Thermal acclimation and habitat-dependent differences in temperature robustness of a crustacean motor circuit.

Introduction: At the cellular level, acute temperature changes alter ionic conductances, ion channel kinetics, and the activity of entire neuronal circuits. This can result in severe consequences for neural function, animal behavior and survival. In poikilothermic animals, and particularly in aquatic species whose core temperature equals the surrounding water temperature, neurons experience rather rapid and wide-ranging temperature fluctuations. Recent work on pattern generating neural circuits in the crustacean stomatogastric nervous system have demonstrated that neuronal circuits can exhibit an intrinsic robustness to temperature fluctuations. However, considering the increased warming of the oceans and recurring heatwaves due to climate change, the question arises whether this intrinsic robustness can acclimate to changing environmental conditions, and whether it differs between species and ocean habitats. Methods: We address these questions using the pyloric pattern generating circuits in the stomatogastric nervous system of two crab species, Hemigrapsus sanguineus and Carcinus maenas that have seen a worldwide expansion in recent decades. Results and discussion: Consistent with their history as invasive species, we find that pyloric activity showed a broad temperature robustness (>30°C). Moreover, the temperature-robust range was dependent on habitat temperature in both species. Warm-acclimating animals shifted the critical temperature at which circuit activity breaks down to higher temperatures. This came at the cost of robustness against cold stimuli in H. sanguineus, but not in C. maenas. Comparing the temperature responses of C. maenas from a cold latitude (the North Sea) to those from a warm latitude (Spain) demonstrated that similar shifts in robustness occurred in natural environments. Our results thus demonstrate that neuronal temperature robustness correlates with, and responds to, environmental temperature conditions, potentially preparing animals for changing ecological conditions and shifting habitats.

Quantitative description of neuronal calcium dynamics in C. elegans' thermoreception.

The dynamical mechanisms underlying thermoreception in the nematode C. elegans are studied with a mathematical model for the amphid finger-like ciliated (AFD) neurons. The equations, equipped with Arrhenius temperature factors, account for the worm's thermotaxis when seeking environments at its cultivation temperature, and for the AFD's calcium dynamics when exposed to both linearly ramping and oscillatory temperature stimuli. Calculations of the peak time for calcium responses during simulations of pulse-like temperature inputs are consistent with known behavioral time scales of C. elegans.

The mechanoreceptor pezo-1 is required for normal crawling locomotion in the nematode C. elegans.

The discovery in 2010 of the PIEZO family of mechanoreceptors revolutionized our understanding of the role of proprioceptive feedback in mammalian physiology. Much remains to be elucidated. This study looks at the role this receptor plays in normal locomotion. Like humans, the nematode C. elegans expresses PIEZO-type channels (encoded by the pezo-1 gene) throughout its somatic musculature. Here we use the unbiased automated behavioral software Tierpsy to characterize the effects that mutations removing PEZO-1 from body wall musculature have on C. elegans crawling. We find that loss of PEZO-1 results in disrupted locomotion and posture, consistent with phenotypes associated with loss of PIEZO2 in human musculature. C. elegans is thus an amenable system to study the role of mechanoreception on muscle physiology and function.

Combining Old and New Tricks: The Study of Genes, Neurons, and Behavior in Crayfish.

For over a century the nervous system of decapod crustaceans has been a workhorse for the neurobiology community. Many fundamental discoveries including the identification of electrical and inhibitory synapses, lateral and pre-synaptic inhibition, and the Na+/K+-pump were made using lobsters, crabs, or crayfish. Key among many advantages of crustaceans for neurobiological research is the unique access to large, accessible, and identifiable neurons, and the many distinct and complex behaviors that can be observed in lab settings. Despite these advantages, recent decades have seen work on crustaceans hindered by the lack of molecular and genetic tools required for unveiling the cellular processes contributing to neurophysiology and behavior. In this perspective paper, we argue that the recently sequenced marbled crayfish, Procambarus virginalis, is suited to become a genetic model system for crustacean neuroscience. P. virginalis are parthenogenetic and produce genetically identical offspring, suggesting that germline transformation creates transgenic animal strains that are easy to maintain across generations. Like other decapod crustaceans, marbled crayfish possess large neurons in well-studied circuits such as the giant tail flip neurons and central pattern generating neurons in the stomatogastric ganglion. We provide initial data demonstrating that marbled crayfish neurons are accessible through standard physiological and molecular techniques, including single-cell electrophysiology, gene expression measurements, and RNA-interference. We discuss progress in CRISPR-mediated manipulations of the germline to knock-out target genes using the 'Receptor-mediated ovary transduction of cargo' (ReMOT) method. Finally, we consider the impact these approaches will have for neurophysiology research in decapod crustaceans and more broadly across invertebrates.

