Simple Nervous System Research Articles

A Connectome-Based Digital Twin Caenorhabditis elegans Capable of Intelligent Sensorimotor Behavior

Despite possessing a simple nervous system, the Caenorhabditis elegans exhibits remarkably intelligent behavior. However, the underlying mechanisms involved in sensory processing and decision making, which contribute to locomotion behaviors, remain unclear. In order to investigate the coordinated function of neurons in achieving chemotaxis behavior, we have developed a digital twin of the C. elegans that combines a connectome-based neural network model with a realistic digital worm body. Through training the digital worm using offline chemotaxis behavioral data generated with a PID controller, we have successfully replicated faithful sinusoidal crawling and intelligent chemotaxis behavior, similar to real worms. By ablating individual neurons, we have examined their roles in modulating or contributing to the regulation of behavior. Our findings highlight the critical involvement of 119 neurons in sinusoidal crawling, including B-type, A-type, D-type, and PDB motor neurons, as well as AVB and AVA interneurons, which was experimentally demonstrated. We have also predicted the involvement of DD04 and DD05 neurons and the lack of relevance of DD02 and DD03 neurons in crawling, which have been confirmed through experimentation. Additionally, head motor neurons, sublateral motor neurons, layer 1 interneurons, and layer 1 and layer 5 sensory neurons are expected to play a role in crawling. In summary, we present a novel methodological framework that enables the establishment of an animal model capable of closed-loop control, faithfully replicating realistic animal behavior. This framework holds potential for examining the neural mechanisms of behaviors in other species.

How to create a nervous system aneurysm model in canines? ligation of the lingual artery is a simple and effective method.

Background: The purpose of this research was to establish a safe, effective, and simple nervous system aneurysm model. This method could quickly and stably establish an exact canine tongue aneurysm model. This paper summarizes the technique and key points of the method. Methods: Under the condition of anesthesia by inhaling isoflurane with a mask, we punctured the femoral artery of the canine, and the tip of the catheter was placed in the common carotid artery for intracranial arteriography. The positions of the lingual artery, external carotid artery, and internal carotid artery were identified. Then, the skin near the mandible was cut according to the positioning and separated layer by layer until the bifurcation of the lingual artery and external carotid artery was exposed. The lingual artery were then sutured with 2-0 silk sutures approximately 3mm from the external carotid/lingual artery bifurcation. The final angiographic review showed that the aneurysm model was successfully established. Results: The lingual artery aneurysm was successfully established in all 8 canines. All canines obtained a stable model of nervous system aneurysm and confirmed by DSA angiography. Conclusion: We have established a safe, effective, stable and simple method to establish a canine nervous system aneurysm model with controllable size. In addition, this method has the advantages of no arteriotomy, less trauma, constant anatomical location, and low risk of stroke.

Characterization of the planarian surface electroencephalogram

BackgroundDespite large morphological differences between the nervous systems of lower animals and humans, striking functional similarities have been reported. However, little is known about how these functional similarities translate to cognitive similarities. As a first step towards studying the cognitive abilities of simple nervous systems, we here characterize the ongoing electrophysiological activity of the planarian Schmidtea mediterranea. One previous report using invasive microelectrodes describes that the ongoing neural activity is characterized by a 1/fx power spectrum with the exponent ‘x’ of the power spectrum close to 1. To extend these findings, we aimed to establish a recording protocol to measure ongoing neural activity safely and securely from alive and healthy planarians under different lighting conditions using non-invasive surface electrodes.ResultsAs a replication and extension of the previous results, we show that the ongoing neural activity is characterized by a 1/fx power spectrum, that the exponent ‘x’ in living planarians is close to 1, and that changes in lighting induce changes in neural activity likely due to the planarian photophobia.ConclusionsWe confirm the existence of continuous EEG activity in planarians and show that it is possible to noninvasively record this activity with surface wire electrodes. This opens up broad possibilities for continuous recordings across longer intervals, and repeated recordings from the same animals to study cognitive processes.

