Model Checking Tap Withdrawal in C. Elegans

We present what we believe to be the first formal verification of a biologically realistic (nonlinear ODE) model of a neural circuit in a multicellular organism: Tap Withdrawal (TW) in \emph{C. Elegans}, the common roundworm. TW is a reflexive behavior exhibited by \emph{C. Elegans} in response to vibrating the surface on which it is moving; the neural circuit underlying this response is the subject of this investigation. Specifically, we perform reachability analysis on the TW circuit model of Wicks et al. (1996), which enables us to estimate key circuit parameters. Underlying our approach is the use of Fan and Mitra's recently developed technique for automatically computing local discrepancy (convergence and divergence rates) of general nonlinear systems. We show that the results we obtain are in agreement with the experimental results of Wicks et al. (1995). As opposed to the fixed parameters found in most biological models, which can only produce the predominant behavior, our techniques characterize ranges of parameters that produce (and do not produce) all three observed behaviors: reversal of movement, acceleration, and lack of response

Amphetamine Exposure During Embryogenesis Leads To Long-Term And Transgenerational Increase In Behavioral Response And Decrease In Dopamine Uptake

Amphetamine (AMPH) is widely prescribed for the treatment of ADHD and a highly abused substance in society, yet little is known about the long-term effects of the drug. Here, we used Caenorhabditis elegans (C. elegans) to establish a model for the long-term and transgenerational effects of AMPH exposure on behavior. Furthermore, experiments were conducted to explore the molecular mechanisms of AMPH that were altered by embryonic AMPH exposure. C. elegans have a well characterized behavioral response to AMPH known as Swimming Induced Paralysis (SWIP). For the SWIP test, animals are placed in fluid, which normally induces a thrashing behavior. However, in the presence AMPH, the animals display a time- and dose-dependent paralysis. AMPH increases the levels of dopamine in the synapse by causing reverse transport through the protein known as the dopamine transporter (DAT), and the SWIP behavior has been shown to be dependent on dopaminergic transmission. We exposed embryos to either control solution alone (M9 solution) or 500μM AMPH dissolved in control solution for 15 hours. 4 days later the SWIP test was performed on young adult animals, revealing that animals previously exposed to AMPH as embryos displayed a higher response to AMPH. The progeny of both groups were tested for SWIP as well. Interestingly, the progeny of the animals exposed to AMPH as embryos showed a higher SWIP response with respect to the progeny of control animals, demonstrating that AMPH had both a long-term and transgenerational effect on the animals. Because the SWIP behavior was previously shown to be dependent on dopaminergic transmission, we performed DA uptake assays using primary cell cultures made from F1 generation animals to investigate alterations in DATs ability to uptake dopamine. Results from the uptake assays showed that primary cultures made from the progeny of animals exposed to AMPH as embryos had reduced ability to uptake DA with respect to control cultures. To further investigate the reduced uptake ability following AMPH exposure, a human neuroblastoma cell line (SH-SY5Y) was exposed to 15 hour of AMPH, and 5 days later, a DA uptake assay using a concentration response of DA was carried out. Results showed that the cells had a reduced Vmax with no change to Km, suggesting a reduced amount of DAT in the cells. We investigated changes in histone methylation as a mechanism for the long-term and transgenerational effect observed. Histones are proteins, which DNA wraps around to form the nucleosome, and methylation changes on histones can modify the binding of DNA to histones leading to a change in gene expression. Western blots of whole animal protein revealed a decreased level of histone 3 lysine 4 trimethylation (H3K4me3) in the F1 generation of AMPH exposed animals. Additionally, a reduction in the enzymes responsible for H3K4me2 methylation and H3K4me3 demethylation was observed in F1 progeny of AMPH exposed animals. Suggesting that AMPH exposure during embryogenesis alters methylation of specific histone markers. Taken together, these experiments show that in C. elegans, AMPH exposure causes a long-term and transgenerational alteration in behavioral response to AMPH, which correlates to alterations in DAT uptake ability

