Calahorro F et al. (2018) Invert Neurosci "The presynaptic machinery at the synapse of C. elegans."

Izquierdo PG, Calahorro F

[Invert Neurosci, 2018]

Synapses are specialized contact sites that mediate information flow between neurons and their targets. Important physical interactions across the synapse are mediated by synaptic adhesion molecules. These adhesions regulate formation of synapses during development and play a role during mature synaptic function. Importantly, genes regulating synaptogenesis and axon regeneration are conserved across the animal phyla. Genetic screens in the nematode Caenorhabditis elegans have identified a number of molecules required for synapse patterning and assembly. C. elegans is able to survive even with its neuronal function severely compromised. This is in comparison with Drosophila and mice where increased complexity makes them less tolerant to impaired function. Although this fact may reflect differences in the function of the homologous proteins in the synapses between these organisms, the most likely interpretation is that many of these components are equally important, but not absolutely essential, for synaptic transmission to support the relatively undemanding life style of laboratory maintained C. elegans. Here, we review research on the major group of synaptic proteins, involved in the presynaptic machinery in C. elegans, showing a strong conservation between higher organisms and highlight how C. elegans can be used as an informative tool for dissecting synaptic components, based on a simple nervous system organization.

Calahorro F (2014) Invert Neurosci "Conserved and divergent processing of neuroligin and neurexin genes: from the nematode C. elegans to human."

Calahorro F

[Invert Neurosci, 2014]

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 are implicated in autism spectrum disorder and/or mental retardation. Moreover, some copy number variations and point mutations in neurexin-encoding genes have been linked to neurodevelopmental disorders including autism. Neurexins are subject to extensive alternative splicing, highly regulated in mammals, with a great physiological importance. In addition, neuroligins and neurexins are subjected to proteolytic processes that regulate synaptic transmission modifying pre- and postsynaptic activities and may also regulate the remodelling of spines at specific synapses. Four neuroligin genes exist in mice and five in human, whilst in the nematode Caenorhabditis elegans, there is only one orthologous gene. In a similar manner, in mammals, there are three neurexin genes, each of them encoding two major isoforms named and , respectively. In contrast, there is one neurexin gene in C. elegans that also generates two isoforms like mammals. The complexity of the genetic organization of neurexins is due to extensive processing resulting in hundreds of isoforms. In this review, a wide comparison is made between the genes in the nematode and human with a view to better understanding the conservation of processing in these synaptic proteins in C. elegans, which may serve as a genetic model to decipher the synaptopathies underpinning neurodevelopmental disorders such as autism.

Calahorro, F. et al. (2017) International Worm Meeting "Modulation of food-dependent sensory integration in C. elegans."

O'Connor, V., Holden-Dye, L., Keefe, F., Dillon, J., Calahorro, F.

[International Worm Meeting, 2017]

Multisensory inputs are integrated before generating the decision making that drives appropriate behavioural outputs leading possibly a processing and a final integration. Dysfunctional behaviour associated with a number of disorders including autism spectrum disorders, schizophrenia, and anxiety appears to disrupt decision making at the level of the post sensory integrating circuits. The molecular and cellular mechanisms underlying the neural processing underlying decision-making behaviours are beginning to be elucidated. In the nematode C. elegans decision-making is mediated by layers of interneurons controlling the interplay of synergistic and antagonistic multimodal inputs from sensory neurons. Food is a potent modulator of decision-making and context-dependent feeding and locomotion are underpinned by a number of well understood microcircuits. We have investigated food- dependent behaviours in mutants deficient in nlg-1, the homologue of the human neuroligin implicated in autism. We identify that deficiency of this gene indirectly impacts on the feeding and other food dependent behaviours. These findings add to the understanding of the pharyngeal and extra pharyngeal circuits that integrate the decision to feed. The cellular basis of this food dependent behaviour reinforces a top down route that imposes regulation of pharyngeal dependent regulation of feeding. These data highlight details of how neuroligin organizes sub-circuits in C. elegans. Further it resolves how the behavioural expression of the molecular determinants of neuroligin supports in function in C. elegans and parallels observations in higher organisms, at level of integrating circuits. Further in this paradigm we highlight the ability to rescue of nlg-1 deficiencies with human homologues thus providing a platform to investigate autism related mechanisms.

Calahorro F et al. (2021) Toxicol Rep "Impact of drug solvents on C. elegans pharyngeal pumping."

