The Impact of Mild Traumatic Brain injury on Neuronal Networks and Neurobehavior

Despite its enormous incidence, mild traumatic brain injury is not well understood. One aspect that needs more definition is how the mechanical energy during injury affects neural circuit function. Recent developments in cellular imaging probes provide an opportunity to assess the dynamic state of neural networks with single-cell resolution. In this dissertation, we developed imaging methods to assess the state of dissociated cortical networks exposed to mild injury. We probed the microarchitecture of an injured cortical circuit subject to two different injury levels, mild stretch (10% peak) and mild/moderate (35%). We found that mild injury produced a transient increase in calcium activity that dissipated within 1 h after injury. Alternatively, mild/moderate mechanical injury produced immediate disruption in network synchrony, loss in excitatory tone, and increased modular topology, suggesting a threshold for repair and degradation. The more significant changes in network behavior at moderate stretch are influenced by NMDA receptor activation and subsequent proteolytic changes in the neuronal populations. With the ability to analyze individual neurons in a circuit before and after injury, we identified several biomarkers that confer increased risk or protection from mechanical injury. We found that pre-injury connectivity and NMDA receptor subtype composition (NR2A and NR2B content) are important predictors of node loss and remodeling. Mechanistically, stretch injury caused a reduction in voltage-dependent Mg2+ block of the NR2B-cotaning NMDA receptors, resulting in increased uncorrelated activity both at the single channel and network level. The reduced coincidence detection of the NMDA receptor and overactivation of these receptors further impaired network function and plasticity. Given the demonstrated link between NR2B-NMDARs and mitochondrial dysfunction, we discovered that neuronal de-integration from the network is mediated through mitochondrial signaling. Finally, we bridged these network level studies with an investigation of changes in neurobehavior following blast-induced traumatic brain injury (bTBI), a form of mild TBI. We first developed and validated an open-source toolbox for automating the scoring of several common behavior tasks to study the deficits that occur following bTBI. We then specifically evaluated the role of neuronal transcription factor Elk-1 in mediating deficits following blast by exposing Elk-1 knockout mouse to equivalent blast pressure loading. Our systems-level behavior analysis showed that bTBI creates a complex change in behavior, with an increase in anxiety and loss of habituation in object recognition. Moreover, we found these behavioral deficits were eliminated in Elk-1 knockout animals exposed to blast loading. Together, we merged information from different perspectives (in silico, in vitro, and in vivo) and length scales (single channels, single-cells, networks, and animals) to study the impact of mild traumatic brain injury on neuronal networks and neurobehavior

Current approaches and future role of high content imaging in safety sciences and drug discovery

High content imaging combines automated microscopy with image analysis approaches to simultaneously quantify multiple phenotypic and/or functional parameters in biological systems. The technology has become an important tool in the fields of safety sciences and drug discovery, because it can be used for mode-of-action identification, determination of hazard potency and the discovery of toxicity targets and biomarkers. In contrast to conventional biochemical endpoints, high content imaging provides insight into the spatial distribution and dynamics of responses in biological systems. This allows the identification of signaling pathways underlying cell defense, adaptation, toxicity and death. Therefore high content imaging is considered a promising technology to address the challenges for the Toxicity testing in the 21st century approach. Currently high content imaging technologies are frequently applied in academia for mechanistic toxicity studies and in pharmaceutical industry for the ranking and selection of lead drug compounds or to identify/confirm mechanisms underlying effects observed in vivo. A recent workshop gathered scientists working on high content imaging in academia, pharmaceutical industry and regulatory bodies with the objective to compile the state-of-the-art of the technology in the different institutions. They defined technical and methodological gaps, addressed the need for quality control, suggested control compounds and acceptance criteria, highlighted cell sources and new readouts and discussed future requirements for regulatory implementation. This review summarizes the discussion, proposed solutions and recommendations of the specialists contributing to the workshop.JRC.I.5-Systems Toxicolog

Microglia modulate blood flow, neurovascular coupling, and hypoperfusion via purinergic actions