Methods for Modulating and Measuring Neuromuscular Exertion in C. elegans.

The nematode C. elegans has been used widely to study the genetic and cellular basis of behavior. Yet the laboratory conditions under which it is typically studied offer only a narrow glimpse into the richness of natural behaviors this remarkable animal evolved over 500 million years of evolution. For example, burrowing behavior naturally occurs in the wild, but it remains understudied. Our group studies burrowing in an attempt to expand our understanding of the natural behavioral repertoire of C. elegans. Aside from being an interesting and tractable behavior, burrowing is experimentally useful and permits the titration of the muscular output exerted by C. elegans. Here we describe several burrowing assays that allow the modulation of muscular exertion. We used these to study both adaptive and pathological muscular processes such as muscle hypertrophy and dystrophy, respectively. We believe these assays will be of use for researchers studying the production of locomotion under normal and disease-challenged conditions.

The dynamic range of voltage-dependent gap junction signaling is maintained by Ih-induced membrane potential depolarization.

Like their chemical counterparts, electrical synapses show complex dynamics such as rectification and voltage dependence that interact with other electrical processes in neurons. The consequences arising from these interactions for the electrical behavior of the synapse, and the dynamics they create, remain largely unexplored. Using a voltage-dependent electrical synapse between a descending modulatory projection neuron (MCN1) and a motor neuron (LG) in the crustacean stomatogastric ganglion, we find that the influence of the hyperpolarization-activated inward current (Ih) is critical to the function of the electrical synapse. When we blocked Ih with CsCl, the apparent voltage dependence of the electrical synapse shifted by 18.7 mV to more hyperpolarized voltages, placing the dynamic range of the electrical synapse outside of the range of voltages used by the LG motor neuron (-60.2 mV to -44.9 mV). With dual electrode current- and voltage-clamp recordings, we demonstrate that this voltage shift is not due to a change in the properties of the gap junction itself, but is a result of a sustained effect of Ih on the presynaptic MCN1 axon terminal membrane potential. Ih-induced depolarization of the axon terminal membrane potential increased the electrical postsynaptic potentials and currents. With Ih present, the axon terminal resting membrane potential is depolarized, shifting the dynamic range of the electrical synapse toward the functional range of the motor neuron. We thus demonstrate that the function of an electrical synapse is critically influenced by a voltage-dependent ionic current (Ih).NEW & NOTEWORTHY Electrical synapses and voltage-gated ionic currents are often studied independently from one another, despite mounting evidence that their interactions can alter synaptic behavior. We show that the hyperpolarization-activated inward ionic current shifts the voltage dependence of electrical synaptic transmission through its depolarizing effect on the membrane potential, enabling it to lie within the functional membrane potential range of a motor neuron. Thus, the electrical synapse's function critically depends on the voltage-gated ionic current.

Distinct mechanoreceptor pezo-1 isoforms modulate food intake in the nematode Caenorhabditis elegans.