Holden-Dye L, O'Connor V, Calahorro F

[Toxicol Rep, 2021]

<i>Caenorhabditis elegans</i> provides a multi-cellular model organism for toxicology and drug discovery. These studies usually require solvents such as dimethyl sulfoxide (DMSO), ethanol or acetone as a vehicle. This raises the need to carefully consider whether the chemical vehicles used in these screens are anodyne towards <i>C. elegans</i>. Here, we use pharyngeal pumping as a bioassay to assess this. Pharyngeal pumping is a visually scoreable behaviour that is controlled by environmental cues activating sensory and integrative neural signalling to coordinate pharyngeal activity. As such it serves as a rich bioassay to screen for chemical modulation. We found that while pumping was insensitive to high concentrations of the widely used drug solvents ethanol and acetone, it was perturbed by concentrations of DMSO above 0.5 % v/v encompassing concentrations used as drug vehicle. This was manifested as an inhibition of pharyngeal pump rate followed by a slow recovery in the continued presence of the solvent. The inhibition was not observed in a neuroligin mutant, <i>nlg-1</i>, consistent with DMSO acting at the level of sensory processing that modulates pumping. We found that <i>bus-17</i> mutants, which have enhanced cuticle penetration to drugs are more sensitive to DMSO. The effect of DMSO is accompanied by a progressive morphological disruption in which internal membrane-like structures of varying size accumulate. These internal structures are seen in all three genotypes investigated in this study and likely arise independent of the effects on pharyngeal pumping. Overall, these results highlight sensory signalling and strain dependent vehicle sensitivity. Although we define concentrations at which this can be mitigated, it highlights the need to consider time-dependent vehicle effects when evaluating control responses in <i>C. elegans</i> chemical biology.

Calahorro, F. et al. (2019) International Worm Meeting "Automated extraction of food-dependent sub-behaviours in freely moving C.elegans."

Calahorro, F., James, C.

[International Worm Meeting, 2019]

Caenorhabditis elegansrequires a gradual regulation of behaviours for spatial navigation, as well as for fine tuned modulation of multiple sensory-dependent cues from the environment. Food-related behaviours in C. elegansare modulated through signalling molecules and precise neural circuits. Different subsets of complex microcircuits in the nervous system, governs the interplay of locomotion strategies during food searching (1,2). Integration of distinct sensory cues is essential for behavioural decision-making. Thus, decision-making in C.elegans is an extremely coordinated process involving layers of sensory neurons, interneurons and motorneurons in the head and ventral nerve cord to generate an appropriate behaviour driven by output muscles (3). Rhythmic behaviours in C.elegansare relatively easy to conduct and observe. Ideally it requires a video recording in order to identify features within particular behaviours, this is a time consuming process. In view of this, we have developed a freely moving tracking toolbox, TrakBox, enabling a real-time analysis of complex behaviours in C. elegans. As a proof-of-concept we have assessed the ability and effectiveness of TrakBox to extract and analyse behavioural information of C. elegansin freely moving worms, verifying its use by analysing a well defined behavioural signature in mod-1mutant. By comparing with known exploratory features in mod-1previously published findings (extracted 'manually') (4), TrakBox was able to show an accurate annotation of sub-behaviours associated with locomotion and exploration, with high levels of accuracy. We also analysed tracks recorded at different duration due to the flexibility of TrakBox which allows long-term monitoring, illustrating the ability to automatically analyse very long tracks. In addition, we quantified features such as reversal events and fractions of time in dwelling/roaming, in chemotaxis assay to further demonstrate the capabilities of the TrakBox analysis. We found that under different track durations, short and long traces, the analysis showed an effective extraction of sub-behaviours. Thus, with TrakBox, we have been able to show alterations of exploratory behaviours in mod-1mutant as previously described, highlighting its capability for the rapid and effective identification of phenotypes in freely moving C. elegans. 1LeDoux, J. E. (2000). Annu Rev Neurosci 23: 155-184.2Barkus, C., S. B. McHugh, et al. (2010). Eur J Pharmacol 626(1): 49-56.3Shtonda, B. B. and L. Avery (2006). J Exp Biol 209(Pt 1): 89-102. 4Flavell, S. W., N. Pokala, et al. (2013). Cell 154(5): 1023-1035.

Calahorro, F. et al. (2019) International Worm Meeting "TrakBox: A low-cost tracking system for monitoring freely moving C.elegans."

Calahorro, F., James, C.