Microglia, the main immunocompetent cells of the brain, regulate neuronal function, but their contribution to cerebral blood flow (CBF) regulation has remained elusive. Here, we identify microglia as important modulators of CBF both under physiological conditions and during hypoperfusion. Microglia establish direct, dynamic purinergic contacts with cells in the neurovascular unit that shape CBF in both mice and humans. Surprisingly, the absence of microglia or blockade of microglial P2Y12 receptor (P2Y12R) substantially impairs neurovascular coupling in mice, which is reiterated by chemogenetically induced microglial dysfunction associated with impaired ATP sensitivity. Hypercapnia induces rapid microglial calcium changes, P2Y12R-mediated formation of perivascular phylopodia, and microglial adenosine production, while depletion of microglia reduces brain pH and impairs hypercapnia-induced vasodilation. Microglial actions modulate vascular cyclic GMP levels but are partially independent of nitric oxide. Finally, microglial dysfunction markedly impairs P2Y12R-mediated cerebrovascular adaptation to common carotid artery occlusion resulting in hypoperfusion. Thus, our data reveal a previously unrecognized role for microglia in CBF regulation, with broad implications for common neurological diseases

Microglia: Development of a human in vitro model, analysis of motility and effects of phagocytosis in the hippocampal neurogenic niche

135 p.This PhD Thesis discusses several topics related to microglia, the resident macrophages of the brain parenchyma. They are derived from yolk sack primitive macrophages that colonize the brain early during embryonic development. Microglia contribute to the correct development and functioning of the central nervous system with their multiple functions, including their role as phagocytes clearing the brain parenchyma from apoptotic cells and protein aggregates. Microglia are also involved in most neurological and neurodegenerative diseases, especially since genome-wide association studies have found that many microglia-specific genes are significant risk factors for neurodegenerative diseases. This PhD Thesis focusses on two aspects of microglial physiology. First, on the development of human models of microglia and their importance in the use of microglia as a therapeutic target in neurodegenerative diseases. Finally, on microglial process motility and phagocytosis, two essential functions of homeostatic microglia that can become compromised in pathology. This thesis defines a method for the semi-automated analysis of microglia in 3D and how inflammation affects microglial motility. This thesis also shows how an impairment of phagocytosis in the adult neurogenic niche negatively affects the neurogenic cascade suggesting a crosstalk between microglia and neural progenitor cells

Cannabidiol Exerts a Neuroprotective and Glia-Balancing Effect in the Subacute Phase of Stroke

Pharmacological agents limiting secondary tissue loss and improving functional outcomes after stroke are still limited. Cannabidiol (CBD), the major non-psychoactive component of Cannabis sativa, has been proposed as a neuroprotective agent against experimental cerebral ischemia. The effects of CBD mostly relate to the modulation of neuroinflammation, including glial activation. To investigate the effects of CBD on glial cells after focal ischemia in vivo, we performed time-lapse imaging of microglia and astroglial Ca2+ signaling in the somatosensory cortex in the subacute phase of stroke by in vivo two-photon laser-scanning microscopy using transgenic mice with microglial EGFP expression and astrocyte-specific expression of the genetically encoded Ca2+ sensor GCaMP3. CBD (10 mg/kg, intraperitoneally) prevented ischemia-induced neurological impairment, reducing the neurological deficit score from 2.0 ± 1.2 to 0.8 ± 0.8, and protected against neurodegeneration, as shown by the reduction (more than 70%) in Fluoro-Jade C staining (18.8 ± 7.5 to 5.3 ± 0.3). CBD reduced ischemia-induced microglial activation assessed by changes in soma area and total branch length, and exerted a balancing effect on astroglial Ca2+ signals. Our findings indicate that the neuroprotective effects of CBD may occur in the subacute phase of ischemia, and reinforce its strong anti-inflammatory property. Nevertheless, its mechanism of action on glial cells still requires further studies

Specific In Vivo Staining of Astrocytes in the Whole Brain after Intravenous Injection of Sulforhodamine Dyes

Fluorescent staining of astrocytes without damaging or interfering with normal brain functions is essential for intravital microscopy studies. Current methods involved either transgenic mice or local intracerebral injection of sulforhodamine 101. Transgenic rat models rarely exist, and in mice, a backcross with GFAP transgenic mice may be difficult. Local injections of fluorescent dyes are invasive. Here, we propose a non-invasive, specific and ubiquitous method to stain astrocytes in vivo. This method is based on iv injection of sulforhodamine dyes and is applicable on rats and mice from postnatal age to adulthood. The astrocytes staining obtained after iv injection was maintained for nearly half a day and showed no adverse reaction on astrocytic calcium signals or electroencephalographic recordings in vivo. The high contrast of the staining facilitates the image processing and allows to quantify 3D morphological parameters of the astrocytes and to characterize their network. Our method may become a reference for in vivo staining of the whole astrocytes population in animal models of neurological disorders