Two PIEZO mechanosensitive cation channels, PIEZO1 and PIEZO2, have been identified in mammals, where they are involved in numerous sensory processes. While structurally similar, PIEZO channels are expressed in distinct tissues and exhibit unique properties. How different PIEZOs transduce force, how their transduction mechanism varies, and how their unique properties match the functional needs of the tissues they are expressed in remain all-important unanswered questions. The nematode Caenorhabditis elegans has a single PIEZO ortholog (pezo-1) predicted to have 12 isoforms. These isoforms share many transmembrane domains but differ in those that distinguish PIEZO1 and PIEZO2 in mammals. We used transcriptional and translational reporters to show that putative promoter sequences immediately upstream of the start codon of long pezo-1 isoforms predominantly drive green fluorescent protein (GFP) expression in mesodermally derived tissues (such as muscle and glands). In contrast, sequences upstream of shorter pezo-1 isoforms resulted in GFP expression primarily in neurons. Putative promoters upstream of different isoforms drove GFP expression in different cells of the same organs of the digestive system. The observed unique pattern of complementary expression suggests that different isoforms could possess distinct functions within these organs. We used mutant analysis to show that pharyngeal muscles and glands require long pezo-1 isoforms to respond appropriately to the presence of food. The number of pezo-1 isoforms in C. elegans, their putative differential pattern of expression, and roles in experimentally tractable processes make this an attractive system to investigate the molecular basis for functional differences between members of the PIEZO family of mechanoreceptors.

Neuronal membrane glycoprotein (nmgp-1) gene deficiency affects chemosensation-related behaviors, dauer exit and egg-laying in Caenorhabditis elegans.

The nervous system monitors the environment to maintain homeostasis, which can be affected by stressful conditions. Using mammalian models of chronic stress, we previously observed altered brain levels of GPM6A, a protein involved in neuronal morphology. However, GPM6A's role in systemic stress responses remains unresolved. The nematode Caenorhabditis elegans expresses a GPM6A ortholog, the neuronal membrane glycoprotein 1 (NMGP-1). Because of the shared features between nematode and mammalian nervous systems and the vast genetic tools available in C. elegans, we used the worm to elucidate the role of GPM6A in the stress response. We first identified nmgp-1 expression in different amphid and phasmid neurons. To understand the nmgp-1 role, we characterized the behavior of nmgp-1(RNAi) animals and two nmgp-1 mutant alleles. Compared to control animals, mutant and RNAi-treated worms exhibited increased recovery time from the stress-resistant dauer stage, altered SDS chemosensation and reduced egg-laying rate resulting in egg retention (bag-of-worms phenotype). Silencing of nmgp-1 expression induced morphological abnormalities in the ASJ sensory neurons, partly responsible for dauer exit. These results indicate that nmgp-1 is required for neuronal morphology and for behaviors associated with chemosensation. Finally, we propose nmgp-1 mutants as a tool to screen drugs for human nervous system pathologies.

Affordable Caenorhabditis elegans tracking system for classroom use.

For decades the nematode C. elegans has served as an outstanding research organism owing to its unsurpassed experimental amenability. This advantage has also made this tiny worm an attractive vehicle for science instruction across higher learning institutions. However, the prohibitive cost associated with the automated behavioral assessment of these animals remains an obstacle preventing their full adoption in undergraduate and high school settings. To improve this situation, we developed an inexpensive worm tracking system for use by high school interns and undergraduate students. Over the past two years this tracker has been successfully used by undergraduate students in our introductory Cell and Molecular lab (BSC220) at Illinois State University. Here we describe and demonstrate the use of our inexpensive worm tracking system.

Physiologists turned Geneticists: Identifying transcripts and genes for neuronal function in the Marbled Crayfish, Procambarus virginalis.

The number of undergraduate researchers interested in pursuing neurophysiological research exceeds the research laboratory positions and hands-on course experiences available because these types of experiments often require extensive experience or expensive equipment. In contrast, genetic and molecular tools can more easily incorporate undergraduates with less time or training. With the explosion of newly sequenced genomes and transcriptomes, there is a large pool of untapped molecular and genetic information which would greatly inform neurophysiological processes. Classically trained neurophysiologists often struggle to make use of newly available genetic information for themselves and their trainees, despite the clear advantage of combining genetic and physiological techniques. This is particularly prevalent among researchers working with organisms that historically had no or only few genetic tools available. Combining these two fields will expose undergraduates to a greater variety of research approaches, concepts, and hands-on experiences. The goal of this manuscript is to provide an easily understandable and reproducible workflow that can be applied in both lab and classroom settings to identify genes involved in neuronal function. We outline clear learning objectives that can be acquired by following our workflow and assessed by peer-evaluation. Using our workflow, we identify and validate the sequence of two new Gamma Aminobutyric Acid A (GABAA) receptor subunit homologs in the recently published genome and transcriptome of the marbled crayfish, Procambarus virginalis. Altogether, this allows undergraduate students to apply their knowledge of the processes of gene expression to functional neuronal outcomes. It also provides them with opportunities to contribute significantly to physiological research, thereby exposing them to interdisciplinary approaches.

