Lumbriculus variegatus: A novel organism for in vivo pharmacology research

For years animal models in science have been invaluable and highly beneficial to the advancement of medicine and pharmacology. Many in vivo models are protected by the Animals (Scientific Procedures) Act 1986, and there is framework put in place to replace, reduce and refine the number of animals used in research. This means there is a call for an in vivo model that will give us insight into specific pharmacological processes, while reducing the need for vertebrate animal models in research. Here we present the fresh freshwater invertebrate, Lumbriculus variegatus, as a novel in vivo model for pharmacology research.Here we have developed two assays to measure the behavioural effects of drugs on L. variegatus when exposed to specific compounds: the stereotypical movement assay, which measures the worms stereotyped behaviours in response to stimuli, and the free locomotion assay, which measures L. variegatus unstimulated movement. We report the effects of compounds with diverse pharmacodynamic properties on L. variegatus using these assays, these include ion channel blockers, neurotransmitters and their antagonists, and drugs of abuse. Alongside this, we have also developed techniques to extract and quantify protein and DNA from this organism.Our results show that ion channel blockers, lidocaine and quinine, reduced both stimulated and unstimulated movement in L. variegatus. Stereotypical movement and free locomotion were both significantly affected when L. variegatus were exposed to ≥20 mM of dopamine and ≥50 μM of dopamine antagonist haloperidol. However, dose-dependent effects were only observed for stimulated movement when exposed to GABA, and changes were observed only at the highest concentration of 500 mM when exposed to glycine. Both stimulated and unstimulated movement was reduced when L. variegatus was exposed to ≥250 mM. L. variegatus also displayed a dose-dependent response to DNP and were unable to recover after 24 hours at 50 μM. These toxic effects were reversed by 10 and 25 μM of haloperidol, and 25 μM of sulpiride. We successfully extracted and quantified both protein and DNA from this organism.We recognise that the experiments we have conducted on L. variegatus throughout this project may not replicate the complexity of higher animals, and experiments utilising invertebrates will not fully replace studies in vertebrate species. L. variegatus have the potential to replace smaller invertebrate models where specialist equipment is needed to visualise them. An advantage of using L. variegatus for pharmacology is that they possess unique stereotypical behaviours that can be easily quantified without the need for specialist equipment. Alongside this, there is no call for special husbandry as with rodents and other larger models, therefore L. variegatus can be cultured in most laboratories, including research and educational institutions

Flexible motor sequence generation during stereotyped escape responses

Complex animal behaviors arise from a flexible combination of stereotyped motor primitives. Here we use the escape responses of the nematode Caenorhabditis elegans to study how a nervous system dynamically explores the action space. The initiation of the escape responses is predictable: the animal moves away from a potential threat, a mechanical or thermal stimulus. But the motor sequence and the timing that follow are variable. We report that a feedforward excitation between neurons encoding distinct motor states underlies robust motor sequence generation, while mutual inhibition between these neurons controls the flexibility of timing in a motor sequence. Electrical synapses contribute to feedforward coupling whereas glutamatergic synapses contribute to inhibition. We conclude that C. elegans generates robust and flexible motor sequences by combining an excitatory coupling and a winner-take-all operation via mutual inhibition between motor modules

Longitudinal Studies Of Caenorhabditis Elegans Aging And Behavior Using A Microfabricated Multi-Well Device

The roundworm C. elegans is a powerful model organism for dissecting the genetics of behavior and aging. The central genetic pathways regulating lifespan, such as insulin signaling, were first identified in worms. C. elegans is also the only animal for which a full map of all neural synpatic connections, or connectome, exists. However, current manual and automated methods are unable to efficiently monitor and quantify behavioral phenotypes which unfold over long time scales. Therefore, it has been difficult to study phenotypes such as long-term behavior states and behavioral changes with age in worms. To address these limitations, here I describe a novel device, called the WorMotel, to longitudinally monitor behavior in up to 240 single C. elegans on time scales encompassing the worm\u27s maximum lifespan of two months. The WorMotel is fabricated from polydimethylsiloxane from a 3-D printed negative mold. Each device consists of 240 individual wells, each of which houses a single worm atop agar and bacterial food. I use custom software to quantify movement between frames to longitudinally monitor behavior for each animal. I first describe the application of the WorMotel to the automation of lifespan measurements in C. elegans, the characterization of intra-strain and inter-strain variability in behavioral decline, the relationship between behavior and lifespan, and the scaling of behavioral decline with increasing stress. I then describe the application of the WorMotel to quantify locomotive behavioral states and their modulation by the presence or absence of food as well as biogenic amine neurotransmitters. Using the WorMotel in combination with genetics and pharmacology, I outline a neural circuit by which the biogenic amines serotonin and octopamine regulate locomotion state to signal animals to adopt behavior appropriate to a fed and fasting state, respectively. I include protocols for construction of custom imaging rigs and requirements for long-term imaging as an appendix. The WorMotel is a powerful tool that can facilitate discovery and understanding of the mechanisms underlying long-term phenotypes such as behavioral states and aging

