wrk-1 and rig-5 control pioneer and follower axon navigation in the ventral nerve cord of Caenorhabditis elegans in a nid-1 mutant background.

During nervous system development, neurons send out axons, which must navigate large distances to reach synaptic targets. Axons grow out sequentially. The early outgrowing axons, pioneers, must integrate information from various guidance cues in their environment to determine the correct direction of outgrowth. Later outgrowing follower axons can at least in part navigate by adhering to pioneer axons. In Caenorhabditis elegans, the right side of the largest longitudinal axon tract, the ventral nerve cord, is pioneered by the AVG axon. How the AVG axon navigates is only partially understood. In this study, we describe the role of two members of the IgCAM family, wrk-1 and rig-5, in AVG axon navigation. While wrk-1 and rig-5 single mutants do not show AVG navigation defects, both mutants have highly penetrant pioneer and follower navigation defects in a nid-1 mutant background. Both mutations increase the fraction of follower axons following the misguided pioneer axon. We found that wrk-1 and rig-5 act in different genetic pathways, suggesting that we identified two pioneer-independent guidance pathways used by follower axons. We assessed general locomotion, mechanosensory responsiveness, and habituation to determine whether axonal navigation defects impact nervous system function. In rig-5 nid-1 double mutants, we found no significant defects in free movement behavior; however, a subpopulation of animals shows minor changes in response duration habituation after mechanosensory stimulation. These results suggest that guidance defects of axons in the motor circuit do not necessarily lead to major movement or behavioral defects but impact more complex behavioral modulation.

Neuroligin Plays a Role in Ethanol-Induced Disruption of Memory and Corresponding Modulation of Glutamate Receptor Expression.

Exposure to alcohol causes deficits in long-term memory formation across species. Using a long-term habituation memory assay in Caenorhabditis elegans, the effects of ethanol on long-term memory (> 24 h) for habituation were investigated. An impairment in long-term memory was observed when animals were trained in the presence of ethanol. Cues of internal state or training context during testing did not restore memory. Ethanol exposure during training also interfered with the downregulation of AMPA/KA-type glutamate receptor subunit (GLR-1) punctal expression previously associated with long-term memory for habituation in C. elegans. Interestingly, ethanol exposure alone had the opposite effect, increasing GLR-1::GFP punctal expression. Worms with a mutation in the C. elegans ortholog of vertebrate neuroligins (nlg-1) were resistant to the effects of ethanol on memory, as they displayed both GLR-1::GFP downregulation and long-term memory for habituation after training in the presence of ethanol. These findings provide insights into the molecular mechanisms through which alcohol consumption impacts memory.

Rapid assessment of the temporal function and phenotypic reversibility of neurodevelopmental disorder risk genes in Caenorhabditis elegans.

Recent studies have indicated that some phenotypes caused by decreased function of select neurodevelopmental disorder (NDD) risk genes can be reversed by restoring gene function in adulthood. However, few of the hundreds of risk genes have been assessed for adult phenotypic reversibility. We developed a strategy to rapidly assess the temporal requirements and phenotypic reversibility of NDD risk gene orthologs using a conditional protein degradation system and machine-vision phenotypic profiling in Caenorhabditis elegans. We measured how degrading and re-expressing orthologs of EBF3, BRN3A and DYNC1H1 at multiple periods throughout development affect 30 morphological, locomotor, sensory and learning phenotypes. We found that phenotypic reversibility was possible for each gene studied. However, the temporal requirements of gene function and degree of rescue varied by gene and phenotype. This work highlights the critical need to assess multiple windows of degradation and re-expression and a large number of phenotypes to understand the many roles a gene can have across the lifespan. This work also demonstrates the benefits of using a high-throughput model system to prioritize NDD risk genes for re-expression studies in other organisms.

ccd-5, a novel cdk-5 binding partner, regulates pioneer axon guidance in the ventral nerve cord of Caenorhabditis elegans.