Distribution, cellular localization, and colocalization of several peptide neurotransmitters in the central nervous system of Aplysia.

Neuropeptides are widely used as neurotransmitters in vertebrates and invertebrates. In vertebrates, a detailed understanding of their functions as transmitters has been hampered by the complexity of the nervous system. The marine mollusk Aplysia, with a simpler nervous system and many large, identified neurons, presents several advantages for addressing this question and has been used to examine the roles of tens of peptides in behavior. To screen for other peptides that might also play roles in behavior, we observed immunoreactivity in individual neurons in the central nervous system of adult Aplysia with antisera raised against the Aplysia peptide FMRFamide and two mammalian peptides that are also found in Aplysia, cholecystokinin (CCK) and neuropeptide Y (NPY), as well as serotonin (5HT). In addition, we observed staining of individual neurons with antisera raised against mammalian somatostatin (SOM) and peptide histidine isoleucine (PHI). However, genomic analysis has shown that these two peptides are not expressed in the Aplysia nervous system, and we have therefore labeled the unknown peptides stained by these two antibodies as XSOM and XPHI There was an area at the anterior end of the cerebral ganglion that had staining by antisera raised against many different transmitters, suggesting that this may be a modulatory region of the nervous system. There was also staining for XSOM and, in some cases, FMRFamide in the bag cell cluster of the abdominal ganglion. In addition, these and other studies have revealed a fairly high degree of colocalization of different neuropeptides in individual neurons, suggesting that the peptides do not just act independently but can also interact in different combinations to produce complex functions. The simple nervous system of Aplysia is advantageous for further testing these ideas.

Peptide-driven control of somersaulting in Hydra vulgaris

The cnidarian Hydra vulgaris has a simple nervous system with a few hundred neurons in distributed networks. Yet Hydra can perform somersaults, a complex acrobatic locomotion. To understand the neural mechanisms of somersaulting we used calcium imaging and found that rhythmical potential 1 (RP1) neurons activate before somersaulting. Decreasing RP1 activity or ablating RP1 neurons reduced somersaulting, while two-photon activation of RP1 neurons induced somersaulting. Hym-248, a peptide synthesized by RP1 cells, selectively generated somersaulting. We conclude that RP1 activity, via release of Hym-248, is necessary and sufficient for somersaulting. We propose a circuit model to explain the sequential unfolding of this locomotion, using integrate-to-threshold decision making and cross-inhibition. Our work demonstrates that peptide-based signaling is used by simple nervous systems to generate behavioral fixed action patterns.

Brainstem neural mechanisms controlling locomotion with special reference to basal vertebrates.

Over the last 60 years, the basic neural circuitry responsible for the supraspinal control of locomotion has progressively been uncovered. Initially, significant progress was made in identifying the different supraspinal structures controlling locomotion in mammals as well as some of the underlying mechanisms. It became clear, however, that the complexity of the mammalian central nervous system (CNS) prevented researchers from characterizing the detailed cellular mechanisms involved and that animal models with a simpler nervous system were needed. Basal vertebrate species such as lampreys, xenopus embryos, and zebrafish became models of choice. More recently, optogenetic approaches have considerably revived interest in mammalian models. The mesencephalic locomotor region (MLR) is an important brainstem region known to control locomotion in all vertebrate species examined to date. It controls locomotion through intermediary cells in the hindbrain, the reticulospinal neurons (RSNs). The MLR comprises populations of cholinergic and glutamatergic neurons and their specific contribution to the control of locomotion is not fully resolved yet. Moreover, the downward projections from the MLR to RSNs is still not fully understood. Reporting on discoveries made in different animal models, this review article focuses on the MLR, its projections to RSNs, and the contribution of these neural elements to the control of locomotion. Excellent and detailed reviews on the brainstem control of locomotion have been recently published with emphasis on mammalian species. The present review article focuses on findings made in basal vertebrates such as the lamprey, to help direct new research in mammals, including humans.