Microfluidic-based Tools and Methods for Complex Chemosensory-based Behavioral Studies in C. elegans

There is a great interest in studying behavior and the underlying biological basis for behaviors in small model organisms. Some properties of C. elegans that greatly facilitate genetic investigations are its small size, relatively simplistic ‘brain’, complex repertoire of behaviors, and ease of isogenic population studies. In order to take full advantage of these characteristics, it is desirable to have methods for analyzing behaviors of large populations of animals in well-controlled environments. One set of behaviors extensively used to investigate numerous phenomena in neurobiology within C. elegans deals with the navigation of chemical environments (chemotaxis). Studies based on C. elegans chemotaxis are used in investigating chemosensation, innate preferences, learning, memory, and more. We have improved upon previous microfluidic and computer-vision technologies to advance C. elegans chemosensation and chemotaxis studies to answer more sophisticated biological questions. One developed method is a microfluidic device capable of monitoring animal neuronal activity in vivo while delivering multiple chemical stimuli to animals at sub-second speeds in any desired order without cross-contamination. This method facilitates investigations as to how complex environmental stimulus changes are encoded within a simple, well-characterized nervous system at relevant behavioral timescales. The second developed method is a microfluidic platform and accompanying software capable of tracking a population of C. elegans freely navigating well-controlled, spatial chemical environments over long timescales. Via this method, complete behavioral and stimulus experience history profiles can be generated for each animal within a population. This enables correlations to be made between acute chemotaxis behaviors and animal stimulus histories and provides additional insight as to how a series of acute chemotaxis behaviors results in long-term preference choices. We demonstrate the power and utility of the hardware and software developed for chemosensory-based behavioral studies by investigating starvation associative learning in C. elegans. Within our developed platform, we recapitulated wild-type (WT) learning phenotypes, recapitulated a previously known learning-defective phenotype of the insulin/IGF-1 signaling (IIS) pathway mutant, ins-1, and then utilized behavioral analyses and developed platform capabilities to test multiple hypotheses to explain the observed phenotypic differences between WT and ins-1. The developed capabilities and findings in this work should facilitate further elucidation of the role of INS-1 in the regulation of starvation plasticity. Taken together, the developed technologies in this thesis will allow for more powerful and sophisticated experiments for investigating chemosensory-based phenomena in neurobiology.Ph.D