OpenWorm: overview and recent advances in integrative biological simulation of Caenorhabditis elegans

The adoption of powerful software tools and computational methods from the software industry by the scientific research community has resulted in a renewed interest in integrative, large-scale biological simulations. These typically involve the development of computational platforms to combine diverse, process-specific models into a coherent whole. The OpenWorm Foundation is an independent research organization working towards an integrative simulation of the nematode Caenorhabditis elegans, with the aim of providing a powerful new tool to understand how the organism's behaviour arises from its fundamental biology. In this perspective, we give an overview of the history and philosophy of OpenWorm, descriptions of the constituent sub-projects and corresponding open-science management practices, and discuss current achievements of the project and future directions. This article is part of a discussion meeting issue ‘Connectome to behaviour: modelling C. elegans at cellular resolution’

Investigating Biofumigation for the Control of Plant-Parasitic Nematodes

The white potato cyst nematode, Globodera pallida, is an important pest of potato in all potato-growing regions of the world and is of particular importance to UK agriculture, found in 48-64 % of UK potato fields and incurring costs related to management and yield losses. Biofumigation is a pest management practice that seeks to exploit the production of bioactive compounds, isothiocyanates, from disrupted brassica tissues incorporated into soil. Aspects of biofumigation as they relate to control of G. pallida were investigated. The xenobiotic metabolism of G. pallida juveniles in response to contact with isothiocyanates was investigated through RNAseq analysis of nematodes exposed to Dazomet, an isothiocyanate generator. The roles of genes implicated in this response were investigated and their up-regulation confirmed, identifying several genes directly implicated in detoxification of xenobiotic compounds, presenting targets for development of future controls. A screening system for evaluation of novel biofumigant crops was developed, utilising Caenorhabditis elegans reporter lines that indicated the presence of isothiocyanates through induced expression of green fluorescent protein (GFP). Attempts to generate novel C. elegans reporters for G. pallida genes were unsuccessful, but progress was made towards generation of transgenic root-knot nematodes, a step towards a plant-parasitic nematode model system. The volatile emissions given off by brassicas as they grow were measured and a number of bioactive compounds were identified. New estimates of the contributions of brassicas to atmospheric methyl bromide concentrations were generated. A system was developed to test the toxicity of volatile compounds as given off by the above- and belowground biomass of brassicas, and toxicity was observed in C. elegans adults and G. pallida juveniles and encysted eggs. The approaches taken to investigate biofumigation are novel and support expansion of the scope of future biofumigation research in line with the findings presented

Cell Size Checkpoint Control by the Retinoblastoma Tumor Suppressor Pathway

Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB) tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription

Improvements in optical techniques to investigate the behavior and neuronal network dynamics over long timescales

Developments in optical technology have produced an important shift in experimental neuroscience from electrophysiological methods for observation and stimulation to all-optical solutions. One expects this trend to continue as future developments continue to deliver, and improve upon, the original promises of the technology: 1) minimally invasive actuation and recording of neurons, and 2) a drastic increase in targets that can be treated simultaneously. Moreover, as the high costs of the technology are reduced, one may expect its larger-scale adoption in the neuroscience community. In this thesis, I describe the development and implementation of two alloptical solutions for the analysis of behavior, neuronal signaling, and stimulation, which improve on previous state-of-the-art: (1) A minimally-invasive, high signal-to-noise twophoton microscopy setup capable of simultaneous, live-imaging of a large subset of sensory neurons post activation, and (2) a low-cost tracking solution to stimulate and record behavior. I begin this thesis with a review of recent advances in optical neuroscience techniques for the study of neuronal networks with the focus on work done in Caenorhabditis elegans. Then, in chapter 2, I describe my implementation of a two-photon temporal focusing microscopy setup and show significant improvements through the use of a high power/ high pulse repetition rate excitation system, enabling live imaging with high resolution for extended periods of time. I model temperature increase during a physiological imaging scenario for different repetition rates at fixed peak intensities and find range centered around 1 MHz to be optimal. Lastly, I describe the low-cost tracking setup with the ability to stimulate and record behavior over the course of hours. The setup is capable of two-color stimulation of optogenetic proteins over the area of the behavioral arena in combination with volatile chemicals. To showcase the utility of the system, I demonstrate behavioral analysis of integration of contradictory cues. In summary, I present a set of techniques for the interrogation of neural networks from animal behavior to neuronal activity, over timescales of potentially hours and days. These techniques can be used to address a new dimension of scientific questions.Okinawa Institute of Science and Technology Graduate Universit