Response to comment on "Magnetosensitive neurons mediate geomagnetic orientation in Caenorhabditis elegans".

Many animals can orient using the earth's magnetic field. In a recent study, we performed three distinct behavioral assays providing evidence that the nematode Caenorhabditis elegans orients to earth-strength magnetic fields (Vidal-Gadea et al., 2015). A new study by Landler et al. suggests that C. elegans does not orient to magnetic fields (Landler et al., 2018). They also raise conceptual issues that cast doubt on our study. Here, we explain how they appear to have missed positive results in part by omitting controls and running assays longer than prescribed, so that worms switched their preferred migratory direction within single tests. We also highlight differences in experimental methods and interpretations that may explain our different results and conclusions. Together, these findings provide guidance on how to achieve robust magnetotaxis and reinforce our original finding that C. elegans is a suitable model system to study magnetoreception.

Magnetic orientation in C. elegans relies on the integrity of the villi of the AFD magnetosensory neurons.

The magnetic field of the earth provides many organisms with sufficient information to successfully navigate through their environments. While evidence suggests the widespread use of this sensory modality across many taxa, it remains an understudied sensory modality. We have recently showed that the nematode C. elegans orients to earth-strength magnetic fields using the first pair of described magnetosensory neurons, AFDs. The AFD cells are a pair of ciliated sensory neurons crowned by fifty villi known to be implicated in temperature sensation. We investigated the potential importance of these subcellular structures for the performance of magnetic orientation. We show that ciliary integrity and villi number are essential for magnetic orientation. Mutants with impairments AFD cilia or villi structure failed to orient to magnetic fields. Similarly, C. elegans larvae possessing immature AFD neurons with fewer villi were also unable to orient to magnetic fields. Larvae of every stage however retained the ability to orient to thermal gradients. To our knowledge, this is the first behavioral separation of magnetic and thermal orientation in C. elegans. We conclude that magnetic orientation relies on the function of both cilia and villi in the AFD neurons. The role of villi in multiple sensory transduction pathways involved in the sensory transduction of vectorial stimuli further supports the likely role of the villi of the AFD neurons as the site for magnetic field transduction. The genetic and behavioral tractability of C. elegans make it a promising system for uncovering potentially conserved molecular mechanisms by which animals across taxa detect and orient to magnetic fields.

Magnetosensitive neurons mediate geomagnetic orientation in Caenorhabditis elegans.

Many organisms spanning from bacteria to mammals orient to the earth's magnetic field. For a few animals, central neurons responsive to earth-strength magnetic fields have been identified; however, magnetosensory neurons have yet to be identified in any animal. We show that the nematode Caenorhabditis elegans orients to the earth's magnetic field during vertical burrowing migrations. Well-fed worms migrated up, while starved worms migrated down. Populations isolated from around the world, migrated at angles to the magnetic vector that would optimize vertical translation in their native soil, with northern- and southern-hemisphere worms displaying opposite migratory preferences. Magnetic orientation and vertical migrations required the TAX-4 cyclic nucleotide-gated ion channel in the AFD sensory neuron pair. Calcium imaging showed that these neurons respond to magnetic fields even without synaptic input. C. elegans may have adapted magnetic orientation to simplify their vertical burrowing migration by reducing the orientation task from three dimensions to one.

Humidity sensation requires both mechanosensory and thermosensory pathways in Caenorhabditis elegans.

