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Alterations in the extracellular matrix (ECM) are common in epilepsy, yet whether they are cause or consequence of disease is unknow. Using Theiler’s virus infection model of acquired epilepsy we findde novoexpression of chondroitin sulfate proteoglycans (CSPGs), a major ECM component, in dentate gyrus (DG) and amygdala exclusively in mice with seizures. Preventing synthesis of CSPGs specifically in DG and amygdala by deletion of major CSPG aggrecan reduced seizure burden. Patch-clamp recordings from dentate granule cells (DGCs) revealed enhanced intrinsic and synaptic excitability in seizing mice that was normalized by aggrecan deletion.In situexperiments suggest that DGCs hyperexcitability results from negatively charged CSPGs increasing stationary cations (K+, Ca2+) on the membrane thereby depolarizing neurons, increasing their intrinsic and synaptic excitability. We show similar changes in CSPGs in pilocarpine-induced epilepsy suggesting enhanced CSPGs in the DG and amygdala may be a common ictogenic factor and novel therapeutic potential.

The rodent visual thalamus has served as a powerful model to elucidate the cellular and molecular mechanisms that underlie sensory circuit formation and function. Despite significant advances in our understanding of the role of axon-target interactions and neural activity in orchestrating circuit formation in visual thalamus, the role of non-neuronal cells, such as astrocytes, is less clear. In fact, we know little about the transcriptional identity and development of astrocytes in mouse visual thalamus. To address this gap in knowledge, we studied the expression of canonical astrocyte molecules in visual thalamus using immunostaining, in situ hybridization, and reporter lines. While our data suggests some level of heterogeneity of astrocytes in different nuclei of the visual thalamus, the majority of thalamic astrocytes appeared to be labelled in Aldh1l1-EGFP mice. This led us to use this transgenic line to characterize the neonatal and postnatal development of these cells in visual thalamus. Our data show that not only have the entire cohort of astrocytes migrated into visual thalamus by eye-opening but they also have acquired their adult-like morphology, even while retinogeniculate synapses are still maturing. Furthermore, ultrastructural, immunohistochemical, and functional approaches revealed that by eye-opening, thalamic astrocytes ensheath retinogeniculate synapses and are capable of efficient uptake of glutamate. Taken together, our results reveal that the morphological, anatomical, and functional development of astrocytes in visual thalamus occurs prior to eye-opening and the emergence of experience-dependent visual activity. This article is protected by copyright. All rights reserved.

Background: Vascular pericytes stabilize blood vessels and contribute to their maturation, while playing other key roles in microvascular function. Nevertheless, relatively little is known about involvement of their precursors in the earliest stages of vascular development, specifically during vasculogenesis. Methods: We combined high-power, time-lapse imaging with transcriptional profiling of emerging pericytes and endothelial cells in reporter mouse and cell lines. We also analyzed conditional transgenic animals deficient in Cx43/Gja1 (connexin 43/gap junction alpha-1) expression within Ng2+ cells. Results: A subset of Ng2-DsRed+ cells, likely pericyte/mural cell precursors, arose alongside endothelial cell differentiation and organization and physically engaged vasculogenic endothelium in vivo and in vitro. We found no overlap between this population of differentiating pericyte/mural progenitors and other lineages including hemangiogenic and neuronal/glial cell types. We also observed cell-cell coupling and identified Cx43-based gap junctions contributing to pericyte–endothelial cell precursor communication during vascular assembly. Genetic loss of Cx43/Gja1 in Ng2+ pericyte progenitors compromised embryonic blood vessel formation in a subset of animals, while surviving mutants displayed little-to-no vessel abnormalities, suggesting a resilience to Cx43/Gja1 loss in Ng2+ cells or potential compensation by additional connexin isoforms. Conclusions: Together, our data suggest that a distinct pericyte lineage emerges alongside vasculogenesis and directly communicates with the nascent endothelium via Cx43 during early vessel formation. Cx43/Gja1 loss in pericyte/mural cell progenitors can induce embryonic vessel dysmorphogenesis, but alternate connexin isoforms may be able to compensate. These data provide insight that may reshape the current framework of vascular development and may also inform tissue revascularization/vascularization strategies.