Analysis of RNAi strategies against migratory parasitic nematodes of banana

The main objective of the research project is to studythe potentialof inducing an effective host resistance againstmigratory endo-parasitic nematodes through RNA- mediated interferencetechnology (RNAi).The study is mainly targeted at the migratoryendo-parasitic nematodes Radopholus similis and Pratylenchus coffeae ,which are the most important root pathogens of banana in the tropics.Since these nematodes invade the roots and move inter- andintra-cellularly through the root by disintegrating the cell wall asthey move along, the invasion and subsequent establishment of thenematode inthe plant can be prevented by knocking down nematode genesthat encode cell-wall degrading enzymes (e.g ?-1, 4-endoglucanase). Inaddition to genes specific to plant-parasitic nematodes such as ?-1,4-endoglucanase, genes that affect the parasitic life of the nematodeeitherdirectly or indirectly are also being targeted for silencing. Bydownregulating genes that play important roles in the embryonic andlarval development or nematode locomotion, the complete progression ofpathogenesis can be adversely affected. Based on this hypothesis, thisstudy aims to develop an alternative control strategy against migratoryendo-parasitic nematodes by knocking down the above mentionedparasitism or housekeeping genes. To achieve these goals, dsRNA will bedelivered to nematodes through in vitro and in planta routes. Thenematodes will be subjected to in vitro RNAi by soaking with dsRNA ofselected parasitism genes of the nematodes and these treated nematodesare then used to infect a host plant to investigate specific genes thataffect nematode parasitism. As a control strategy against the migratoryendo-parasitic nematodes, we attempt to engineer host plants, mainlybanana and chick pea to produce dsRNA of the nematode s essential genesbased on the hypothesis that the in planta-produced dsRNA can triggerRNAi response in nematodes that feed on these transgenic plants.nrpages: 250status: publishe

Neural Interactome: Interactive Simulation of a Neuronal System

Connectivity and biophysical processes determine the functionality of neuronal networks. We, therefore, developed a real-time framework, called Neural Interactome1,2, to simultaneously visualize and interact with the structure and dynamics of such networks. Neural Interactome is a cross-platform framework, which combines graph visualization with the simulation of neural dynamics, or experimentally recorded multi neural time series, to allow application of stimuli to neurons to examine network responses. In addition, Neural Interactome supports structural changes, such as disconnection of neurons from the network (ablation feature). Neural dynamics can be explored on a single neuron level (using a zoom feature), back in time (using a review feature), and recorded (using presets feature). The development of the Neural Interactome was guided by generic concepts to be applicable to neuronal networks with different neural connectivity and dynamics. We implement the framework using a model of the nervous system of Caenorhabditis elegans (C. elegans) nematode, a model organism with resolved connectome and neural dynamics. We show that Neural Interactome assists in studying neural response patterns associated with locomotion and other stimuli. In particular, we demonstrate how stimulation and ablation help in identifying neurons that shape particular dynamics. We examine scenarios that were experimentally studied, such as touch response circuit, and explore new scenarios that did not undergo elaborate experimental studies

Intron detention as a mechanism to tightly control the timing of neural differentiation

