discrete sensory modalities with respect to their role in lipid metabolism. In previous work our lab has identified instructive roles for serotonin signaling as a surrogate for food availability, as well as oxygen sensing, in the control of whole body metabolism. In this study, we now identify a role for a pair of pheromone-sensing neurons in regulating fat metabolism in C. elegans, which has emerged as a tractable and highly informative model to study the neurobiology of metabolism. A genetic screen revealed that GPA-3, a member of the Gα family of G proteins, regulates body fat content in the intestine, the major metabolic organ for C. elegans. Genetic and reconstitution studies revealed that the potent body fat phenotype of gpa-3 null mutants is controlled from a pair of neurons called ADL(L/R). We show that cAMP functions as the second messenger in the ADL neurons, and regulates body fat stores via the neurotransmitter acetylcholine, from downstream neurons. We find that the pheromone ascr#3, which is detected by the ADL neurons, regulates body fat stores in a GPA-3-dependent manner. We define here a third sensory modality, pheromone sensing, as a major regulator of body fat metabolism. The pheromone ascr#3 is an indicator of population density, thus we hypothesize that pheromone sensing provides a salient 'denominator' to evaluate the amount of food available within a population and to accordingly adjust metabolic rate and body fat levels