During nervous system development, axons navigate complex environments to reach synaptic targets. Early extending axons must interact with guidance cues in the surrounding tissue, while later extending axons can interact directly with earlier "pioneering" axons, "following" their path. In Caenorhabditis elegans, the AVG neuron pioneers the right axon tract of the ventral nerve cord. We previously found that aex-3, a rab-3 guanine nucleotide exchange factor, is essential for AVG axon navigation in a nid-1 mutant background and that aex-3 might be involved in trafficking of UNC-5, a receptor for the guidance cue UNC-6/netrin. Here, we describe a new gene in this pathway: ccd-5, a putative cdk-5 binding partner. ccd-5 mutants exhibit increased navigation defects of AVG pioneer as well as interneuron and motor neuron follower axons in a nid-1 mutant background. We show that ccd-5 acts in a pathway with cdk-5, aex-3, and unc-5. Navigation defects of follower interneuron and motoneuron axons correlate with AVG pioneer axon defects. This suggests that ccd-5 mostly affects pioneer axon navigation and that follower axon defects are largely a secondary consequence of pioneer navigation defects. To determine the consequences for nervous system function, we assessed various behavioral and movement parameters. ccd-5 single mutants have no significant movement defects, and nid-1 ccd-5 double mutants are less responsive to mechanosensory stimuli compared with nid-1 single mutants. These surprisingly minor defects indicate either a high tolerance for axon guidance defects within the motor circuit and/or an ability to maintain synaptic connections among commonly misguided axons.

Neurobiology: From genome and connectome to understanding behavior.

Many forms of synaptic plasticity are mediated by changes in the abundance, density, and expression levels of postsynaptic ionotropic receptors. A new study identifies the endogenous ligands of five 'orphan' aminergic ligand-gated ion channels in Caenorhabditis elegans, functionally characterizes these channels, and explores the role of one of them in a simple form of learning.

Gaining an understanding of behavioral genetics through studies of foraging in Drosophila and learning in C. elegans.

The pursuit of understanding behavior has led to investigations of how genes, the environment, and the nervous system all work together to produce and influence behavior, giving rise to a field of research known as behavioral neurogenetics. This review focuses on the research journeys of two pioneers of aspects of behavioral neurogenetic research: Dr. Marla Sokolowski and Dr. Catharine Rankin as examples of how different approaches have been used to understand relationships between genes and behavior. Marla Sokolowski's research is centered around the discovery and analysis of foraging, a gene responsible for the natural behavioral polymorphism of Drosophila melanogaster larvae foraging behavior. Catharine Rankin's work began with demonstrating the ability to learn in Caenorhabditis elegans and then setting out to investigate the mechanisms underlying the "simplest" form of learning, habituation. Using these simple invertebrate organisms both investigators were able to perform in-depth dissections of behavior at genetic and molecular levels. By exploring their research and highlighting their findings we present ways their work has furthered our understanding of behavior and contributed to the field of behavioral neurogenetics.

Peel-1 negative selection promotes screening-free CRISPR-Cas9 genome editing in Caenorhabditis elegans.

Improved genome engineering methods that enable automation of large and precise edits are essential for systematic investigations of genome function. We adapted peel-1 negative selection to an optimized Dual-Marker Selection (DMS) cassette protocol for CRISPR-Cas9 genome engineering in Caenorhabditis elegans and observed robust increases in multiple measures of efficiency that were consistent across injectors and four genomic loci. The use of Peel-1-DMS selection killed animals harboring transgenes as extrachromosomal arrays and spared genome-edited integrants, often circumventing the need for visual screening to identify genome-edited animals. To demonstrate the applicability of the approach, we created deletion alleles in the putative proteasomal subunit pbs-1 and the uncharacterized gene K04F10.3 and used machine vision to automatically characterize their phenotypic profiles, revealing homozygous essential and heterozygous behavioral phenotypes. These results provide a robust and scalable approach to rapidly generate and phenotype genome-edited animals without the need for screening or scoring by eye.

The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases.

Since Caenorhabditis elegans was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using C. elegans to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use C. elegans to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in C. elegans, allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution in vivo. Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease) in C. elegans and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.

The role of neuropeptides in learning: Insights from C. elegans.