The life-long persistence of adult neural stem cells (NSCs) requires the accurate balance between their self-renewal, proliferation and differentiation. Recent studies analyzing the transcriptomic dynamics that take place during adult neurogenesis have reveal that post-transcriptional regulation may have a more relevant role than anticipated in the control of NSC biology and function. In this thesis, we have uncovered a novel regulatory mechanism that refines the control of Scratch1 expression during adult neurogenesis. We have shown that this gene is expressed in the adult subependymal zone (SEZ), both in NSCs, transient amplifying progenitors (TAPs) and neuroblasts, exhibiting increasing levels as NSCs progress into the lineage; and fulfilling several functions during the process of differentiation. On the one hand, this transcription factor acts downstream of p53, repressing the transcription of it targets Bbc3 (Puma) and Cdkn1a (p21), which in turn protects the differentiating cells form undergoing apoptosis and, at the same time, stimulates their proliferation. On the other hand, Scratch1 promotes neuronal differentiation, favoring neurogenesis at the expense of gliogenesis. Additionally, we have also determined that Scracth1 starts to be expressed when stem cells acquire the neural identity, but its transcripts are retained in the nucleus of NSCs due to intron detention. This event of regulated splicing is possible thanks to the progressive enlargement of the polypyrimidine tract of its only intron during the evolution of vertebrates, leading to the generation of a suboptimal splice site in mammals. By contrast, in response to the neural differentiation signal, Scratch1 mRNA is m6A modified and consequently spliced and exported to the cytoplasm, where it can be translated. Finally, we have shown that intron detention in this context is not specific for Scratch1 mRNA, but rather it also controls the translational availability of multiple transcripts associated with adult NSC differentiation. Interestingly, a complementary regulation of intron detention occurs in mRNAs from genes involved in the maintenance of stemness. Thus, intron detention is a novel mechanism to fine-tune neurogenesis in the adult NSC niche. El mantenimiento de la población de células madres neurales (NSCs) durante toda la vida del individuo requiere un balance preciso entre su auto-renovación, proliferación y diferenciación. Estudios recientes en los que se han analizado los cambios transcripcionales que tienen lugar durante la neurogénesis adulta han revelado que la regulación post-transcripcional podría tener un peso mayor en el control de la biología y la función de las NSCs de lo que se pensaba previamente. En esta tesis, hemos identificado un nuevo mecanismo de regulación de la expresión del factor de transcripción Scratch1 durante la neurogénesis adulta. Hemos mostrado que este gen se expresa en la zona subependimaria (SEZ) adulta, tanto en NSCs como en progenitores (TAPs) y neuroblastos, presentando niveles crecientes de expresión a medida que las NSCs progresan en el linaje. Además, Scratch1 lleva a cabo diversas funciones durante el proceso de diferenciación. Por una parte, actúa por debajo de p53, reprimiendo la transcripción de sus dianas Bbc3 (Puma) y Cdkn1a (p21), y protegiendo a las células en diferenciación de sufrir apoptosis a la vez que estimula su proliferación. Por otra parte, Scratch1 promueve la diferenciación neuronal, favoreciendo la neurogénesis a expensas de la gliogénesis. Además, hemos determinado que Scratch1 empieza expresarse cuando las células madre adquieren la identidad neural, aunque sus tránscritos permanecen dentro del núcleo de las NSCs debido a un procesamiento deficiente del único intrón en su mRNA. Este evento de splicing regulado es posible gracias a la expansión progresiva de la serie de polipirimidinas (PPT) durante la evolución de los vertebrados, dando lugar a la generación de un sitio de splicing subóptimo en mamíferos. En respuesta a la señal de diferenciación, se induce la metilación (m6A) del mRNA y los tránscritos son procesados y exportados al citoplasma, donde pueden ser traducidos. Por último, hemos encontrado que la detención de intrones no solo regula la expresión de Scratch1, sino que también controla la localización subcelular y la disponibilidad para ser traducidos de múltiples tránscritos relacionados con la diferenciación de las NSCs. Más aun, el control de la retención de intrones se realiza de forma contraria en los transcritos correspondientes a genes relacionados con las células madre. Por lo tanto, aquí definimos un nuevo mecanismo que controla de forma precisa la neurogénesis adulta