2,675 research outputs found

    Investigating the roles of cell adhesion molecules in synapse formation and function

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    Recent findings have revealed a crucial contribution of the adhesion molecule neuroligin-1 to the precise organization and regulation of intercellular synaptic connections within the central nervous system, and disruption of neuroligin-1 signaling in vivo fosters cognitive abnormalities. Despite considerable recent progress, several uncertainties remain regarding the exact synaptic function of neuroligin-1. Principle among these uncertainties is whether neuroligin-1 primarily promotes initiation of de novo synaptic connections or maturation of functional, pre-existent connections. To begin to address this, experiments must be devised that are capable of dissociating activity-dependent and -independent effects of neuroligin-1 signaling on pre- and postsynaptic compartments. An additional uncertainty is how and when synapses containing neuroligin-1 are specified as either excitatory or inhibitory. Elucidating these synapse specification cascades will prove crucial in defining the contribution of neuroligin-1 to overall network balances of excitation and inhibition that guide proper cognitive development. A final uncertainty is how alternate adhesion complexes may coordinate with neuroligin-1 to initiate or maintain synaptic connections. Differentiating redundant from complementary functions among adhesion systems will help reconcile unresolved discrepancies between in vitro and in vivo experiments and ultimately provide a clearer understanding of synapse formation and function in vivo. Herein I detail significant new findings clarifying each of these uncertainties. Utilizing a specific transfection protocol, I first demonstrate that neuroligin-1 is capable of robustly inducing presynaptic differentiation independent of proper postsynaptic development and synaptic activity. Second, employing both multi-molecular perturbations and a delimited biological model of the synapse, I show that the postsynaptic scaffolding molecule PSD95 specifically acts downstream of neuroligin-1-mediated synapse initiation. Third, the model synapse is again employed to differentiate between separate synaptic functions of neuroligin-1 and alternate adhesion molecule SynCAM1. Building from these distinct synaptic functions, I provide preliminary evidence that SynCAM1 matures inactive neuroligin-1-initiated synapses. Fourth, I present the first direct evidence that neuroligin-1 contributes to dendritic morphogenesis in mammalian neurons, consistent with recent findings within the Xenopus system. Collectively, these results evince a robust capacity of neuroligin-1 in initial stages of synaptogenesis and contribute to a new theory of neuroligin-1 function in both activity-dependent synapse initiation and activity-dependent synapse maturation

    A method for the three-dimensional reconstruction of Neurobiotin(TM)-filled neurons and the location of their synaptic inputs

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    Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit a1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses

    Acute Synaptic Activity Causes Differential miRNA Expression in the Drosophila melanogaster Larval Central Nervous System

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    The primary goal of this thesis was to determine if spaced synaptic stimulation induced the differential expression of microRNAs (miRNAs) in the Drosophila melanogaster central nervous system (CNS). Prior to attaining this goal, we needed to identify and validate a spaced stimulation paradigm that could induce the formation of new synaptic growth at a model synapse, the larval neuromuscular junction (NMJ). Both Channelrhodopsin- and high potassium-based stimulation paradigms adapted from (Ataman, et al. 2008) were tested. Once validation of these paradigms was complete, we sought to characterize the miRNA expression profile of the larval CNS by miRNA array. Following attainment of these data, we used quantitative real-time PCR (RT-qPCR) to determine if acute synaptic stimulation caused the differential expression of neuronal miRNAs. We found that upon high potassium spaced training in a wild type (Canton S) genotype, 5 miRNAs showed significant differential expression when normalized to a validated reference gene, the U1 snRNA. Moreover, absolute quantification of our RT-qPCR study implicated one miRNA: miR-958 as being significantly regulated by activity. Investigation into potential targets for miR-958 revealed it to be a potential regular of Dlar, a protein tyrosine phosphatase implicated in synapse development. This investigation provides the foundation to directly test our underlying hypothesis that, following spaced training, differential expression of miRNAs alters the translation of proteins required to induce and maintain these structural changes at the synapse

