115 research outputs found

    Neural Circuit Dependence of Acute and Subacute Nociception in C. Elegans

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    Nociception, the detection and avoidance of harmful cues, is a crucial system in all organisms. Animals use nociceptive systems to escape from substances that decrease survival, and can also modulate the threshold for avoidance behaviors to weigh the attractive features of an environment against its harmful features. To allow regulation, the nociception system of mammals incorporates multiple feedback and feedforward loops in its central and peripheral pathways. The nociception system of the roundworm Caenorhabditis elegans shares many features of the mammalian circuit. Both neural circuits feature a direct path from sensory neurons to motor neurons that is connected by a single class of interneuron, bypassing the higher processing centers. Both neural circuits also feature higher processing pathways that receive information from sensory neurons and provide further input onto the direct pathway. While the anatomical wiring of the C. elegans nervous system has been known for decades, how sensory neurons access different downstream paths in the circuit is less clear. One possible route of differential access of sensory input to downstream neurons is through different dynamics of activation. The temporal dimension of neural circuits cannot be deduced by anatomical wiring, but must be measured directly. In my thesis, I have characterized and manipulated the dynamic properties of a classical nociceptor in C. elegans, the polymodal sensory neuron ASH, and asked how these properties instruct downstream circuits and behavior. I thus first elucidated ASH calcium activation dynamics using simple step responses and using a newly developed systems identification approach for C. elegans. Using both long pulses and rapidly fluctuating “white noise” sequences of different nociceptive stimuli, I deduced their ASH activation profiles and linear temporal filters describing how the neuron summates the history of stimulus encounter. This analysis demonstrated that ASH calcium responses to natural stimuli include both linear features and multiple nonlinear components. Mutations in G protein-coupled sensory signaling disrupt both fast linear filtering and sustained responses to nociceptive stimuli. Mutations in a voltage gated calcium channel alter the temporal qualities of the ASH response in a pattern suggesting a role of this channel in sensory adaptation. In the course of these studies, I discovered several additional classes of sensory neurons that respond to nociceptive stimuli with robust calcium responses, even though past studies did not demonstrate a role for these neurons in nociceptive behavior. To gain experimental control over the dynamic activity that initiates nociceptive signaling, I ectopically expressed the pheromone receptors SRG-34 and SRG-36 in ASH and activated this system with their endogenous ligand, the ascaroside C3. ASH does not normally detect C3, but when it expresses either of these receptors it generates robust calcium responses to C3. These calcium signals have distinct temporal dynamics: SRG-34 mediated calcium signals are fast rising and fast adapting, while SRG-36 mediated calcium signals increase slowly during stimulation with little adaptation. Expression of SRG-34 or SRG-36 in ASH caused animals to avoid C3. Remarkably, time-aligned histograms of C3-induced avoidance behavior during stimulus onset, presence, and removal closely followed the dynamics of ASH calcium activity at these same time points, with a fast onset and adaptation for SRG-34 and a slow, sustained avoidance of SRG-36. ASH can directly activated the backward command motor neuron AVA or indirectly activate AVA through other neuronal pathways, including the intermediate interneuron AIB. Selectively silencing the AIB interneuron with the a chemical genetics system using the histamine-gated chloride channel resulted in complete loss of nociceptive avoidance behaviors induced by slow-ramping SRG-36 receptor in ASH, but had less of an effect on SRG-34 avoidance. Selectively silencing the AVA backward command interneuron reduced reversals, but spared or increased other avoidance behaviors for both SRG-34 and SRG-36. These results indicate that downstream interneurons are engaged in different ways, and to different degrees, depending on the mechanism of ASH activation. I next monitored the activity of AIB and AVA neurons in freely-moving ASH:srg-34 or ASH:srg-36 animals responding to C3. In ASH:srg-34 animals, AIB and AVA begin increasing activity upon C3 onset. In ASH:srg-36 worms, AIB increased activity before AVA. Together with my AIB silencing results, these observations suggest that AIB accumulates signals from ASH over time to promote AVA activity. Using a coherent type-1 feed forward loop with a calcium slope-determined AND or OR logic, I modeled features of AIB contribution to nociceptive behaviors in response to different ASH temporal dynamics. These findings suggest that feedforward excitation loops, a motif seen in C. elegans and mammalian nervous systems, can result in behaviorally-salient consequences in response to different sensory neuron calcium dynamics

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

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    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