The Long-Rage Directional Behavior of the Nematode C. Elegans

Like any mobile organism, C. elegans relies on sensory cues to find food. In the absence of such cues, animals might display defined search patterns or other stereotyped behavior. The motion of C. elegans has previously been characterized as a sinusoid whose direction can be modulated by gradual steering or by sharp turns, reversals and omega bends. However, such a fine-grained behavioral description does not by itself predict the longrange features of the animals’ pattern of movement. Using large (24 cm x 24 cm) Petri dishes, we characterized the movement pattern of C. elegans in the absence of stimuli. To collect trajectories over such a large surface, we devised an imaging setup employing an array of consumer flatbed scanners. We have confirmed quantitatively the results obtained with the scanner-array setup with a camera imaging setup, in a more stringently homogeneous environment. Wild-type worms display striking behavior in the absence of food. The majority (~60%) of the animals’ paths displays persistence in the direction of motion over length scales that are 50-100 times the body-length of C. elegans. The overall direction of movement differs from animal to animal, suggesting that the directed motion we observe might not be interpreted as a taxis to an external cue in the experimental environment. Interestingly, animals appear to exhibit directionality at large scales despite nondirectional motion at smaller scales. We quantified the extent of local directional persistence by computing the autocorrelation function of the velocities. Unexpectedly, correlations in the direction of motion decay over time scales that are much faster than the scales over which directional persistence appears to be maintained. We sought to establish quantitatively that the worm motion is, in fact, biased. To determine whether a null, random walk-like model of locomotion could account for directional behavior, we generated synthetic trajectories drawing from the same angle and step distributions of individual trajectories, and quantified the probabilities of obtaining larger net displacements than the experimental. Such a model fails to reproduce the experimental results. Moreover, the mean square displacements computed for the data display non-diffusive behavior, further demonstrating that the observed directional persistence cannot be explained by a simple random-walk model. To corroborate the hypothesis of biased movement in a model-independent fashion, we employed a geometrical characterization of the trajectories. Isotropic, unbiased walks result in paths that display a random distribution of turning angles between consecutive segments. In contrast, parsing of the worm’s trajectories yields different results depending on the segmentation scale adopted. In fact, increasing the segment size results in increasingly narrow turning angle distributions, centered around the zero. This suggests the emergence of directional coherence at long time scales. In order to investigate whether directional persistence is attained by a sensory mechanism, we analyzed the paths displayed by animals with impaired sensory function. Animals mutant for che-2, which display disrupted ciliary morphology and pleiotropic behavioral defects, exhibited non-directional behavior. Surprisingly however, daf-19 mutants, which lack sensory cilia altogether, displayed residual directionality, albeit at a lower penetrance (~20%) than the wild-type. This result suggests that directionality might implicate sensory modalities that do not require ciliary function, such as AFD-mediated thermosensation or URX-mediated oxygen sensation. Alternatively, the behavior of daf-19 mutants might imply that neural activity, but not sensory inputs, are required to achieve directed motion. Mutations in osm-9, a TRPV channel implicated in several avoidance behaviors in the worm, did not result in an observable phenotype. In contrast, mutations in tax-2/tax-4, a cGMP-gated channel required to transduce a number of sensory stimuli, resulted in loss of directionality. However, specific mutations targeting the signal transduction pathways for thermotaxis, olfaction, phototaxis, and aerotaxis, upstream of TAX-4, did not disrupt directional behavior. To get further insight into the nature of the stimulus directing the animals’ behavior, if any, we performed rescue experiments of TAX-4 function in specific subsets of neurons. In agreement with the results obtained by genetic lesions in the signal transduction pathways for thermotaxis and odortaxis, no rescue of directional behavior was observed when expressing TAX-4 in the thermosensory neuron AFD, or in the olfactory neurons AWB and AWC. Partial rescue of wild-type behavior was obtained by expression of TAX-4 in a set of five cells, which comprised the oxygen-sensing AQR, PQR and URX neurons as well as the ASJ and ASK sensory neurons, which transduce chemical stimuli and responses to dauer pheromone. To address the concern that the animals’ motion might be directed to a chemosensory cue within the plate, we investigated the correlation between path directions displayed by animals that were assayed on a same plate. We did not observe a detectable correlation between path headings, indicating that the worm is not chemotaxing to a plate-specific cue. In conclusion, our results indicate that the motion of C. elegans cannot be assimilated to a random walk, and that directional persistence arises at long times despite local nondirectional behavior. In addition, although we have not conclusively ruled out a sensorybased explanation, the genetic and phenomenological evidence gathered foreshadows the intriguing possibility that C. elegans might be achieving directional motion by relying solely on self-based information

The effect of silver nanoparticles on synaptic responses in the lateral giant escape circuit of the crayfish

The lateral giant neuron in the crayfish is a neuron that controls the reflexive escape response; the context and experience-dependent tuning of this response is critical for survival. Serotonin modulates synaptic responses in this neuron, and social experiences (stress) changes the modulatory role of serotonin. I investigated the mechanisms of induced synaptic response changes and serotonergic modulatory changes after silver nanoparticle exposure to ask if 1) contaminant induced stress changes serotonergic modulation in a manner consistent with social stress and 2) shed light on potential neurotoxic effects of widely used, but poorly understood nanomaterials. Our data show that stress induced by exposure to silver nanoparticles changes excitability of this neuron. Integrating these data concerning environmental stress with prior knowledge of behavioral and electrophysiological correlates of social stress we can ask if changes induced in synaptic responses and modulation induced by stressors of different kinds are generalizable

Dopaminergic and Activity-Dependent Modulation of Mechanosensory Responses in Drosophila Melanogaster Larvae