Rapid and widespread testing of severe acute respiratory coronavirus 2 (SARS-CoV-2) is essential for an effective public health response aimed at containing and mitigating the coronavirus disease 2019 (COVID-19) pandemic. Successful health policy implementation relies on early identification of infected individuals and extensive contact tracing. However, rural communities, where resources for testing are sparse or simply absent, face distinctive challenges to achieving this success. Accordingly, we report the development of an academic, public land grant University laboratory-based detection assay for the identification of SARS-CoV-2 in samples from various clinical specimens that can be readily deployed in areas where access to testing is limited. The test, which is a quantitative reverse transcription polymerase chain reaction (RT-qPCR)-based procedure, was validated on samples provided by the state laboratory and submitted for FDA Emergency Use Authorization. Our test exhibits comparable sensitivity and exceeds specificity and inclusivity values compared to other molecular assays. Additionally, this test can be re-configured to meet supply chain shortages, modified for scale up demands, and is amenable to several clinical specimens. Test development also involved 3D engineering critical supplies and formulating a stable collection media that allowed samples to be transported for hours over a dispersed rural region without the need for a cold-chain. These two elements that were critical when shortages impacted testing and when personnel needed to reach areas that were geographically isolated from the testing center. Overall, using a robust, easy-to-adapt methodology, we show that an academic laboratory can supplement COVID-19 testing needs and help local health departments assess and manage outbreaks. This additional testing capacity is particularly germane for smaller cities and rural regions that would otherwise be unable to meet the testing demand., Here, the authors report the development of a versatile academic, SARSCoV-2 RT-qPCR molecular diagnostic test that uses 3D printed technology for sample collection, is implemented in rural setting in the US state of Virginia and validated in its population.

Given the important functions that glutamate serves in excitatory neurotransmission, understanding the regulation of glutamate in physiological and pathological states is critical to devising novel therapies to treat epilepsy. Exclusive expression of pyruvate carboxylase and glutamine synthetase in astrocytes positions astrocytes as essential regulators of glutamate in the central nervous system (CNS). Additionally, astrocytes can significantly alter the volume of the extracellular space (ECS) in the CNS due to their expression of the bi-directional water channel, aquaporin-4, which are enriched at perivascular endfeet. Rapid ECS shrinkage has been observed following epileptiform activity and can inherently concentrate ions and neurotransmitters including glutamate. This review highlights our emerging knowledge on the various potential contributions of astrocytes to epilepsy, particularly supporting the notion that astrocytes may be involved in seizure initiation via failure of homeostatic responses that lead to increased ambient glutamate. We also review the mechanisms whereby ambient glutamate can influence neuronal excitability, including via generation of the glutamate receptor subunit GluN2B-mediated slow inward currents, as well as indirectly affect neuronal excitability via actions on metabotropic glutamate receptors that can potentiate GluN2B currents and influence neuronal glutamate release probabilities. Additionally, we discuss evidence for upregulation of System xc-, a cystine/glutamate antiporter expressed on astrocytes, in epileptic tissue and changes in expression patterns of glutamate receptors. NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [1R01CA227149-01A1, 1R01NS036692-01A1]; DODUnited States Department of Defense [W81XWH-18-1-0521] This work was supported by NIH grants 1R01CA227149-01A1 and 1R01NS036692-01A1, and DOD grant W81XWH-18-1-0521.