Learning is critical for survival as it provides the capacity to adapt to a changing environment. At the molecular and cellular level, learning leads to alterations within neural circuits that include synaptic rewiring and synaptic plasticity. These changes are mediated by signalling molecules known as neuromodulators. One such class of neuromodulators are neuropeptides, a diverse group of short peptides that primarily act through G protein-coupled receptors. There has been substantial progress in recent years on dissecting the role of neuropeptides in learning circuits using compact yet powerful invertebrate model systems. We will focus on insights gained using the nematode Caenorhabditis elegans, with its unparalleled genetic tractability, compact nervous system of ∼300 neurons, high level of conservation with mammalian systems and amenability to a suite of behavioural analyses. Specifically, we will summarise recent discoveries in C. elegans on the role of neuropeptides in non-associative and associative learning.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

Habituation in high-throughput genetic model organisms as a tool to investigate the mechanisms of neurodevelopmental disorders.

Alterations in habituation, a highly conserved form of non-associative learning, are suspected to contribute to a range of the complex behavioural phenotypes present in multiple neurodevelopmental disorders. While progress has been made in understanding the genetics of these disorders through the application of next-generation sequencing and related technologies, the pathogenicity of genetic variants and causes of learning and memory impairments can be difficult to determine from sequencing data alone. High-throughput genetic model organisms such as the roundworm Caenorhabditis elegans, fruit fly Drosophila melanogaster, and zebrafish Danio rerio offer low-cost and efficient methods to investigate the functions of identified neurodevelopmental disorder risk genes and the functional consequences of specific disorder-associated variants. Here, we review ways assessing habituation has been used in the genotype-first approach to first validate neurodevelopmental disorder candidate genes and now to systematically characterize large candidate gene lists. We then discuss exciting ways habituation, in combination with other techniques, can be used as a tool to assess the pathogenicity of putative genes and genetic variants, uncover and confirm molecular networks, and identify potential therapeutic avenues.

But can they learn? My accidental discovery of learning and memory in C. elegans.

I did not set out to study C. elegans. My undergraduate and graduate training was in Psychology. My postdoctoral work involved studying learning and memory in 1 mm diameter juvenile Aplysia californica. As a starting Assistant Professor when I attempted to continue my studies on Aplysia I encountered barriers to carrying out that work; at about the same time I was introduced to Caenorhabditis elegans and decided to investigate whether they could learn and remember. My laboratory was the first to demonstrate conclusively that C. elegans could learn and in the years since then my lab and many others have demonstrated that C. elegans is capable of a variety of forms of learning and memory.

Synaptic vesicle fusion is modulated through feedback inhibition by dopamine auto-receptors.

Mechanisms of synaptic vesicular fusion and neurotransmitter clearance are highly controlled processes whose finely-tuned regulation is critical for neural function. This modulation has been suggested to involve pre-synaptic auto-receptors; however, their underlying mechanisms of action remain unclear. Previous studies with the well-defined C. elegans nervous system have used functional imaging to implicate acid sensing ion channels (ASIC-1) to describe synaptic vesicle fusion dynamics within its eight dopaminergic neurons. Implementing a similar imaging approach with a pH-sensitive fluorescent reporter and fluorescence resonance after photobleaching (FRAP), we analyzed dynamic imaging data collected from individual synaptic termini in live animals. We present evidence that constitutive fusion of neurotransmitter vesicles on dopaminergic synaptic termini is modulated through DOP-2 auto-receptors via a negative feedback loop. Integrating our previous results showing the role of ASIC-1 in a positive feedback loop, we also put forth an updated model for synaptic vesicle fusion in which, along with DAT-1 and ASIC-1, the dopamine auto-receptor DOP-2 lies at a modulatory hub at dopaminergic synapses. Our findings are of potential broader significance as similar mechanisms are likely to be used by auto-receptors for other small molecule neurotransmitters across species.

Systematic phenomics analysis of autism-associated genes reveals parallel networks underlying reversible impairments in habituation.