    Sensory Coding and Olfactory Integration in Caenorhabditis Elegans

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    Animals must sense their external environments to guide meaningful behavior. The nematode Caenorhabditis elegans, for example, uses volatile cues to navigate toward food from a distance. How does an animal integrate the olfactory information from its environment? Here, I ask how multiple sensory neurons drive and shape one interneuron’s activity. C. elegans senses several odors, including the bacterial metabolite diacetyl, using the AWA sensory neurons. AWA forms chemical and electrical synapses onto several interconnected interneurons, which contribute to chemotaxis toward attractive odors like diacetyl. One AWA target is the interneuron AIA, which is connected to AWA via a putative electrical synapse. Both AWA and AIA are robustly activated by diacetyl, but the reliability of their responses decreases at low concentrations. AIA relies on AWA for its reliable response to diacetyl. However, directly activating AWA is not sufficient to evoke reliable AIA responses. Instead, AIA responses to optogenetic AWA stimulation had high and variable latencies and low probabilities. AIA responses, when they did occur, had stereotyped on-dynamics to all concentrations of diacetyl tested, to AWA optogenetic stimulation, and to several additional attractive odors, suggesting all-or-none AIA activation to sensory input. In animals lacking chemical synaptic transmission, AIA responses to direct AWA optogenetic stimulation were fast and reliable, resembling those evoked by diacetyl. AWA-to-AIA communication is thus regulated by inhibitory synaptic input from surrounding neurons. This inhibition comes from a small set of glutamatergic sensory neurons that work together to gate AIA responses to AWA activation. Consistently, two of these glutamatergic sensory neurons directly sense and are inhibited by diacetyl. Their responses are less reliable, or even non-existent, at low concentrations of diacetyl. The difference in the reliability of AIA responses to different diacetyl concentrations may be explained by differences in the composition of the upstream sensory responses. Reliable AIA responses appear to require both activation from AWA through an electrical synapse and the release of inhibition from glutamatergic sensory neurons through chemical synapses. AIA acts as a coincidence detector, and its activity represents a readout of global sensory state, providing insight into how AIA represents “food” signals that are sensed by multiple sensory neurons

    Feed-Forward Propagation of Temporal and Rate Information between Cortical Populations during Coherent Activation in Engineered In Vitro Networks.

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    Transient propagation of information across neuronal assembles is thought to underlie many cognitive processes. However, the nature of the neural code that is embedded within these transmissions remains uncertain. Much of our understanding of how information is transmitted among these assemblies has been derived from computational models. While these models have been instrumental in understanding these processes they often make simplifying assumptions about the biophysical properties of neurons that may influence the nature and properties expressed. To address this issue we created an in vitro analog of a feed-forward network composed of two small populations (also referred to as assemblies or layers) of living dissociated rat cortical neurons. The populations were separated by, and communicated through, a microelectromechanical systems (MEMS) device containing a strip of microscale tunnels. Delayed culturing of one population in the first layer followed by the second a few days later induced the unidirectional growth of axons through the microtunnels resulting in a primarily feed-forward communication between these two small neural populations. In this study we systematically manipulated the number of tunnels that connected each layer and hence, the number of axons providing communication between those populations. We then assess the effect of reducing the number of tunnels has upon the properties of between-layer communication capacity and fidelity of neural transmission among spike trains transmitted across and within layers. We show evidence based on Victor-Purpura's and van Rossum's spike train similarity metrics supporting the presence of both rate and temporal information embedded within these transmissions whose fidelity increased during communication both between and within layers when the number of tunnels are increased. We also provide evidence reinforcing the role of synchronized activity upon transmission fidelity during the spontaneous synchronized network burst events that propagated between layers and highlight the potential applications of these MEMs devices as a tool for further investigation of structure and functional dynamics among neural populations

    The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates

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    Differentiation of the presynaptic terminal is a complex and rapid event that normally occurs in spatially specific axonal regions distant from the soma; thus, it is believed to be dependent on intra-axonal mechanisms. However, the full nature of the local events governing presynaptic assembly remains unknown. Herein, we investigated the involvement of the ubiquitin-proteasome system (UPS), the major degradative pathway, in the local modulation of presynaptic differentiation. We found that proteasome inhibition has a synaptogenic effect on isolated axons. In addition, formation of a stable cluster of synaptic vesicles onto a postsynaptic partner occurs in parallel to an on-site decrease in proteasome degradation. Accumulation of ubiquitinated proteins at nascent sites is a local trigger for presynaptic clustering. Finally, proteasome-related ubiquitin chains (K11 and K48) function as signals for the assembly of presynaptic terminals. Collectively, we propose a new axon-intrinsic mechanism for presynaptic assembly through local UPS inhibition. Subsequent on-site accumulation of proteins in their polyubiquitinated state triggers formation of presynapses.info:eu-repo/semantics/publishedVersio

    The development of optical projection tomography instrumentation and its application to in vivo three dimensional imaging of zebrafish