Vascular pericytes (PCs) stabilize blood vessels and contribute to their maturation, while also playing key roles in microvascular function. Nevertheless, relatively little is known about their involvement in the earliest stages of vascular development, specifically during vasculogenesis. We combined high-power, time-lapse imaging with transcriptional profiling of PCs and endothelial cells (ECs) in reporter mouse and cell lines. PCs emerged alongside EC differentiation, and physically engaged the vasculogenic endothelium in vivo and in vitro. We observed PC-EC coupling and identified Connexin43 (Cx43)-based gap junctions as key elements in PC-EC communication during vessel assembly. We found no overlap between differentiating PCs and other lineages including hemangiogenic and neuronal/glial cell types. Together, our data suggest that a distinct PC lineage emerges prior to vasculogenesis and participates in early vessel formation through direct communication with nascent endothelium via Cx43 – providing insight that reshapes the current framework of vascular development and informs tissue vascularization strategies.

SummaryPericytes (PCs), cells that extend along capillaries to contribute stability and other critical functions to established vasculature, are attracting attention from various fields involving vascular-related pathologies. Here, we demonstrate primary evidence of PC communication with endothelial cells (ECs) prior to tube coalescence. Observations of apparent PCs during early embryogenesis urged development of a mouse embryonic stem cell line (DR-ESCs), enabling unique dual-reporter investigations into earliest PC-EC interactions. Live imaging of differentiating DR-ESCs corroborated emergence of a PC lineage, which preceded EC differentiation, and further revealed highly dynamic PC-EC interactions during coordinated vessel formation. We show direct PC-EC communication via cell microinjection and dye-transfer, and RNA-seq analysis indicates a PC-EC coupling mechanism via gap junction Connexin43 (Cx43), exclusively up-regulated throughout DR-ESC differentiation. High resolution imaging of embryonic and postnatal mouse vasculature substantiates Cx43 plaques at PC-EC borders. These findings indicate a new role for PCs during vasculogenesis via Cx43-mediated communication with ECs.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Objective: Currently prescribed antiepileptic drugs (AEDs) are ineffective in treating approximately 30% of epilepsy patients. Sulfasalazine (SAS) is an US Food and Drug Administration (FDA)–approved drug for the treatment of Crohn disease that has been shown to inhibit the cystine/glutamate antiporter system xc‐ (SXC) and decrease tumor‐associated seizures. This study evaluates the effect of SAS on distinct pharmacologically induced network excitability and determines whether it can further decrease hyperexcitability when administered with currently prescribed AEDs. Methods: Using in vitro cortical mouse brain slices, whole‐cell patch‐clamp recordings were made from layer 2/3 pyramidal neurons. Epileptiform activity was induced with bicuculline (bic), 4‐aminopyridine (4‐AP) and magnesium‐free (Mg2+‐free) solution to determine the effect of SAS on epileptiform events. In addition, voltagesensitive dye (VSD) recordings were performed to characterize the effect of SAS on the spatiotemporal spread of hyperexcitable network activity and compared to currently prescribed AEDs. Results: SAS decreased evoked excitatory postsynaptic currents (eEPSCs) and increased the decay kinetics of evoked inhibitory postsynaptic currents (eIPSCs) in layer 2/3 pyramidal neurons. Although application of SAS to bic and Mg2+‐free–induced epileptiform activity caused a decrease in the duration of epileptiform events, SAS completely blocked 4‐AP–induced epileptiform events. In VSD recordings, SAS decreased VSD optical signals induced by 4‐AP. Co‐application of SAS with the AED topiramate (TPM) caused a significantly further decrease in the spatiotemporal spread of VSD optical signals. Significance: Taken together this study provides evidence that inhibition of SXC by SAS can decrease network hyperexcitability induced by three distinct pharmacologic agents in the superficial layers of the cortex. Furthermore, SAS provided additional suppression of 4‐AP–induced network activity when administered with the currently prescribed AED TPM. These findings may serve as a foundation to assess the potential for SAS or other compounds that selectively target SXC as an adjuvant treatment for epilepsy. Foundation for the National Institutes of Health, Grant/Award Number: 5R01‐NS036692, R01‐NS052634 and R01CA227149