Subcellular Imaging of Neuronal Calcium Handling In Vivo.

Calcium (Ca2+) imaging has been largely used to examine neuronal activity, but it is becoming increasingly clear that subcellular Ca2+ handling is a crucial component of intracellular signaling. The visualization of subcellular Ca2+ dynamics in vivo,where neurons can be studied in their native, intact circuitry, has proven technically challenging in complex nervous systems. The transparency and relatively simple nervous system of the nematode Caenorhabditis elegans enable the cell-specific expression and in vivo visualization of fluorescent tags and indicators. Among these are fluorescent indicators that have been modified for use in the cytoplasm as well as various subcellular compartments, such as the mitochondria. This protocol enables non-ratiometric Ca2+ imaging in vivo with a subcellular resolution that permits the analysis of Ca2+ dynamics down to the level of individual dendritic spines and mitochondria. Here, two available genetically encoded indicators with different Ca2+ affinities are used to demonstrate the use of this protocol for measuring relative Ca2+ levels within the cytoplasm or mitochondrial matrix in a single pair of excitatory interneurons (AVA). Together with the genetic manipulations and longitudinal observations possible in C. elegans, this imaging protocol may be useful for answering questions regarding how Ca2+ handling regulates neuronal function and plasticity.

The Expression of miRNAs Involved in Long-Term Memory Formation in the CNS of the Mollusk Helix lucorum

Mollusks are unique animals with a relatively simple central nervous system (CNS) containing giant neurons with identified functions. With such simple CNS, mollusks yet display sufficiently complex behavior, thus ideal for various studies of behavioral processes, including long-term memory (LTM) formation. For our research, we use the formation of the fear avoidance reflex in the terrestrial mollusk Helix lucorum as a learning model. We have shown previously that LTM formation in Helix requires epigenetic modifications of histones leading to both activation and inactivation of the specific genes. It is known that microRNAs (miRNAs) negatively regulate the expression of genes; however, the role of miRNAs in behavioral regulation has been poorly investigated. Currently, there is no miRNAs sequencing data being published on Helix lucorum, which makes it impossible to investigate the role of miRNAs in the memory formation of this mollusk. In this study, we have performed sequencing and comparative bioinformatics analysis of the miRNAs from the CNS of Helix lucorum. We have identified 95 different microRNAs, including microRNAs belonging to the MIR-9, MIR-10, MIR-22, MIR-124, MIR-137, and MIR-153 families, known to be involved in various CNS processes of vertebrates and other species, particularly, in the fear behavior and LTM. We have shown that in the CNS of Helix lucorum MIR-10 family (26 miRNAs) is the most representative one, including Hlu-Mir-10-S5-5p and Hlu-Mir-10-S9-5p as top hits. Moreover, we have shown the involvement of the MIR-10 family in LTM formation in Helix. The expression of 17 representatives of MIR-10 differentially changes during different periods of LTM consolidation in the CNS of Helix. In addition, using comparative analysis of microRNA expression upon learning in normal snails and snails with deficient learning abilities with dysfunction of the serotonergic system, we identified a number of microRNAs from several families, including MIR-10, which expression changes only in normal animals. The obtained data can be used for further fundamental and applied behavioral research.

2-D sex images elicit mate copying in fruit flies

Although the environment is three-dimensional (3-D), humans are able to extract subtle information from two-dimensional (2-D) images, particularly in the domain of sex. However, whether animals with simpler nervous systems are capable of such information extraction remains to be demonstrated, as this ability would suggest a functional generalisation capacity. Here, we performed mate-copying experiments in Drosophila melanogaster using 2-D artificial stimuli. Mate copying occurs when naïve females observe the mating success of potential mates and use that social information to build their own mating preference. By replacing live demonstrations with (i) photos or (ii) simplified images of copulating pairs, we found that even crudely simplified images of sexual intercourse still elicit mate copying, suggesting that Drosophila is able to extract sex-related information even from a degraded image. This new method constitutes a powerful tool to further investigate mate copying in that species and sexual preferences in general.

