Brain-wide bidirectional neuropeptide modulation of individual neuron classes regulates a developmental decision.

Secreted neuromodulators, like biogenic amines and neuropeptides, can reconfigure circuit functions both locally and at a distance and establish global brain states that alter circuit outputs over prolonged timescales.1-3 Despite their diversity and ubiquitous presence, many studies on neuromodulation tend to focus on dissecting the function and site of action of individual neuropeptides. Here, we take a different approach by conducting a systems-level investigation of neuropeptide receptor signaling function and cell-type-specific distribution in the context of the Caenorhabditis elegans diapause entry developmental decision. C. elegans diapause entry is controlled by sensory perception of external factors and is regulated by neuropeptide signaling.4-8 We performed a comprehensive functional screen of neuropeptide receptor mutants for pheromone-induced diapause entry phenotypes and integrated these results with published C. elegans single-cell RNA-seq data to reveal that almost all neuron classes expressed at least one receptor with a role in diapause entry.9 Our receptor expression analysis also identified four highly modulated neural hubs with no previously reported roles in diapause entry that are distributed throughout the animal's body, possibly as a means of synchronizing the whole-organism transition into the appropriate larval morph. Furthermore, most neuron classes expressed unique neuropeptide receptor repertoires that have opposing effects on the diapause entry decision. We propose that brain-wide antagonistic neuropeptide modulation of individual neuron classes by distinct neuropeptide receptor subsets could serve as a strategy against overmodulation and that this motif might generalize to other decision-making paradigms in other organisms.

Interneuron control of C. elegans developmental decision-making.

Natural environments are highly dynamic, and this complexity challenges animals to accurately integrate external cues to shape their responses. Adaptive developmental plasticity enables organisms to remodel their physiology, morphology, and behavior to better suit the predicted future environment and ultimately enhance their ecological success.1 Understanding how an animal generates a neural representation of current and forecasted environmental conditions and converts these circuit computations into a predictive adaptive physiological response may provide fundamental insights into the molecular and cellular basis of decision-making over developmentally relevant timescales. Although it is known that sensory cues usually trigger the developmental switch and that downstream inter-tissue signaling pathways enact the alternative developmental phenotype, the integrative neural mechanisms that transduce external inputs into effector pathways are less clear.2,3 In adverse environments, Caenorhabditis elegans larvae can enter a stress-resistant diapause state with arrested metabolism and reproductive physiology.4 Amphid sensory neurons feed into both rapid chemotactic and short-term foraging mode decisions, mediated by amphid and pre-motor interneurons, as well as the long-term diapause entry decision. Here, we identify amphid interneurons that integrate pheromone cues and propagate this information via a neuropeptidergic pathway to influence larval developmental fate, bypassing the pre-motor system. AIA interneuron-derived FLP-2 neuropeptide signaling promotes reproductive growth, and AIA activity is suppressed by pheromones. FLP-2 signaling is inhibited by upstream glutamatergic transmission via the metabotropic receptor MGL-1 and mediated by the broadly expressed neuropeptide G-protein-coupled receptor NPR-30. Thus, metabotropic signaling allows the reuse of parts of a sensory system for a decision with a distinct timescale.

A conserved behavioral role for a nematode interneuron neuropeptide receptor.

Neuropeptides are evolutionarily conserved modulators of many aspects of animal behavior and physiology, and expand the repertoire of processes that can be controlled by a limited number of neurons. Deciphering the neuropeptidergic codes that govern distinct processes requires systematic functional analyses of neuropeptides and their cognate receptors. Even in well-studied model organisms like Caenorhabditis elegans, however, such efforts have been precluded by a lack of mutant reagents. Here, we generated and screened 21 C. elegans neuropeptide G-protein coupled receptor mutants with no pre-existing reagents for the touch-evoked escape response, and implicated six receptors expressed in diverse neuron classes representing multiple circuit levels in this behavior. We further characterized the mutant with the most severe phenotype, frpr-14, which was defective in multiple behavioral paradigms. We leveraged this range of phenotypes to reveal that FRPR-14 modulation of different precommand interneuron classes, AVH and AIB, can drive distinct behavioral subsets, demonstrating cellular context-dependent roles for FRPR-14 signaling. We then show that Caenorhabditis briggsae CBR-FRPR-14 modulates an AVH-like interneuron pair to regulate the same behaviors as C. elegans but to a smaller extent. Our results also suggest that differences in touch-evoked escape circuit architecture between closely related species results from changes in neuropeptide receptor expression pattern, as opposed to ligand-receptor pairing. This study provides insights into the principles utilized by a compact, multiplexed nervous system to generate intraspecific behavioral complexity and interspecific variation.

Genetic reduction of tyramine ß hydroxylase suppresses Tau toxicity in a Drosophila model of tauopathy.

Tauopathies are a class of neurodegenerative diseases characterized by the abnormal phosphorylation and accumulation of the microtubule-associated protein, Tau. These diseases are associated with degeneration and dysfunction of the noradrenergic system, a critical regulator of memory, locomotion, and the fight or flight response. Though Tau pathology accumulates early in noradrenergic neurons, the relationship between noradrenaline signaling and tauopathy pathogenesis remains unclear. The fruit fly, Drosophila melanogaster, is a valuable model organism commonly used to investigate factors that promote Tau-mediated degeneration. Moreover, Drosophila contain the biogenic amine, octopamine, which is the functional homolog to noradrenaline. Using a Drosophila model of tauopathy, we conducted a candidate modifier screen targeting tyramine ß hydroxylase (tßh), the enzyme that controls the production of octopamine in the fly, to determine if levels of this enzyme modulate Tau-induced degeneration in the fly eye. We found that genetic reduction of tßh suppresses Tau toxicity, independent of Tau phosphorylation. These findings show that reduction of tßh, a critical enzyme in the octopaminergic pathway, suppresses Tau pathogenicity and establishes an interaction that can be further utilized to determine the relationship between noradrenergic-like signaling and Tau toxicity in Drosophila.

Biology and genome of a newly discovered sibling species of Caenorhabditis elegans.

A 'sibling' species of the model organism Caenorhabditis elegans has long been sought for use in comparative analyses that would enable deep evolutionary interpretations of biological phenomena. Here, we describe the first sibling species of C. elegans, C. inopinata n. sp., isolated from fig syconia in Okinawa, Japan. We investigate the morphology, developmental processes and behaviour of C. inopinata, which differ significantly from those of C. elegans. The 123-Mb C. inopinata genome was sequenced and assembled into six nuclear chromosomes, allowing delineation of Caenorhabditis genome evolution and revealing unique characteristics, such as highly expanded transposable elements that might have contributed to the genome evolution of C. inopinata. In addition, C. inopinata exhibits massive gene losses in chemoreceptor gene families, which could be correlated with its limited habitat area. We have developed genetic and molecular techniques for C. inopinata; thus C. inopinata provides an exciting new platform for comparative evolutionary studies.

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

cGAL, a temperature-robust GAL4-UAS system for Caenorhabditis elegans.

The GAL4-UAS system is a powerful tool for manipulating gene expression, but its application in Caenorhabditis elegans has not been described. Here we systematically optimize the system's three main components to develop a temperature-optimized GAL4-UAS system (cGAL) that robustly controls gene expression in C. elegans from 15 to 25 °C. We demonstrate this system's utility in transcriptional reporter analysis, site-of-action experiments and exogenous transgene expression; and we provide a basic driver and effector toolkit.