Perineuronal nets (PNNs) are extracellular matrix assemblies of highly negatively charged proteoglycans that wrap around fast-spiking parvalbumin (PV) expressing interneurons in the cerebral cortex. PNNs play important roles in neuronal plasticity and modulate biophysical properties of the enclosed interneurons. Various central nervous system diseases including schizophrenia, Alzheimer disease and epilepsy present with qualitative alteration in PNNs, however prior studies failed to quantitatively assess such changes at single PNN level and correlate them with functional changes in disease. We describe a method to quantify the structural integrity of PNNs using high magnification image analysis of Wisteria Floribunda Agglutinin (WFA)-labeled PNNs in combination with cell-type-specific marker such as PV and NeuN. A polyline intensity profile of WFA along the entire perimeter of cell shows alternate segments with and without WFA labeling, indicating the intact chondroitin sulfate proteoglycan (CSPG) and holes of PNN respectively. This line intensity profile defines CSPG peaks, where intact PNN is present, and CSPG valleys (holes) where the PNN is missing. The average number of peaks reflect the integrity of the lattice assembly of PNN. The average size of PNN holes can be readily computed using image analysis software. Furthermore, degradation of PNNs using a bacterial-derived enzyme, Chondroitinase ABC (ChABC), allows to experimentally manipulate PNNs in situ brain slices during which biophysical properties can be assessed by patch-clamp recordings. We describe optimized experimental parameters to degrade PNNs in brain slices before as well as during recordings to study the possible change in function in real time. Our protocols provide effective and appropriate methods to modulate and quantify the PNN's experimental manipulations.

Epilepsy is a neurological disorder afflicting ~65 million people worldwide. It is caused by aberrant synchronized firing of populations of neurons primarily due to imbalance between excitatory and inhibitory neurotransmission. Hence, the historical focus of epilepsy research has been neurocentric. However, the past two decades have enjoyed an explosion of research into the role of glia in supporting and modulating neuronal activity, providing compelling evidence of glial involvement in the pathophysiology of epilepsy. The mechanisms by which glia, particularly astrocytes and microglia, may contribute to epilepsy and consequently could be harnessed therapeutically are discussed in this Review.

Brain tumor patients commonly present with epileptic seizures. We show that tumor-associated seizures are the consequence of impaired GABAergic inhibition due to an overall loss of peritumoral fast spiking interneurons (FSNs) concomitant with a significantly reduced firing rate of those that remain. The reduced firing is due to the degradation of perineuronal nets (PNNs) that surround FSNs. We show that PNNs decrease specific membrane capacitance of FSNs permitting them to fire action potentials at supra-physiological frequencies. Tumor-released proteolytic enzymes degrade PNNs, resulting in increased membrane capacitance, reduced firing, and hence decreased GABA release. These studies uncovered a hitherto unknown role of PNNs as an electrostatic insulator that reduces specific membrane capacitance, functionally akin to myelin sheaths around axons, thereby permitting FSNs to exceed physiological firing rates. Disruption of PNNs may similarly account for excitation-inhibition imbalances in other forms of epilepsy and PNN protection through proteolytic inhibition may provide therapeutic benefits., Brain tumours are associated with epilepsy. Here the authors show, using a mouse model, that the degradation of perineuronal nets around fast spiking interneurons near the tumour contribute to seizures by increasing their membrane capacitance and firing.

It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca(2+) loading. Earlier we have reported that in motor neurons a generic rise in [Ca(2+)]i does not always lead to mitochondrial Ca(2+) loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca(2+) regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca(2+)]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca(2+)]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca(2+)]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca(2+)]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na(+)/Ca(2+) exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca(2+) sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na(+)/Ca(2+) exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.