Neurobiological reduction: From cellular explanations of behavior to interventions.

Scientific reductionism, the view that higher level functions can be explained by properties at some lower-level or levels, has been an assumption of nervous system analyses since the acceptance of the neuron doctrine in the late 19th century, and became a dominant experimental approach with the development of intracellular recording techniques in the mid-20th century. Subsequent refinements of electrophysiological approaches and the continual development of molecular and genetic techniques have promoted a focus on molecular and cellular mechanisms in experimental analyses and explanations of sensory, motor, and cognitive functions. Reductionist assumptions have also influenced our views of the etiology and treatment of psychopathologies, and have more recently led to claims that we can, or even should, pharmacologically enhance the normal brain. Reductionism remains an area of active debate in the philosophy of science. In neuroscience and psychology, the debate typically focuses on the mind-brain question and the mechanisms of cognition, and how or if they can be explained in neurobiological terms. However, these debates are affected by the complexity of the phenomena being considered and the difficulty of obtaining the necessary neurobiological detail. We can instead ask whether features identified in neurobiological analyses of simpler aspects in simpler nervous systems support current molecular and cellular approaches to explaining systems or behaviors. While my view is that they do not, this does not invite the opposing view prevalent in dichotomous thinking that molecular and cellular detail is irrelevant and we should focus on computations or representations. We instead need to consider how to address the long-standing dilemma of how a nervous system that ostensibly functions through discrete cell to cell communication can generate population effects across multiple spatial and temporal scales to generate behavior.

An early midbrain sensorimotor pathway is involved in the timely initiation and direction of swimming in the hatchling Xenopus laevis tadpole.

Vertebrate locomotion is heavily dependent on descending control originating in the midbrain and subsequently influencing central pattern generators in the spinal cord. However, the midbrain neuronal circuitry and its connections with other brainstem and spinal motor circuits has not been fully elucidated. Vertebrates with very simple nervous system, like the hatchling Xenopus laevis tadpole, have been instrumental in unravelling fundamental principles of locomotion and its suspraspinal control. Here, we use behavioral and electrophysiological approaches in combination with lesions of the midbrain to investigate its contribution to the initiation and control of the tadpole swimming in response to trunk skin stimulation. None of the midbrain lesions studied here blocked the tadpole's sustained swim behavior following trunk skin stimulation. However, we identified that distinct midbrain lesions led to significant changes in the latency and trajectory of swimming. These changes could partly be explained by the increase in synchronous muscle contractions on the opposite sides of the tadpole's body and permanent deflection of the tail from its normal position, respectively. We conclude that the tadpole's embryonic trunk skin sensorimotor pathway involves the midbrain, which harbors essential neuronal circuitry to significantly contribute to the appropriate, timely and coordinated selection and execution of locomotion, imperative to the animal's survival.

Neural Network-Based Autonomous Search Model with Undulatory Locomotion Inspired by Caenorhabditis Elegans.

Caenorhabditis elegans (C. elegans) exhibits sophisticated chemotaxis behavior with a unique locomotion pattern using a simple nervous system only and is, therefore, well suited to inspire simple, cost-effective robotic navigation schemes. Chemotaxis in C. elegans involves two complementary strategies: klinokinesis, which allows reorientation by sharp turns when moving away from targets; and klinotaxis, which gradually adjusts the direction of motion toward the preferred side throughout the movement. In this study, we developed an autonomous search model with undulatory locomotion that combines these two C. elegans chemotaxis strategies with its body undulatory locomotion. To search for peaks in environmental variables such as chemical concentrations and radiation in directions close to the steepest gradients, only one sensor is needed. To develop our model, we first evolved a central pattern generator and designed a minimal network unit with proprioceptive feedback to encode and propagate rhythmic signals; hence, we realized realistic undulatory locomotion. We then constructed adaptive sensory neuron models following real electrophysiological characteristics and incorporated a state-dependent gating mechanism, enabling the model to execute the two orientation strategies simultaneously according to information from a single sensor. Simulation results verified the effectiveness, superiority, and realness of the model. Our simply structured model exploits multiple biological mechanisms to search for the shortest-path concentration peak over a wide range of gradients and can serve as a theoretical prototype for worm-like navigation robots.

