See Elegans: Simple-to-use, accurate, and automatic 3D detection of neural activity from densely packed neurons.

In the emerging field of whole-brain imaging at single-cell resolution, which represents one of the new frontiers to investigate the link between brain activity and behavior, the nematode Caenorhabditis elegans offers one of the most characterized models for systems neuroscience. Whole-brain recordings consist of 3D time series of volumes that need to be processed to obtain neuronal traces. Current solutions for this task are either computationally demanding or limited to specific acquisition setups. Here, we propose See Elegans, a direct programming algorithm that combines different techniques for automatic neuron segmentation and tracking without the need for the RFP channel, and we compare it with other available algorithms. While outperforming them in most cases, our solution offers a novel method to guide the identification of a subset of head neurons based on position and activity. The built-in interface allows the user to follow and manually curate each of the processing steps. See Elegans is thus a simple-to-use interface aimed at speeding up the post-processing of volumetric calcium imaging recordings while maintaining a high level of accuracy and low computational demands. (Contact: enrico.lanza@iit.it).

The recurrent pathogenic Pro890Leu substitution in CLTC causes a generalized defect in synaptic transmission in Caenorhabditis elegans.

De novo CLTC mutations underlie a spectrum of early-onset neurodevelopmental phenotypes having developmental delay/intellectual disability (ID), epilepsy, and movement disorders (MD) as major clinical features. CLTC encodes the widely expressed heavy polypeptide of clathrin, a major component of the coated vesicles mediating endocytosis, intracellular trafficking, and synaptic vesicle recycling. The underlying pathogenic mechanism is largely unknown. Here, we assessed the functional impact of the recurrent c.2669C > T (p.P890L) substitution, which is associated with a relatively mild ID/MD phenotype. Primary fibroblasts endogenously expressing the mutated protein show reduced transferrin uptake compared to fibroblast lines obtained from three unrelated healthy donors, suggesting defective clathrin-mediated endocytosis. In vitro studies also reveal a block in cell cycle transition from G0/G1 to the S phase in patient's cells compared to control cells. To demonstrate the causative role of the p.P890L substitution, the pathogenic missense change was introduced at the orthologous position of the Caenorhabditis elegans gene, chc-1 (p.P892L), via CRISPR/Cas9. The resulting homozygous gene-edited strain displays resistance to aldicarb and hypersensitivity to PTZ, indicating defective release of acetylcholine and GABA by ventral cord motor neurons. Consistently, mutant animals show synaptic vesicle depletion at the sublateral nerve cords, and slightly defective dopamine signaling, highlighting a generalized deficit in synaptic transmission. This defective release of neurotransmitters is associated with their secondary accumulation at the presynaptic membrane. Automated analysis of C. elegans locomotion indicates that chc-1 mutants move slower than their isogenic controls and display defective synaptic plasticity. Phenotypic profiling of chc-1 (+/P892L) heterozygous animals and transgenic overexpression experiments document a mild dominant-negative behavior for the mutant allele. Finally, a more severe phenotype resembling that of chc-1 null mutants is observed in animals harboring the c.3146 T > C substitution (p.L1049P), homologs of the pathogenic c.3140 T > C (p.L1047P) change associated with a severe epileptic phenotype. Overall, our findings provide novel insights into disease mechanisms and genotype-phenotype correlations of CLTC-related disorders.

Phenotypic Assessment of Pathogenic Variants in GNAO1 and Response to Caffeine in C. elegans Models of the Disease.

De novo mutations affecting the G protein α o subunit (Gαo)-encoding gene (GNAO1) cause childhood-onset developmental delay, hyperkinetic movement disorders, and epilepsy. Recently, we established Caenorhabditis elegans as an informative experimental model for deciphering pathogenic mechanisms associated with GNAO1 defects and identifying new therapies. In this study, we generated two additional gene-edited strains that harbor pathogenic variants which affect residues Glu246 and Arg209-two mutational hotspots in Gαo. In line with previous findings, biallelic changes displayed a variable hypomorphic effect on Gαo-mediated signaling that led to the excessive release of neurotransmitters by different classes of neurons, which, in turn, caused hyperactive egg laying and locomotion. Of note, heterozygous variants showed a cell-specific dominant-negative behavior, which was strictly dependent on the affected residue. As with previously generated mutants (S47G and A221D), caffeine was effective in attenuating the hyperkinetic behavior of R209H and E246K animals, indicating that its efficacy is mutation-independent. Conversely, istradefylline, a selective adenosine A2A receptor antagonist, was effective in R209H animals but not in E246K worms, suggesting that caffeine acts through both adenosine receptor-dependent and receptor-independent mechanisms. Overall, our findings provide new insights into disease mechanisms and further support the potential efficacy of caffeine in controlling dyskinesia associated with pathogenic GNAO1 mutations.