All terrestrial animals must find a proper level of moisture to ensure their health and survival. The cellular-molecular basis for sensing humidity is unknown in most animals, however. We used the model nematode Caenorhabditis elegans to uncover a mechanism for sensing humidity. We found that whereas C. elegans showed no obvious preference for humidity levels under standard culture conditions, worms displayed a strong preference after pairing starvation with different humidity levels, orienting to gradients as shallow as 0.03% relative humidity per millimeter. Cell-specific ablation and rescue experiments demonstrate that orientation to humidity in C. elegans requires the obligatory combination of distinct mechanosensitive and thermosensitive pathways. The mechanosensitive pathway requires a conserved DEG/ENaC/ASIC mechanoreceptor complex in the FLP neuron pair. Because humidity levels influence the hydration of the worm's cuticle, our results suggest that FLP may convey humidity information by reporting the degree that subcuticular dendritic sensory branches of FLP neurons are stretched by hydration. The thermosensitive pathway requires cGMP-gated channels in the AFD neuron pair. Because humidity levels affect evaporative cooling, AFD may convey humidity information by reporting thermal flux. Thus, humidity sensation arises as a metamodality in C. elegans that requires the integration of parallel mechanosensory and thermosensory pathways. This hygrosensation strategy, first proposed by Thunberg more than 100 y ago, may be conserved because the underlying pathways have cellular and molecular equivalents across a wide range of species, including insects and humans.

The evolutionary transition to sideways-walking gaits in brachyurans was accompanied by a reduction in the number of motor neurons innervating proximal leg musculature.

The forwards-walking portly crab, Libinia emarginata is an ancient brachyuran. Its phylogenetic position and behavioral repertoire make it an excellent candidate to reveal the adaptations, which were required for brachyuran crabs to complete their transition to sideways-walking from their forwards-walking ancestors. Previously we showed that in common with other forwards-walking (but distantly related) crustaceans, L. emarginata relies more heavily on its more numerous proximal musculature to propel itself forward than its sideways-walking closer relatives. We investigated if the proximal musculature of L. emarginata is innervated by a greater number of motor neurons than that of sideways-walking brachyurans. We found the distal musculature of spider crabs is innervated by a highly conserved number of motor neurons. However, innervation of its proximal musculature is more numerous than in closely-related (sideways-walking) species, resembling in number and morphology those described for forwards-walking crustaceans. We propose that transition from forward- to sideways-walking in crustaceans involved a decreased role for the proximal leg in favor of the more distal merus-carpus joint.

Conserved role of dopamine in the modulation of behavior.

Dopamine is an ancient signaling molecule. It is responsible for maintaining the adaptability of behavioral outputs and is found across taxa. The following is a summary of the role of dopamine and the mechanisms of its function and dysfunction. We discuss our recent findings on dopaminergic control of behaviors in C. elegans and discuss its potential implications for work in the fields of C. elegans and Parkinson research.

Coordination of behavioral hierarchies during environmental transitions in Caenorhabditis elegans.

For animals inhabiting multiple environments, the ability to select appropriate behaviors is crucial as their adaptability is often context dependent. Caenorhabditis elegans uses distinct gaits to move on land and in water. Gait transitions can potentially coordinate behaviors associated with distinct environments. We investigated whether land and water differentially affect the behavioral repertoire of C. elegans. Swimming worms interrupted foraging, feeding, egg-laying and defecation. Exogenous dopamine induced bouts of these land-associated behaviors in water. Our finding that worms do not drink fluid while immersed may explain why higher drug doses are required in water than on land to elicit the same effects. C. elegans is a valid model to study behavioral hierarchies and how environmental pressures alter their balance.

Caenorhabditis elegans selects distinct crawling and swimming gaits via dopamine and serotonin.

Many animals, including humans, select alternate forms of motion (gaits) to move efficiently in different environments. However, it is unclear whether primitive animals, such as nematodes, also use this strategy. We used a multifaceted approach to study how the nematode Caenorhabditis elegans freely moves into and out of water. We demonstrate that C. elegans uses biogenic amines to switch between distinct crawling and swimming gaits. Dopamine is necessary and sufficient to initiate and maintain crawling after swimming. Serotonin is necessary and sufficient to transition from crawling to swimming and to inhibit a set of crawl-specific behaviors. Further study of locomotory switching in C. elegans and its dependence on biogenic amines may provide insight into how gait transitions are performed in other animals.