Excessive activation of AMPA receptor has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). However, it is not clear why motor neurons are preferentially sensitive to AMPA receptor mediated excessive [Ca2+]i rise and excitotoxicity. In the present study we examined whether palmitoylation regulates Ca2+ permeability of AMPA receptor and excitotoxicity in cultured spinal cord neurons. We adapted chronic 2-bromopalmitate (2-BrP) treatment to achieve depalmitoylation and examined its effect on the cytotoxicity in spinal cord neurons exposed to AMPA. The change in AMPA induced signaling and cytotoxicity in motor neurons and other spinal neurons under identical conditions of exposure to AMPA was studied. 2-BrP treatment inhibited AMPA induced rise in [Ca2+]i and cytotoxicity in both types of neurons but the degree of inhibition was significantly higher in motor neurons as compared to other spinal neurons. The AMPA induced [Na+]i rise was moderately affected in both type of neurons on depalmitoylation. Depalmitoylation reduced the expression levels of AMPA receptor subunits (GluR1 and GluR2) and also PSD-95 but stargazin levels remained unaffected. Our results demonstrate that 2-BrP attenuates AMPA receptor activated Ca2+ signaling and cytotoxicity preferentially in motor neurons and suggest that AMPA receptor modulation by depalmitoylation could play a significant role in preventing motor neuron degeneration.

It is well established that motor neurons are highly vulnerable to glutamate induced excitotoxicity. The selective vulnerability of these neurons has been attributed to AMPA receptor mediated excessive rise in cytosolic calcium and consequent mitochondrial Ca(2+) loading. Earlier we have reported that in motor neurons a generic rise in [Ca(2+)]i does not always lead to mitochondrial Ca(2+) loading and membrane depolarization but it occurs upon AMPA receptor activation. The mechanism of such specific mitochondrial involvement upon AMPA receptor activation is not known. The present study examines the mitochondrial Ca(2+) regulation and oxidative stress in spinal cord neurons upon AMPA subtype of glutamate receptor activation. Stimulating the spinal neurons with AMPA exhibited a sharp rise in [Ca(2+)]m in both motor and other spinal neurons that was sustained up to the end of recording time of 30min. The rise in [Ca(2+)]m was substantially higher in motor neurons than in other spinal neurons which could be due to the differential mitochondrial homeostasis in two types of neurons. To examine this possibility, we measured AMPA induced [Ca(2+)]m loading in the presence of mitochondrial inhibitors. In both cell types the AMPA induced [Ca(2+)]m loading was blocked by mitochondrial calcium uniporter blocker ruthenium red. In motor neurons it was also inhibited substantially by CGP37157 and cyclosporine-A, the blockers of Na(+)/Ca(2+) exchanger and mitochondrial permeability transition pore (MPTP) respectively, whereas no effect of these agents was observed in other spinal neurons. Thus in motor neurons the Ca(2+) sequestration by mitochondria occurs through mitochondrial calcium uniporter as well as due to reversal of Na(+)/Ca(2+) exchanger, in contrast the latter pathway does not contribute in other spinal neurons. The ROS formation was inhibited by nitric oxide synthase (NOS) inhibitor L-NAME in both types of neurons, however the mitochondrial complex-I inhibitor rotenone suppressed the ROS formation only in motor neurons. It appears that activation of cytoplasmic nNOS leads to ROS formation in both types of spinal neurons but mitochondria is the major source of ROS in motor neurons. Spinal neurons exhibited a significant time dependent fall in glutathione (GSH) level. The GSH level in motor neurons did not recover even at 24h after AMPA exposure, whereas the other spinal neurons exhibited a tendency to maintain the GSH after a certain level suggesting that the oxidative stress is arrested in other spinal neurons but it continues to increase in motor neurons. Thus our results demonstrate that upon AMPA receptor stimulation the motor neurons employ some additional pathways for regulation of mitochondrial calcium and oxidative stress as compared to other spinal neurons. It is suggested that such differential signaling mechanisms in motor neurons could be crucial for their selective vulnerability to excitotoxicity.