A central theme of this dissertation is nervous system plasticity. Activity-dependent plasticity and dopaminergic modulation are two processes by which neural circuits adapt their function to developmental and environmental changes. These processes are involved in basic cognitive functions and can contribute to neurological disorder. An important goal in modern neurobiology is understanding how genotypic variation influences plasticity, and leveraging the quantitative genetics resources in model organisms is a valuable component of this endeavor. To this end I investigated activity-dependent plasticity and dopaminergic modulation in Drosophila melanogaster larvae using neurobiological and genetic approaches. Larval mechanosensory behavior is described in Chapter 2. The behavioral experiments in that chapter provide a system to study mechanisms of plasticity and decision-making, while the electrophysiological characterization shows that sensory-motor output depends on neural activity levels of the circuit. This system is used to investigate activity-dependent plasticity in Chapter 3, i.e., habituation to repetitive tactile stimuli. In Chapter 4, those assays are combined with pharmacological manipulations, genetic manipulations, and other experimental paradigms to investigate dopaminergic modulation. Bioinformatics analyses were used in Chapter 5 to characterize natural genetic variation and the influence of single nucleotide polymorphisms on dopamine-related gene expression. The impact and suggested future directions based on this work are discussed in Chapter 6. Dopamine also modulates cardiomyocytes. Chapter 7 describes biochemical pathways that mediate dopaminergic modulation of heart rate. The final two chapters describe neurobiology research endeavors that are separate from my work on dopamine. Experiments that have helped characterize a role for Serf, a gene that codes for a small protein with previously unknown function, are described in Chapter 8. In the final chapter I describe optogenetic behavioral and electrophysiology preparations that are being integrated into high school classrooms and undergraduate physiology laboratories. Assessment of student motivation and learning outcomes in response to those experiments is also discussed

Neural Circuitry Underlying Nociceptive Escape Behavior in Drosophila

Rapid and efficient escape behaviors in response to noxious sensory stimuli are essential for protection and survival. In Drosophila larvae, the class III (cIII) and class IV (cIV) dendritic arborization (da) neurons detect low-threshold mechanosensory and noxious stimuli, respectively. Their axons project to modality-specific locations in the neuropil, reminiscent of vertebrate dorsal horn organization. Despite extensive characterization of nociceptors across organisms, how noxious stimuli are transformed to the coordinated behaviors that protect animals from harm remains poorly understood. In larvae, noxious mechanical and thermal stimuli trigger an escape behavior consisting of sequential C-shape body bending followed by corkscrew-like rolling, and finally an increase in forward locomotion (escape crawl). The downstream circuitry controlling the sequential coordination of escape responses is largely unknown. This work identifies a population of interneurons in the nerve cord, Down-and-Back (DnB) neurons, that are activated by noxious heat, promote nociceptive behavior, and are required for robust escape responses to noxious stimuli. Activation of DnB neurons can trigger both rolling, and the initial C-shape body bend independent of rolling, revealing modularity in the initial nociceptive responses. Electron microscopic circuit reconstruction shows that DnBs receive direct input from nociceptive and mechanosensory neurons, are presynaptic to pre-motor circuits, and link indirectly to a population of command-like neurons (Goro) that control rolling. DnB activation promotes activity in Goro neurons, and coincident inactivation of Goro neurons prevents the rolling sequence but leaves intact body bending motor responses. Thus, activity from nociceptors to DnB interneurons coordinates modular elements of nociceptive escape behavior. The impact of DnB neurons may not be restricted to synaptic partners, as DnB presynaptic sites accumulate dense-core vesicles, suggesting aminergic or peptidergic signaling. Anatomical analyses show that DnB neurons receive spatially segregated input from cIII mechanosensory and cIV nociceptive neurons. However, DnB neurons do not seem to promote or be required for gentle-touch responses, suggesting a modulatory role for cIII input. Behavioral experiments suggest that cIII input presented prior to cIV input can enhance nociceptive behavior. Moreover, weak co-activation of DnB and cIII neurons can also enhance nociceptive responses, particularly C-shape bending. These results indicate that timing and level of cIII activation might determine its modulatory role. Taken together, these studies describe a novel nociceptive circuit, which integrates nociceptive and mechanosensory inputs, and controls modular motor pathways to promote robust escape behavior. Future work on this circuit could reveal neural mechanisms for sequence transitions, peptidergic modulation of nociception, and developmental mechanisms that control convergence of sensory afferents onto common synaptic partners

2018 - The Twenty-third Annual Symposium of Student Scholars

The full program book from the Twenty-third Annual Symposium of Student Scholars, held on April 19, 2018. Includes abstracts from the presentations and posters.https://digitalcommons.kennesaw.edu/sssprograms/1020/thumbnail.jp