[International Worm Meeting, 2019]

Quantification and fine analysis of features in sub-behaviours underpinning global-position and locomotion patterns are key to dissect and map the global brain dynamics leading behaviours and decisions between alternative behaviours in C.elegans. For this purpose, we have developed TrakBox, a low-cost tracking system to track a wide range of C.elegansbehaviours, for flexible periods of time, and to automatically analyse in real-time. The tracking device consists of two parts: i) the low-cost hardware which comprises an off-the-shelf USB microscope mounted on a 3D printed tracking platform, and ii) bespoke software that allows the tracking of single nematodes over long periods of time, and which performs automatic analysis of behaviour. TrakBox has the capability to accommodate plates of various sizes (as well as multi-well plates), provides its own white (LED) light source. Inverse kinematics is used to translate the nematode's xand ycoordinates to arm-angles and the arm is moved to keep the worm in the centre of the field of view. Video images are captured at around 3 frames per second, and advanced image processing techniques are used to isolate the nematode on each frame and determine the location of the worm's centre of mass. Advanced signal processing filters and corrects the worm position over time and constantly updates a series of behaviour parameters calculated 'on-the-fly'. The TrakBox software records and derives a number of parameters over the course of the worm tracking - primarily through the worm xand ycoordinates. Instantaneous velocity is calculated as well as the direction vector, this allows for the detection and localisation of reversals in the recordings. Worm dwelling versus roaming times are calculated based on worm movements being below a specified location threshold (for dwelling) - the software also distinguishes quiescent behaviour from dwelling behaviour. The times the worm performs reversals is automatically calculated and indicated. Nematode behaviour can be observed over long periods of time as the nematode exhibits different behaviour patterns. Worm tracks can be saved and re-analysed at a later date by the end-user, where different analysis parameters can be used to generate new results.

Calahorro, F. et al. (2017) International Worm Meeting "TrakBox: A new modular option for behavioural monitoring of freely moving C. elegans."

James, C., Calahorro, F., O'Connor, V., Holden-Dye, L.

[International Worm Meeting, 2017]

Tracking the movement of C. elegans is a well-stablished route to identify and refine understanding of microcircuits that control behaviour. Over several years a number of different experimental platforms and associated software for video analysis have been developed to facilitate this (1). To contribute to this we have designed a relatively low cost modular system for real-time tracking and analysis of worm locomotory behaviour. TrakBox (EMbody Biosignals Ltd) (2) is assembled from 3-D printed components in which a robotic arm moves a USB camera to maintain the animal being tracked within a target site. Reverse kinematics is used to decode the coordinates of the worm as it moves around the plate with continuous tracking being possible for at least 24 h. Videos may also be captured but are not required for the behavioural analyses. Advanced signal processing filters and corrects the worm position over time and constantly updates a series of behaviour parameters calculated 'on the fly'. TrakBox software derives parameters over the entire course of tracking that describe worm position, instantaneous velocity, instantaneous direction of travel, dwelling and roaming times and number of reversals. The user may define the behavioural parameters of interest and they may be displayed as a graphical representation. As a proof of concept we compared the behaviour of N2 and a mod-1 mutant. mod-1 encodes a serotonin-gated chloride channel and a loss of function mutant exhibits increased exploration and extended roaming time (3). TrakBox efficiently extracted this phenotype by analysing roaming/dwelling fractions of time, velocity and reversal events and their duration. Thus, we show TrakBox permits discrete real-time analysis of C. elegans locomotory behaviour that circumvents the need for post-hoc analysis of videos. This experimental platform also has applications for investigation of the effects of drugs on behaviour, may be applied to analysis of other microscopic nematodes including pest species, have uses in field studies and provide an affordable option for class room demonstration of C. elegans biology. (1) Husson, S. J. et al. Keeping track of worm trackers (September 10, 2012), WormBook, ed. (2) http://embody-biosignals.com/ (3) Flavell, S. W., N. Pokala, E. Z. Macosko, D. R. Albrecht, J. Larsch and C. I. Bargmann (2013). "Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans." Cell 154(5): 1023-1035

Calahorro F et al. (2011) Invert Neurosci "Caenorhabditis elegans as an experimental tool for the study of complex neurological diseases: Parkinson's disease, Alzheimer's disease and autism spectrum disorder."

Calahorro F, Ruiz-Rubio M

[Invert Neurosci, 2011]