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    OPT is a three dimensional (3D) imaging technique that can produce 3D reconstructions of transparent samples, requiring only a widefield imaging system and sample rotation. OPT can be readily applied to chemically cleared samples, or to live transparent organisms such as nematodes or zebrafish. For preclinical imaging, there is a trade-off between optical accessibility and biological relevance to humans. Adult Danio rerio (zebrafish) represent a promising compromise, with greater homology to humans than smaller animals, and superior optical accessibility than mice. However, their size and physiology present a number of imaging challenges including non-negligible absorption and optical scattering, and limited time for image data acquisition if the fish are to be recovered for longitudinal studies. A key goal of this PhD thesis research was to develop OPT to address these challenges and improve in vivo imaging capabilities for this model organism. This thesis builds on previous work at Imperial where angularly multiplexed OPT using compressed sensing was developed and applied to in vivo imaging of a cancer-burdened adult zebrafish, with a sufficiently short OPT data acquisition time to allow recovery of the fish after anaesthesia. The previous cross-sectional study of this work was extended to a longitudinal study of cancer progression that I undertook. The volume and quality of data acquired in the longitudinal study presented a number of data processing challenges, which I addressed with improved automation of the data processing pipeline and with the demonstration that convolutional neural networks (CNN) could replace the iterative compressed sensing algorithm previously used to suppress artifacts when reconstructing undersampled OPT data sets. To address the issue of high attenuation through the centre of an adult zebrafish, I developed conformal-high-dynamic-range (C-HDR) OPT and demonstrated that it could provide sufficient dynamic range for brightfield imaging of such optically thick samples, noting that transmitted light images can provide anatomical context for fluorescence image data. To reduce the impact of optical scattering in OPT, I developed a parallelised quasi-confocal version of OPT called slice-illuminated OPT (slice-OPT) to reject scattered photons and demonstrated this with live zebrafish. To enable 3D imaging with short wave infrared (SWIR) light, without the requirement of an expensive Ge or InGaAs camera, I implemented a single pixel camera and demonstrated single-pixel OPT (SP-OPT) for the first time.Open Acces

    Set up of a light sheet fluorescence microscope for cellular studies

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    Light-sheet fluorescence microscopy (LSFM) has been present in cell biology laboratories for quite some time, mainly as custom-made systems, with imaging applications ranging from single cells (in the μm scale) to small organisms (mm). Such microscopes distinguish themselves for having very low phototoxicity levels and high spatial and temporal resolution, properties that render it ideal for 3D characterization of cell motility in migration and traction force studies. Cellular motion has proven to be essential in biological processes such as tumor metastasis and tissue development. Experimental setups make extensive use of microdevices (bioMEMS) that are providing higher degrees of empirical complexity. The following report details the process of setting-up a functional LSFM device for imaging cell motion in microfluidic devices. It begins with a brief summary of fluorescence imaging and current techniques, important to understand why single-plane illumination microscopy (SPIM) was chosen among other light-sheet methods. Then, the whole SPIM set-up process is described, containing explanations about the physical and material properties of the hardware used, the intricacies of the control system, and important procedures. These procedures include: calibration of the microscope, sample preparation in microdevices, and image acquisition from the software provided. Real fluorescence images acquired serve as evidence of the functionality of the instrument. The current limitations are highlighted, and pointers on how to improve or enhance the device are given. The report contains many diagrams, tables and pictures to aid in the understanding of important concepts. In the Annex, a comprehensive table listing the project costs by category is attached. This table includes links to the manufacturers and providers. The aim of this writing is to serve as an exhaustive guideline and be of reproducible use for researchers aiming to build SPIM systems for similar applications.Ingeniería Biomédic

    An Assessment of miRNA Manipulation on Senescence and Ageing Phenotypes in vitro and in vivo

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    Ageing has been widely described as a progressive functional deterioration of tissues that causes diminished organ function and increased mortality risk. It has been established that the proportion of senescent cells in tissues rises with age in many organs and in age-related illnesses, suggesting that cellular senescence plays a significant role in the functional decline related to ageing. Correspondingly, it has previously been shown in animal models that eliminating senescent cells might mitigate the deleterious consequences of ageing. As a key regulator of several cellular mechanisms, there are microRNAs (miRNAs) known to be associated with senescence. However, miRNAs that may directly trigger or reverse senescence remain to be elucidated. Here, the first goal of thesis was to identify the miRNA profile of proliferating, senescent, and rescued senescent endothelial cells to determine miRNAs that may be causal or influential of cellular senescence. I found that miR-361-5p not only associated with senescence but also reduced the load of senescent cells in vitro in human endothelial cells upon induction in late passage cells. Secondly, C. elegans was used to examine the role of miR-361-5p targeted genes on ageing in vivo. I found that 56% of genes which were dysregulated in vitro adversely affected healthspan and/or lifespan in vivo. Finally, a previous finding from our lab (Holly et al., 2015) identified three miRNAs-associated with human ageing and senescence in human primary fibroblasts of which miR-15b-5p may reduce senescence markers and secretory phenotypes (SASP) in the human dermal fibroblast cells. This thesis presents new miRNAs (miR-361-5p and miR-15b-5p) which may be involved in the aetiology of senescence and may be used in future in ageing intervention
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