Oligodendroglial GABAergic Signaling: More Than Inhibition!

GABA is the main inhibitory neurotransmitter in the CNS acting at two distinct types of receptor: ligand-gated ionotropic GABAA receptors and G protein-coupled metabotropic GABAB receptors, thus mediating fast and slow inhibition of excitability at central synapses. GABAergic signal transmission has been intensively studied in neurons in contrast to oligodendrocytes and their precursors (OPCs), although the latter express both types of GABA receptor. Recent studies focusing on interneuron myelination and interneuron-OPC synapses have shed light on the importance of GABA signaling in the oligodendrocyte lineage. In this review, we start with a short summary on GABA itself and neuronal GABAergic signaling. Then, we elaborate on the physiological role of GABA receptors within the oligodendrocyte lineage and conclude with a description of these receptors as putative targets in treatments of CNS diseases

Deletion of LRP1 From Astrocytes Modifies Neuronal Network Activity in an in vitro Model of the Tripartite Synapse

The low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor that binds over 40 potential ligands and is involved in processes such as cell differentiation, proliferation, and survival. LRP1 is ubiquitously expressed in the organism and enriched among others in blood vessels, liver, and the central nervous system (CNS). There, it is strongly expressed by neurons, microglia, immature oligodendrocytes, and astrocytes. The constitutive LRP1 knockout leads to embryonic lethality. Therefore, previous studies focused on conditional LRP1-knockout strategies and revealed that the deletion of LRP1 causes an increased differentiation of neural stem and precursor cells into astrocytes. Furthermore, astrocytic LRP1 is necessary for the degradation of Aβ and the reduced accumulation of amyloid plaques in Alzheimer’s disease. Although the role of LRP1 in neurons has intensely been investigated, the function of LRP1 with regard to the differentiation and maturation of astrocytes and their functionality is still unknown. To address this question, we generated an inducible conditional transgenic mouse model, where LRP1 is specifically deleted from GLASTpositive astrocyte precursor cells. The recombination with resulting knockout events was visualized by the simultaneous expression of the fluorescent reporter tdTomato. We observed a significantly increased number of GLT-1 expressing astrocytes in LRP1- depleted astrocytic cultures in comparison to control astrocytes. Furthermore, we investigated the influence of astrocytic LRP1 on neuronal activity and synaptogenesis using the co-culture of hippocampal neurons with control or LRP1-depleted astrocytes. These analyses revealed that the LRP1-deficient astrocytes caused a decreased number of single action potentials as well as a negatively influenced neuronal network activity. Moreover, the proportion of pre- and postsynaptic structures was significantly altered in neurons co-cultured with LPR1-depleted astrocytes. However, the number of structural synapses was not affected. Additionally, the supernatant of hippocampal neurons co-cultured with LRP1-deficient astrocytes showed an altered set of cytokines in comparison to the control condition, which potentially contributed to the altered neuronal transmission and synaptogenesis. Our results suggest astrocytic LRP1 as a modulator of synaptic transmission and synaptogenesis by altering the expression of the glutamate transporter on the cell surface on astrocytes and the release of cytokines in vitro

Acute brain injuries trigger microglia as an additional source of the proteoglycan NG2

NG2 is a type I transmembrane glycoprotein known as chondroitin sulfate proteoglycan 4 (CSPG4). In the healthy central nervous system, NG2 is exclusively expressed by oligodendrocyte progenitor cells and by vasculature pericytes. A large body of immunohistochemical studies showed that under pathological conditions such as acute brain injuries and experimental autoimmune encephalomyelitis (EAE), a number of activated microglia were NG2 immuno-positive, suggesting NG2 expression in these cells. Alternative explanations for the microglial NG2 labeling consider the biochemical properties of NG2 or the phagocytic activity of activated microglia. Reportedly, the transmembrane NG2 proteoglycan can be cleaved by a variety of proteases to deposit the NG2 ectodomain into the extracellular matrix. The ectodomain, however, could also stick to the microglial surface. Since microglia are phagocytic cells engulfing debris of dying cells, it is difficult to identify a genuine expression of NG2. Recent studies showing (1) pericytes giving rise to microglial after stroke, and (2) immune cells of NG2-EYFP knock-in mice lacking NG2 expression in an EAE model generated doubts for the de novo expression of NG2 in microglia after acute brain injuries. In the current study, we took advantage of three knock-in mouse lines (NG2-CreERT2, CX3CR1-EGFP and NG2-EYFP) to study NG2 expression indicated by transgenic fluorescent proteins in microglia after tMCAO (transient middle cerebral artery occlusion) or cortical stab wound injury (SWI). We provide strong evidence that NG2-expressing cells, including OPCs and pericytes, did not differentiate into microglia after acute brain injuries, whereas activated microglia did express NG2 in a disease-dependent manner. A subset of microglia continuously activated the NG2 gene at least within the first week after tMCAO, whereas within 3 days after SWI a limited number of microglia at the lesion site transiently expressed NG2. Immunohistochemical studies demonstrated that these microglia with NG2 gene activity also synthesized the NG2 protein, suggesting activated microglia as an additional source of the NG2 proteoglycan after acute brain injuries