    Comparison of Multiscale Imaging Methods for Brain Research

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    A major challenge in neuroscience is how to study structural alterations in the brain. Even small changes in synaptic composition could have severe outcomes for body functions. Many neuropathological diseases are attributable to disorganization of particular synaptic proteins. Yet, to detect and comprehensively describe and evaluate such often rather subtle deviations from the normal physiological status in a detailed and quantitative manner is very challenging. Here, we have compared side-by-side several commercially available light microscopes for their suitability in visualizing synaptic components in larger parts of the brain at low resolution, at extended resolution as well as at super-resolution. Microscopic technologies included stereo, widefield, deconvolution, confocal, and super-resolution set-ups. We also analyzed the impact of adaptive optics, a motorized objective correction collar and CUDA graphics card technology on imaging quality and acquisition speed. Our observations evaluate a basic set of techniques, which allow for multi-color brain imaging from centimeter to nanometer scales. The comparative multi-modal strategy we established can be used as a guide for researchers to select the most appropriate light microscopy method in addressing specific questions in brain research, and we also give insights into recent developments such as optical aberration corrections

    Neurexin directs partner-specific synaptic connectivity in C. elegans

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    In neural circuits, individual neurons often make projections onto multiple postsynaptic partners. Here, we investigate molecular mechanisms by which these divergent connections are generated, using dyadic synapses in C. elegans as a model. We report that C. elegans nrx-1/neurexin directs divergent connectivity through differential actions at synapses with partnering neurons and muscles. We show that cholinergic outputs onto neurons are, unexpectedly, located at previously undefined spine-like protrusions from GABAergic dendrites. Both these spine-like features and cholinergic receptor clustering are strikingly disrupted in the absence of nrx-1. Excitatory transmission onto GABAergic neurons, but not neuromuscular transmission, is also disrupted. Our data indicate that NRX-1 located at presynaptic sites specifically directs postsynaptic development in GABAergic neurons. Our findings provide evidence that individual neurons can direct differential patterns of connectivity with their post-synaptic partners through partner-specific utilization of synaptic organizers, offering a novel view into molecular control of divergent connectivity

    From Understanding Cellular Function to Novel Drug Discovery: The Role of Planar Patch-Clamp Array Chip Technology

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    All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, thus limiting their use in modeling physiological function. These chips are therefore not most suitable for studies involving neuronal communication. Multielectrode arrays (MEAs), in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high-resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, approaches to chemical patterning for cell placement, and present physiological data from cultured neuronal cells

    SynapseJ: An Automated, Synapse Identification Macro for ImageJ

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    While electron microscopy represents the gold standard for detection of synapses, a number of limitations prevent its broad applicability. A key method for detecting synapses is immunostaining for markers of pre- and post-synaptic proteins, which can infer a synapse based upon the apposition of the two markers. While immunostaining and imaging techniques have improved to allow for identification of synapses in tissue, analysis and identification of these appositions are not facile, and there has been a lack of tools to accurately identify these appositions. Here, we delineate a macro that uses open-source and freely available ImageJ or FIJI for analysis of multichannel, z-stack confocal images. With use of a high magnification with a high NA objective, we outline two methods to identify puncta in either sparsely or densely labeled images. Puncta from each channel are used to eliminate non-apposed puncta and are subsequently linked with their cognate from the other channel. These methods are applied to analysis of a pre-synaptic marker, bassoon, with two different post-synaptic markers, gephyrin and N-methyl-d-aspartate (NMDA) receptor subunit 1 (NR1). Using gephyrin as an inhibitory, post-synaptic scaffolding protein, we identify inhibitory synapses in basolateral amygdala, central amygdala, arcuate and the ventromedial hypothalamus. Systematic variation of the settings identify the parameters most critical for this analysis. Identification of specifically overlapping puncta allows for correlation of morphometry data between each channel. Finally, we extend the analysis to only examine puncta overlapping with a cytoplasmic marker of specific cell types, a distinct advantage beyond electron microscopy. Bassoon puncta are restricted to virally transduced, pedunculopontine tegmental nucleus (PPN) axons expressing yellow fluorescent protein. NR1 puncta are restricted to tyrosine hydroxylase labeled dopaminergic neurons of the substantia nigra pars compacta (SNc). The macro identifies bassoon-NR1 overlap throughout the image, or those only restricted to the PPN-SNc connections. Thus, we have extended the available analysis tools that can be used to study synapses in situ. Our analysis code is freely available and open-source allowing for further innovation
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