Homologous gene regulatory networks control development of apical organs and brains in Bilateria.

Apical organs are relatively simple larval nervous systems. The extent to which apical organs are evolutionarily related to the more complex nervous systems of other animals remains unclear. To identify common developmental mechanisms, we analyzed the gene regulatory network (GRN) controlling the development of the apical organ in sea urchins. We characterized the developmental expression of 30 transcription factors and identified key regulatory functions for FoxQ2, Hbn, Delta/Notch signaling, and SoxC in the patterning of the apical organ and the specification of neurons. Almost the entire set of apical transcription factors is expressed in the nervous system of worms, flies, zebrafish, frogs, and mice. Furthermore, a regulatory module controlling the axial patterning of the vertebrate brain is expressed in the ectoderm of sea urchin embryos. We conclude that GRNs controlling the formation of bilaterian nervous systems share a common origin and that the apical GRN likely resembles an ancestral regulatory program.

Multisensory Integration in Caenorhabditis elegans in Comparison to Mammals.

Multisensory integration refers to sensory inputs from different sensory modalities being processed simultaneously to produce a unitary output. Surrounded by stimuli from multiple modalities, animals utilize multisensory integration to form a coherent and robust representation of the complex environment. Even though multisensory integration is fundamentally essential for animal life, our understanding of the underlying mechanisms, especially at the molecular, synaptic and circuit levels, remains poorly understood. The study of sensory perception in Caenorhabditis elegans has begun to fill this gap. We have gained a considerable amount of insight into the general principles of sensory neurobiology owing to C. elegans’ highly sensitive perceptions, relatively simple nervous system, ample genetic tools and completely mapped neural connectome. Many interesting paradigms of multisensory integration have been characterized in C. elegans, for which input convergence occurs at the sensory neuron or the interneuron level. In this narrative review, we describe some representative cases of multisensory integration in C. elegans, summarize the underlying mechanisms and compare them with those in mammalian systems. Despite the differences, we believe C. elegans is able to provide unique insights into how processing and integrating multisensory inputs can generate flexible and adaptive behaviors. With the emergence of whole brain imaging, the ability of C. elegans to monitor nearly the entire nervous system may be crucial for understanding the function of the brain as a whole.

Single-cell transcriptomics identifies conserved regulators of neuroglandular lineages.

Communication in bilaterian nervous systems is mediated by electrical and secreted signals; however, the evolutionary origin and relation of neurons to other secretory cell types has not been elucidated. Here, we use developmental single-cell RNA sequencing in the cnidarian Nematostella vectensis, representing an early evolutionary lineage with a simple nervous system. Validated by transgenics, we demonstrate that neurons, stinging cells, and gland cells arise from a common multipotent progenitor population. We identify the conserved transcription factor gene SoxC as a key upstream regulator of all neuroglandular lineages and demonstrate that SoxC knockdown eliminates both neuronal and secretory cell types. While in vertebrates and many other bilaterians neurogenesis is largely restricted to early developmental stages, we show that in the sea anemone, differentiation of neuroglandular cells is maintained throughout all life stages, and follows the same molecular trajectories from embryo to adulthood, ensuring lifelong homeostasis of neuroglandular cell lineages.

Brain size and behavioral specialization in the jataí stingless bee (Tetragonisca angustula).