Neural progenitor cell differentiation and migration : Role of glutamate signaling, brain-derived neurotrophic factor, and hypoxia/acidosis

The mammalian central nervous system (CNS) develops from multipotent neural stem or progenitor cells. During development the cells proliferate actively and differentiate into all the different cell types of the brain. Neurogenesis continues in the adult brain but to a much lesser extent than during development. Adult neurogenesis is influenced by many different factors, including insults to the brain and neurodegenerative disease. Neurotransmitters have been implicated as regulators of neurogenesis. The main excitatory neurotransmitter glutamate is linked to neural progenitor cell proliferation and differentiation as well as migration of newborn neurons. Glutamate is also involved in the pathogenesis of several neurological disorders and other factors linked to brain pathogenesis, such as hypoxia and acidosis, are known to influence neural progenitor cells. Elucidating the mechanisms governing stem/progenitor cell behavior during normal and pathological conditions will aid in the development of cell-based therapies for treating insult or disease within the CNS. The aim of this thesis was to study the role of glutamate receptor agonists and antagonists in differentiation and migration of neural progenitors and their progeny to increase the understanding of how this neurotransmitter influences these cells. In addition, the effects of brain-derived neurotrophic factor (BDNF) and the reactivity of the cells to conditions associated with ischemic stroke (hypoxia/acidosis) were studied. By utilizing the neurosphere model we found that differentiating neural progenitors initially mainly expressed and responded to stimuli through metabotropic glutamate receptor 5 (mGluR5) and that the expression and functional response of the receptor corresponded with the distribution of radial glial cells. Ionotropic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate (KA) receptors were also present during early differentiation and expressed mainly by neuron-like cells. The expression of mGluR5 decreased and the expression and functional maturity of AMPA/KA receptors increased with time in culture. Pharmacological blocking studies revealed that radial glial process extension and neuronal motility are regulated through both mGluR5 and AMPA/KA receptors, but that the receptors have opposing effects on these cellular mechanisms. After prolonged differentiation a small subpopulation of neuronal cells responding to stimulation with N-methyl-D-aspartate (NMDA) and gamma amino butyric acid (GABA) appeared. This subpopulation of cells was responsive to motogenic actions mediated by BDNF. In addition, we found that radial glial and neuron-like cells exhibited differences in resting membrane potential and intracellular pH and reacted differently when exposed to hypoxic and acidic conditions. This study contributes new information regarding neural progenitor cell characteristics and behavior when differentiated in the presence of or challenged with factors influencing neurogenesis, both during normal and pathological conditions. These findings may be useful in developing treatment programs for neurological disorders.Det centrala nervsystemet (CNS) hos mammalier utvecklas utgående från multipotenta neurala stamceller. Under utvecklingen delar sig de neurala stamcellerna aktivt och ger upphov till alla de olika celltyperna i hjärnan. Nybildning av nervceller fortsätter i den vuxna hjärnan men i mycket begränsad mån. Denna nybildning påverkas av många olika faktorer, inklusive hjärnskador och neurodegenerativa sjukdomar. Signalsubstanser i hjärnan har föreslagits påverka nybildningen av nervceller. Glutamat är den huvudsakliga excitatoriska signalsubstansen i hjärnan och har länkats till proliferation och differentiering av neurala stamceller samt migration av nybildade nervceller. Glutamat är också involverad i patogenesen av flera olika neurologiska sjukdomar och andra faktorer länkade till patologiska tillstånd i hjärnan, såsom hypoxi och acidos, har också visats påverka neurala stamceller. Klargörande av de mekanismer som styr stamcellernas beteende under normala och patologiska tillstånd kan bidra till utvecklingen av cellbaserade terapiformer för behandling av skador eller sjukdomar i CNS. Målet med denna avhandling var att studera rollen av glutamatreceptoragonister och -antagonister vid differentiering och migration av neurala stamceller och deras derivat för att öka förståelsen för hur denna signalsubstans påverkar dessa celler. Ytterligare studerades hur cellerna påverkas av den neurotrofa faktorn BDNF och förhållanden associerade med slaganfall (hypoxi/acidos). Genom att utnyttja den så kallade neurosfärmodellen fann vi att neurala stamceller vid differentiering initialt huvudsakligen uppvisade funktionella svar vid stimulering via den metabotropa glutamatreceptorn mGluR5 och att uttrycket av mGluR5 korrelerade med distributionen av radiala gliaceller. Funktionella jonotropa AMPA/KA-receptorer fanns också vara närvarande tidigt under differentieringsprocessen, främst i neuroner. Uttrycket av mGluR5 minskade medan uttrycket och den funktionella mognaden av AMPA/KA-receptorer ökade med tiden. Farmakologiska studier visade att både mGluR5 och AMPA/KA-receptorer är involverade i regleringen av neuronala cellers motilitet och radiala gliacellers processutväxt men att receptorerna har motsatta effekter på dessa cellulära mekanismer. Efter en längre periods differentiering kunde en liten subpopulation av celler som uppvisade funktionella svar vid stimulering med NMDA och GABA identifieras. BDNF ökade motiliteten av denna subpopulation. Vi fann ytterligare att radiala gliaceller och neuroner uppisade skillnader i vilomembranpotential och intracellulärt pH samt reagerade olika i hypoxiska och sura förhållanden. Denna studie bidrar med ny information angående neurala stamcellers egenskaper och beteende vid differentiering i närvaro av eller vid stimulering med faktorer som påverkar neurogenes, både under normala och patologiska förhållanden. Dessa upptäckter kan vara av nytta vid utvecklingen av behandlingsprogram för neurologiska skador och sjukdomar

