1,720,994 research outputs found
Volume CLEM and conventional EM: a dual approach to investigate the mechanism of neuronal dysfunction in Angelman Syndrome
No full textAngelman Syndrome (AS) is a rare neurodevelopmental disorder arising from the loss of the maternal copy of UBE3A, a paternally imprinted gene at neuronal level. Similarly to AS, other neurological defects due to an unproper UBE3A dosage are directly associated with alterations in brain development and synapse ultrastructure. The nanometric resolution of electron microscopy (EM) has proven essential for studying their outcome at the network level and establishing meaningful correlations between synapses geometrical features and their function. To explore whether UBE3A loss effect on the developing connectome was cell-autonomous, we in-utero electroporated CRISPR/Cas9 constructs to silence the endogenous UBE3A expression in a subset of layer 2/3 pyramidal neurons of the mouse somatosensory cortex. By creating z-confined NIRB marks we were able to relocate dendrites of interest and analyze spines complexity three-dimensionally from volumetric SBF-SEM datasets. Furthermore, we exploited conventional EM to examine excitatory synapses structure in the cortical circuit of a classic AS model pan-neuronally depleted from UBE3A expression. We identified pre-synaptic compartment defects that seem to diminish over time to make room for later changes in the post-synapse. Overall, the combined use of these two models should help clarify whether the defects originate within the neurons or their wider network
CLEM and Volume EM to study synapse architecture in Angelman Syndrome
No full textAngelman Syndrome (AS) is a neurogenetic disorder characterized by severe intellectual disability, speech impairment, seizure disorder, abnormal sleep patterns, motor dysfunction and unusually happy demeanor.
AS is caused by the loss of the maternal copy of the UBE3A gene, which is biallelically expressed in all cell types, but only maternally expressed in neurons due to epigenetic silencing of the paternal allele.
Studies in AS models suggest that the UBE3A gene, encoding for the ubiquitin-protein ligase E3A, is crucial for synaptic formation and maintenance, neuronal plasticity, learning and memory formation and, because of having only the maternal copy of UBE3A gene, neurons are quite vulnerable to its loss.
During development, the synapse is subjected to fine modification ranging from activity-dependent dendritic spine enlargement to changes in size and shape of the postsynaptic density. Similar modifications are at the bases of synaptic plasticity.
To study the effect of UBE3A inactivation on the functional organization of synapses and neuronal connectivity in AS, we will combine sparse in utero electroporation of layer 2/3 pyramidal neurons of the mouse somatosensory cortex with CRISPR/Cas9-mediated genome editing to inactivate endogenous Ube3a. The fluorescent protein tdTomato will be used as a filler to identify and select electroporated neurons. We will use Serial Block Face Scanning Electron Microscopy and CLEM to generate volumetric dataset to reconstruct electroporated neurons and to quantitatively study synapses in their environment, finally connecting the geometry of synapses with their functional state
vCLEM: bridging the gap between high-resolution structural imaging and functional analysis of synapses
No full textUnderstanding the sophisticated details of synaptic structures is crucial when studying
the mechanisms of neuronal communication and brain organization in health and
disease. Traditional imaging techniques often fail to meet the necessity of providing
both the detailed ultrastructure and the specific protein localization we need for
comprehensive synaptic studies.
Correlative Light and Electron Microscopy (CLEM) addresses this challenge by imaging
the same exact biological structure with two, or more, imaging modalities, thus
combining the strengths of light with the resolution of electron microscopy. Volume
CLEM (vCLEM) adds a higher level of complexity to this already intricated method,
introducing the invaluable benefits of the third dimension.
We use vCLEM to study Angelman Syndrome (AS), a neurogenic disorder resulting from
the loss of the maternal copy of the UBE3A gene. Taking advantage of CRISPR/Cas9-
mediated genome editing endogenous Ube3a is inactivated via sparse in utero
electroporation (IUE) of layer 2/3 pyramidal neurons of the mouse somatosensory
cortex.
The correlative workflow is complex and involves several steps: first, a confocal
microscope is used to identify electroporated fluorescent neurons in the brain section
and acquire images; next, Near Infra-Red Banding (NIRB) is conducted to generate
spatial coordinates for locating the electroporated neurons along the x, y, and z axes;
the sample is processed for the Serial Block-Face Scanning Electron Microscope (SBF-
SEM), which is then exploited to create volumetric datasets.
This approach represents a significant advancement in the study of synapses and
allows us to gain a comprehensive understanding of the effect of the loss of Ube3a gene
on synapse ultrastructure and brain connectivity during development
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The role of Voltage-Gated Sodium Channels in neuronal differentiation and their contribution to Amyotrophic Lateral Sclerosis pathogenesis
No full textVoltage-gated sodium channels (VGSCs) are multimeric protein complexes formed by one alpha subunit and one or two accessory beta subunits. These channels are responsible for the generation of inward Na+ currents. A small fraction of these currents is represented by a non-inactivating component, the persistent Na+ current or INaP. Recent evidence suggests that VGSCs and INaP are important not only in the regulation of excitable cell electrical properties but also in promoting neuronal development and neurite outgrowth. Interestingly, both VGSCs and INaP alterations and defects in neuron morphology and neurite outgrowth were reported in Amyotrophic Lateral Sclerosis (ALS). ALS is fatal neurodegenerative disease causing muscle denervation and wasting, paralysis and death of the patient usually within 5 years from diagnosis. We previously observed defects in motor axons morphology together with motor- and interneuron INaP -dependent hyperexcitability in a zebrafish model expressing an ALS-associated mutant SOD1. To investigate the role of VGSCs related currents in neuronal differentiation and their possible role in the pathogenesis of ALS, we took advantage of the NSC34 cell line, often used as an in vitro model to study motoneuron diseases. We used two NSC34 populations: cells stably expressing G93A mutant SOD1 and cells defective in the expression of the Vesicle Associated Membrane Protein (VAMP)-Associated Protein B (VAPB). We analyzed VGSCs expression and localization through molecular, biochemical and imaging approaches and found interesting correlations and differences between VGSCs expression, localization, function and neurite outgrowth in wild-type and mutant cells. Our study thus confirms the proposed role of VGSCs in tuning neuronal differentiation with a precise temporal pattern. Disruption of this pattern might have relevant implications in ALS pathogenesis
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Numerical prediction of low-frequency ground vibrations induced by high-speed trains at Ledsgaard, Sweden
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