Caenorhabditis elegans provides an efficient drug screening platform for GNAO1-related disorders and highlights the potential role of caffeine in controlling dyskinesia.

Dominant GNAO1 mutations cause an emerging group of childhood-onset neurological disorders characterized by developmental delay, intellectual disability, movement disorders, drug-resistant seizures and neurological deterioration. GNAO1 encodes the α-subunit of an inhibitory GTP/GDP-binding protein regulating ion channel activity and neurotransmitter release. The pathogenic mechanisms underlying GNAO1-related disorders remain largely elusive and there are no effective therapies. Here, we assessed the functional impact of two disease-causing variants associated with distinct clinical features, c.139A > G (p.S47G) and c.662C > A (p.A221D), using Caenorhabditis elegans as a model organism. The c.139A > G change was introduced into the orthologous position of the C. elegans gene via CRISPR/Cas9, whereas a knock-in strain carrying the p.A221D variant was already available. Like null mutants, homozygous knock-in animals showed increased egg laying and were hypersensitive to aldicarb, an inhibitor of acetylcholinesterase, suggesting excessive neurotransmitter release by different classes of motor neurons. Automated analysis of C. elegans locomotion indicated that goa-1 mutants move faster than control animals, with more frequent body bends and a higher reversal rate and display uncoordinated locomotion. Phenotypic profiling of heterozygous animals revealed a strong hypomorphic effect of both variants, with a partial dominant-negative activity for the p.A221D allele. Finally, caffeine was shown to rescue aberrant motor function in C. elegans harboring the goa-1 variants; this effect is mainly exerted through adenosine receptor antagonism. Overall, our findings establish a suitable platform for drug discovery, which may assist in accelerating the development of new therapies for this devastating condition, and highlight the potential role of caffeine in controlling GNAO1-related dyskinesia.

A Shearless Microfluidic Device Detects a Role in Mechanosensitivity for AWCON Neuron in Caenorhabditis elegans.

AWC olfactory neurons are fundamental for chemotaxis toward volatile attractants in Caenorhabditis elegans. Here, it is shown that AWCON responds not only to chemicals but also to mechanical stimuli caused by fluid flow changes in a microfluidic device. The dynamics of calcium events are correlated with the stimulus amplitude. It is further shown that the mechanosensitivity of AWCON neurons has an intrinsic nature rather than a synaptic origin, and the calcium transient response is mediated by TAX-4 cGMP-gated cation channel, suggesting the involvement of one or more "odorant" receptors in AWCON mechano-transduction. In many cases, the responses show plateau properties resembling bistable calcium dynamics where neurons can switch from one stable state to the other. To investigate the unprecedentedly observed mechanosensitivity of AWCON neurons, a novel microfluidic device is designed to minimize the fluid shear flow in the arena hosting the nematodes. Animals in this device show reduced neuronal activation of AWCON neurons. The results observed indicate that the tangential component of the mechanical stress is the main contributor to the mechanosensitivity of AWCON . Furthermore, the microfluidic platform, integrating shearless perfusion and calcium imaging, provides a novel and more controlled solution for in vivo analysis both in micro-organisms and cultured cells.

C. elegans-based chemosensation strategy for the early detection of cancer metabolites in urine samples.

Chemosensory receptors play a crucial role in distinguishing the wide range of volatile/soluble molecules by binding them with high accuracy. Chemosensation is the main sensory modality in organisms lacking long-range sensory mechanisms like vision/hearing. Despite its low number of sensory neurons, the nematode Caenorhabditis elegans possesses several chemosensory receptors, allowing it to detect about as many odorants as mammals. Here, we show that C. elegans displays attraction towards urine samples of women with breast cancer, avoiding control ones. Behavioral assays on animals lacking AWC sensory neurons demonstrate the relevance of these neurons in sensing cancer odorants: calcium imaging on AWC increases the accuracy of the discrimination (97.22%). Also, chemotaxis assays on animals lacking GPCRs expressed in AWC allow to identify receptors involved in binding cancer metabolites, suggesting that an alteration of a few metabolites is sufficient for the cancer discriminating behavior of C. elegans, which may help identify a fundamental fingerprint of breast cancer.