The nematode Caenorhabditis elegans has a very well-defined and genetically tractable nervous system which offers an effective model to explore basic mechanistic pathways that might be underpin complex human neurological diseases. Here, the role C. elegans is playing in understanding two neurodegenerative conditions, Parkinson's and Alzheimer's disease (AD), and a complex neurological condition, autism, is used as an exemplar of the utility of this model system. C. elegans is an imperfect model of Parkinson's disease because it lacks orthologues of the human disease-related genes PARK1 and LRRK2 which are linked to the autosomal dominant form of this disease. Despite this fact, the nematode is a good model because it allows transgenic expression of these human genes and the study of the impact on dopaminergic neurons in several genetic backgrounds and environmental conditions. For AD, C. elegans has orthologues of the amyloid precursor protein and both human presenilins, PS1 and PS2. In addition, many of the neurotoxic properties linked with A amyloid and tau peptides can be studied in the nematode. Autism spectrum disorder is a complex neurodevelopmental disorder characterised by impairments in human social interaction, difficulties in communication, and restrictive and repetitive behaviours. Establishing C. elegans as a model for this complex behavioural disorder is difficult; however, abnormalities in neuronal synaptic communication are implicated in the aetiology of the disorder. Numerous studies have associated autism with mutations in several genes involved in excitatory and inhibitory synapses in the mammalian brain, including neuroligin, neurexin and shank, for which there are C. elegans orthologues. Thus, several molecular pathways and behavioural phenotypes in C. elegans have been related to autism. In general, the nematode offers a series of advantages that combined with knowledge from other animal models and human research, provides a powerful complementary experimental approach for understanding the molecular mechanisms and underlying aetiology of complex neurological diseases.

Calahorro F et al. (2015) Gene Expr Patterns "Analysis of splice variants for the C.elegans orthologue of human neuroligin reveals a developmentally regulated transcript."

Calahorro F, O'Connor V, Holden-Dye L

[Gene Expr Patterns, 2015]

Neuroligins are synaptic adhesion molecules and important determinants of synaptic function. They are expressed at postsynaptic sites and involved in synaptic organization through key extracellular and intracellular protein interactions. They undergo trans-synaptic interaction with presynaptic neurexins. Distinct neuroligins use differences in their intracellular domains to selectively recruit synaptic scaffolds and this plays an important role in how they encode specialization of synaptic function. Several levels of regulation including gene expression, splicing, protein translation and processing regulate the expression of neuroligin function. We have used in silico and cDNA analyses to investigate the mRNA splicing of the Caenorhabditis elegans orthologue nlg-1. Transcript analysis highlights the potential for gene regulation with respect to both temporal expression and splicing. We found nlg-1 splice variants with all the predicted exons are a minor species relative to major splice variants lacking exons 13 and 14, or 14 alone. These major alternatively spliced variants change the intracellular domain of the gene product NLG-1. Interestingly, exon 14 encodes a cassette with two distinct potential functional domains. One is a polyproline SH3 binding domain and the other has homology to a region encoding the binding site for the scaffolding protein gephyrin in mammalian neuroligins. This suggests differential splicing impacts on NLG-1 competence to recruit intracellular binding partners. This may have developmental relevance as nlg-1 exon 14 containing transcripts are selectively expressed in L2-L3 larvae. These results highlight a developmental regulation of C.elegans nlg-1 that could play a key role in the assembly of synaptic protein complexes during the early stages of nervous system development.

Calahorro F et al. (2013) Genes Brain Behav "Human alpha- and beta-NRXN1 isoforms rescue behavioral impairments of Caenorhabditis elegans neurexin-deficient mutants."

Ruiz-Rubio M, Calahorro F

[Genes Brain Behav, 2013]

Neurexins are cell adhesion proteins that interact with neuroligin and other ligands at the synapse. In humans, mutations in neurexin or neuroligin genes have been associated with autism and other mental disorders. The human neurexin and neuroligin genes are orthologous to the Caenorhabditis elegans genes nrx-1 and nlg-1, respectively. Here we show that nrx-1-deficient mutants are defective in exploratory capacity, sinusoidal postural movements and gentle touch response. Interestingly, the exploratory behavioral phenotype observed in nrx-1 mutants was markedly different to nlg-1-deficient mutants; thus, while the former had a 'hyper-reversal' phenotype increasing the number of changes of direction with respect to the wild-type strain, the nlg-1 mutants presented a 'hypo-reversal' phenotype. On the other hand, the nrx-1- and nlg-1-defective mutants showed similar abnormal sinusoidal postural movement phenotypes. The response of these mutant strains to aldicarb (acetylcholinesterase inhibitor), levamisole (ACh agonist) and pentylenetetrazole [gamma-aminobutyric (GABA) receptor antagonist], suggested that the varying behavioral phenotypes were caused by defects in ACh and/or GABA inputs. The defective behavioral phenotypes of nrx-1-deficient mutants were rescued in transgenic strains expressing either human alpha- or beta-NRXN-1 isoforms under the worm nrx-1 promoter. A previous report had shown that human and rat neuroligins were functional in C. elegans. Together, these results suggest that the functional mechanism underpinning both neuroligin and neurexin in the nematode are comparable to human. In this sense the nematode might constitute a simple in vivo model for understanding basic mechanisms involved in neurological diseases for which neuroligin and neurexin are implicated in having a role.