A major challenge facing the genetics of autism spectrum disorders (ASDs) is the large and growing number of candidate risk genes and gene variants of unknown functional significance. Here, we used Caenorhabditis elegans to systematically functionally characterize ASD-associated genes in vivo. Using our custom machine vision system, we quantified 26 phenotypes spanning morphology, locomotion, tactile sensitivity, and habituation learning in 135 strains each carrying a mutation in an ortholog of an ASD-associated gene. We identified hundreds of genotype-phenotype relationships ranging from severe developmental delays and uncoordinated movement to subtle deficits in sensory and learning behaviors. We clustered genes by similarity in phenomic profiles and used epistasis analysis to discover parallel networks centered on CHD8•chd-7 and NLGN3•nlg-1 that underlie mechanosensory hyperresponsivity and impaired habituation learning. We then leveraged our data for in vivo functional assays to gauge missense variant effect. Expression of wild-type NLG-1 in nlg-1 mutant C. elegans rescued their sensory and learning impairments. Testing the rescuing ability of conserved ASD-associated neuroligin variants revealed varied partial loss of function despite proper subcellular localization. Finally, we used CRISPR-Cas9 auxin-inducible degradation to determine that phenotypic abnormalities caused by developmental loss of NLG-1 can be reversed by adult expression. This work charts the phenotypic landscape of ASD-associated genes, offers in vivo variant functional assays, and potential therapeutic targets for ASD.

Is There a Shared Etiology of Olfactory Impairments in Normal Aging and Neurodegenerative Disease?

As we age, our olfactory function declines. In addition to occurring in normal aging, more rapid decrement of olfactory decline has been associated with several neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (PD). It has been argued that since olfactory deficits occur less frequently or are absent in diseases such as progressive supranuclear palsy, corticobasal degeneration, and multiple system atrophy, olfactory deficits can be used for differential diagnoses of AD and PD. The purpose of this review is to provide a survey of current knowledge about the molecular bases and differential patterns of olfactory deficits present in normal aging, AD, and PD. As substantial research has been conducted in this area, the majority of the content of this review focuses on articles published in the past decade. We hypothesize that olfactory deficits in normal aging, AD, and PD may have different underlying causes, and propose the use of model organisms with small, tractable nervous systems and/or easy to manipulate genomes to further investigate the cellular mechanisms responsible for these deficits.

Habituation Is More Than Learning to Ignore: Multiple Mechanisms Serve to Facilitate Shifts in Behavioral Strategy.

Recent work indicates that there are distinct response habituation mechanisms that can be recruited by different stimulation rates and that can underlie different components (e.g., the duration or speed) of a single behavioral response. These findings raise the question: why is "the simplest form of learning" so complicated mechanistically? Beyond evolutionary selection for robustness of plasticity in learning to ignore, it is proposed in this article that multiple mechanisms of habituation have evolved to streamline shifts in ongoing behavioral strategy. Then, speculations are offered regarding the implications of this reconceptualization of habituation for approaching the analysis of mechanisms of more complex forms of learning and memory.

EFHC1, implicated in juvenile myoclonic epilepsy, functions at the cilium and synapse to modulate dopamine signaling.

Neurons throughout the mammalian brain possess non-motile cilia, organelles with varied functions in sensory physiology and cellular signaling. Yet, the roles of cilia in these neurons are poorly understood. To shed light into their functions, we studied EFHC1, an evolutionarily conserved protein required for motile cilia function and linked to a common form of inherited epilepsy in humans, juvenile myoclonic epilepsy (JME). We demonstrate that C. elegans EFHC-1 functions within specialized non-motile mechanosensory cilia, where it regulates neuronal activation and dopamine signaling. EFHC-1 also localizes at the synapse, where it further modulates dopamine signaling in cooperation with the orthologue of an R-type voltage-gated calcium channel. Our findings unveil a previously undescribed dual-regulation of neuronal excitability at sites of neuronal sensory input (cilium) and neuronal output (synapse). Such a distributed regulatory mechanism may be essential for establishing neuronal activation thresholds under physiological conditions, and when impaired, may represent a novel pathomechanism for epilepsy.

High-Throughput Analysis of Behavior Under the Control of Optogenetics in Caenorhabditis elegans.

In this unit, we describe an inexpensive and versatile method for optogenetic stimulation of a large population of genetically engineered Caenorhabditis elegans worms while quantitatively analyzing behavior. A custom light-emitting diode light source is used to deliver blue-light stimuli, causing direct depolarization of neurons expressing the light-gated cation channel Channelrhodopsin-2, which in turn evokes behavioral responses. The behavioral responses are recorded by a high-throughput machine vision-based tracking system, the Multi-Worm Tracker, for detailed analysis. This approach allows researchers to bypass technical obstacles to simultaneously deliver uniform stimuli to a large number of freely behaving animals and investigate the neural underpinnings of behavior. © 2018 by John Wiley & Sons, Inc.