HIV Tat and Morphine-induced Neurodegeneration in a Beclin 1 Hemizygous Mouse Model

Early in infection, HIV crosses the blood-brain barrier and induces neuropathology. Viral presence in the CNS coupled with secretion of neurotoxic proteins causes neuroinflammation, glial dysfunction, excitotoxicity, and neuronal death. Despite advances in combined antiretroviral therapy, HIV-infected patients present with a spectrum of cognitive and psychomotor deficits collectively referred to as HIV-associated neurological disorders (HAND). A subset of HAND patients abuses drugs such as opiates like heroin and morphine show an exacerbation and rapid progression of HIV neuropathology; however, the mechanisms of this synergy are not well understood. Autophagy is a lysosomal degradative process which eliminates and recycles cytosolic components and is implicated in facilitating HIV-1 replication in the CNS and periphery, and in Tat-induced neurodegeneration. When a key initiator of autophagy Beclin 1 was silenced using siRNAs, there was a marked reduction of HIV-1 replication in human microglia and astrocytes and the corresponding inflammatory response. As such, the goal of the current study is to determine if diminished Beclin 1 is neuroprotective against Tat and morphine-induced neurodegeneration using heterozygous Beclin 1 (Becn1+/-) mice. Examination of Tat and morphine-induced inflammatory molecule secretion revealed that Becn1+/- mixed astrocyte and microglia (glia) exhibited attenuated secretion of cytokine IL-6 and chemokines RANTES and MCP-1 compared to control (C57BL/6J) glia, an effect mediated through the μ-opioid receptor. Dysregulation of autophagy-related gene expression and excessive intracellular calcium accumulation were limited in Becn1+/- glia. When determining the effects of Tat-and morphine co-exposure on neuronal survival in vitro, we found Becn1+/- neurons were particularly sensitive to injury, excitotoxicity, and toxic exposures; however, when C57BL/6J neurons were exposed to conditioned media of C57BL/6J and Becn1+/- glia treated with Tat and morphine, neurons treated with Becn1+/- supernatant had better outcomes than those treated with C57BL/6J conditioned media. Furthermore, despite minimal difference between strains in locomotor assessment, we observed significantly greater striatal neuron losses in adult C57BL/6J mice exposed to intrastriatal Tat-and systemic morphine compared to Becn1+/- mice. Our studies demonstrate the potential of targeting Beclin 1 in glia for the prevention of Tat and opiate-induced CNS dysfunction