Social insects are instructive models for understanding the association between investment in brain size and behavioral variability because they show a relatively simple nervous system associated with a large set of complex behaviors. In the jataí stingless bee (Tetragonisca angustula), division of labor relies both on age and body size differences among workers. When young, both minors and soldiers engage in intranidal tasks and move to extranidal tasks as they age. Minors switch to foraging activities, while soldiers take over defensive roles. Nest defense performed by soldiers includes two different tasks: (1) hovering around the nest entrance for the detection and interception of heterospecific bees (a task relying mostly on vision) and (2) standing at the nest entrance tube for inspection of returning foragers and discrimination against conspecific non-nestmates based on olfactory cues. Here, using different-sized individuals (minors and soldiers) as well as same-sized individuals (hovering and standing soldiers) performing distinct tasks, we investigated the effects of both morphological and behavioral variability on brain size. We found a negative allometric growth between brain size and body size across jataí workers, meaning that minors had relatively larger brains than soldiers. Between soldier types, we found that hovering soldiers had larger brain compartments related to visual processing (the optic lobes) and learning (the mushroom bodies). Brain size differences between jataí soldiers thus correspond to behavioral specialization in defense (i.e., vision for hovering soldiers) and illustrate a functional neuroplasticity underpinning division of labor.

Bisphenol A Exposure Induces Sensory Processing Deficits in Larval Zebrafish during Neurodevelopment.

Because of their ex utero development, relatively simple nervous system, translucency, and availability of tools to investigate neural function, larval zebrafish are an exceptional model for understanding neurodevelopmental disorders and the consequences of environmental toxins. Furthermore, early in development, zebrafish larvae easily absorb chemicals from water, a significant advantage over methods required to expose developing organisms to chemical agents in utero. Bisphenol A (BPA) and BPA analogs are ubiquitous environmental toxins with known molecular consequences. All humans have measurable quantities of BPA in their bodies. Most concerning, the level of BPA exposure is correlated with neurodevelopmental difficulties in people. Given the importance of understanding the health-related effects of this common toxin, we have exploited the experimental advantages of the larval zebrafish model system to investigate the behavioral and anatomic effects of BPA exposure. We discovered that BPA exposure early in development leads to deficits in the processing of sensory information, as indicated by BPA’s effects on prepulse inhibition (PPI) and short-term habituation (STH) of the C-start reflex. We observed no changes in locomotion, thigmotaxis, and repetitive behaviors (circling). Despite changes in sensory processing, we detected no regional or whole-brain volume changes. Our results show that early BPA exposure can induce sensory processing deficits, as revealed by alterations in simple behaviors that are mediated by a well-defined neural circuit.

Genomic Study of Histamine Receptor Ligand Binding Sites of the Bivalve Mollusc Crassostrea virginica

Histamine is a biogenic amine found in a wide variety of invertebrates. Histamine is particularly well studied in arthropods and gastropods where it is involved in local immune responses as well as regulating physiological functions in the gut. Histamine also functions as a neurotransmitter, especially for sensory systems. Previous physiology work of our lab found that histamine activates the sensory system of Crassostraea virginica, eliciting a motor response in the gill. Our earlier cell biology and immunofluorescence work also showed the presence of histamine receptors in ganglia and mantle of C. virginica. Recently the genome of C. virginicaand other bivalves have begun to be mapped. By conducting BLAST searches of the NCBI (National Center for Biotechnology Information) database using DNA and protein sequences of C. virginica and other invertebrate and mammalian species we found matches for histamine receptor H1R genes on chromosome 8; H2R on chromosomes 1, 2, 5 and 10; and H3R on chromosome 3. BLASTS of other invertebrates and mammals found matches with very low Expect Values (E Values) and moderately high Percent Identity, signifying similarities of H1R, H2R and H3R of C. virginica to those of other bivalves, gastropods, insects, mice, rats and humans. We hypothesize that the ligand binding sites (LBS) for H1R, H2R and H3R receptors in C. virginica are evolutionarily conserved and will closely match those of other animals. To study this, we conducted searches of the NCBI database for H1R, H2R and H3R receptors LBS of C. virginica and compared them to other animals. We found the LBS for H2R in C. virginica was identified and match some other invertebrates well, but did not match humans of other mammals very well. The LBS for H3R matched some other bivalves, invertebrates as well as humans and other mammals well. The LBS for H1R in C. virginica and other invertebrates we looked at has not yet been identified. The LBS for H1R in humans and other mammals is very highly conserved. This study complements our earlier physiology and cell biology studies demonstrating the presence and function for histamine in C. virginica, and shows that the genome of C. virginica contains genes to produce histamine receptor LBS that are similar to those of other animals where it has been identified. This new information is valuable as it shows that the simple nervous system of histamine can be used to expand studies on histamine neurotransmission.

Genomic Study of GABA Receptor Ligand Binding Sites of the Bivalve Mollusc Crassostrea virginica

GABA (γ‐aminobutyric acid) an inhibitory neurotransmitter in molluscs and other animals has not been well studied in bivalves. In humans, impairment of GABA neurotransmission can cause epilepsy. In the bivalve mollusc Crassostrea virginica, as well as other bivalves, serotonin is an excitatory neurotransmitter that increases beating rates of gill lateral cell cilia. Previously our lab demonstrated in C. virginica serotonin’s increase of cilia beating rates is blocked by applying GABA to the visceral or cerebral ganglia. Additionally, bicuculline methchloride, a GABAa receptor antagonist, blocked the effects of GABA. By using HPLC we previously detected GABA in low ng amounts in cerebral and visceral ganglia of C. virginica. Our immunofluorescence studies showed the presence of GABA neurons in cerebral and visceral ganglia; and that some serotonin neurons had GABA receptors on their soma. Recently the genome of C. virginica and other bivalves has begun to be mapped. By conducting BLAST searches of the NCBI (National Center for Biotechnology Information) database using DNA and protein sequences of C. virginica and other invertebrate and mammalian species we found matches for GABAa and GABAb receptor genes on C. virginicachromosomes 3 and 5, respectively. Various invertebrates had Percent Identity above 60%, while humans and mice had Percent Identity of about 40% to 50% for GABAa and GABAb. We hypothesize that the ligand binding sites (LBS) for GABAa and GABAb receptors in C. virginica are evolutionarily conserved and will closely match those of other animals. To study this, we conducted searches of the NCBI database for GABAa and GABAb LBS of C. virginica and compared them to other animals. We found GABAb LBS contained 4 amino acids (N, L, A, Y) in positions 34, 122, 123, 267 that were highly conserved in LBS of other bivalves, gastropods, insects, mice, rats and humans. GABAa LBS for C. virginica has not yet been identified. We did find LBS of other animals contained 2 amino acids (L, Y) that were highly conserved among the animals in which it has been identified. The C. virginica GABAa receptor does contain L and Y amino acids. The study complements our earlier physiology and cell biology studies demonstrating the presence and a function for GABA in C. virginica and shows the genome of C. virginica contains genes to produce GABA receptor LBS that are similar to those of other animals. This new information is valuable as it shows the simple nervous system of C. virginica can be used to conduct studies on GABA neurotransmission.

C. elegansAttraction Learning in casy‐1Mutants

C. eleganshave a simple nervous system with approximately 300 neurons and exhibit well‐documented learning‐specific behaviors. Several genetic mutations, such as in the casy‐1 genes, have been shown to disrupt these learning behaviors in C. elegans. The casy‐1 gene encodes a calcium‐binding protein that is important for synapse regulation, and has shown deficits in learning behaviors. While casy‐1mutant C. eleganshave specifically shown deficits in aversion learning, their attraction learning is not well characterized. Here, we examined the effects of casy‐1 mutation on attraction learning in C. elegans. To do so, we have quantified C. elegans locomotor behaviors such as reversal frequency, run length, and pause frequency in wild‐type and casy‐1 mutant C. elegansin the context of a salt chemotaxis learning assay. This work helps to elucidate the effect of casy‐1on learning behaviors, which is particularly relevant as this gene is an ortholog of mammalian calsyntenins.