The Paradox of Astroglial Ca2+ Signals at the Interface of Excitation and Inhibition

Astroglial networks constitute a non-neuronal communication system in the brain and are acknowledged modulators of synaptic plasticity. A sophisticated set of transmitter receptors in combination with distinct secretion mechanisms enables astrocytes to sense and modulate synaptic transmission. This integrative function evolved around intracellular Ca2+ signals, by and large considered as the main indicator of astrocyte activity. Regular brain physiology meticulously relies on the constant reciprocity of excitation and inhibition (E/I). Astrocytes are metabolically, physically, and functionally associated to the E/I convergence. Metabolically, astrocytes provide glutamine, the precursor of both major neurotransmitters governing E/I in the central nervous system (CNS): glutamate and γ-aminobutyric acid (GABA). Perisynaptic astroglial processes are structurally and functionally associated with the respective circuits throughout the CNS. Astonishingly, in astrocytes, glutamatergic as well as GABAergic inputs elicit similar rises in intracellular Ca2+ that in turn can trigger the release of glutamate and GABA as well. Paradoxically, as gliotransmitters, these two molecules can thus strengthen, weaken or even reverse the input signal. Therefore, the net impact on neuronal network function is often convoluted and cannot be simply predicted by the nature of the stimulus itself. In this review, we highlight the ambiguity of astrocytes on discriminating and affecting synaptic activity in physiological and pathological state. Indeed, aberrant astroglial Ca2+ signaling is a key aspect of pathological conditions exhibiting compromised network excitability, such as epilepsy. Here, we gather recent evidence on the complexity of astroglial Ca2+ signals in health and disease, challenging the traditional, neuro-centric concept of segregating E/I, in favor of a non-binary, mutually dependent perspective on glutamatergic and GABAergic transmission

L-Type Ca2+ Channels of NG2 Glia Determine Proliferation and NMDA Receptor-Dependent Plasticity

NG2 (nerve/glial antigen 2) glia are uniformly distributed in the gray and white matter of the central nervous system (CNS). They are the major proliferating cells in the brain and can differentiate into oligodendrocytes. NG2 glia do not only receive synaptic input from excitatory and inhibitory neurons, but also secrete growth factors and cytokines, modulating CNS homeostasis. They express several receptors and ion channels that play a role in rapidly responding upon synaptic signals and generating fast feedback, potentially regulating their own properties. Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) induces an intracellular Ca2+ rise initiating a series of cellular activities. We confirmed that NG2 glia express L-type VGCCs in the white and gray matter during CNS development, particularly in the early postnatal stage. However, the function of L-type VGCCs in NG2 glia remains elusive. Therefore, we deleted L-type VGCC subtypes Cav1.2 and Cav1.3 genes conditionally in NG2 glia by crossbreeding NG2-CreERT2 knock-in mice to floxed Cav1.2 and flexed Cav1.3 transgenic mice. Our results showed that ablation of Cav1.2 and Cav1.3 strongly inhibited the proliferation of cortical NG2 glia, while differentiation in white and gray matter was not affected. As a consequence, no difference on myelination could be detected in various brain regions. In addition, we observed morphological alterations of the nodes of Ranvier induced by VGCC-deficient NG2 glia, i.e., shortened paired paranodes in the corpus callosum. Furthermore, deletion of Cav1.2 and Cav1.3 largely eliminated N-methyl-D-aspartate (NMDA)-dependent long-term depression (LTD) and potentiation in the hippocampus while the synaptic input to NG2 glia from axons remained unaltered. We conclude that L-type VGCCs of NG2 glia are essential for cell proliferation and proper structural organization of nodes of Ranvier, but not for differentiation and myelin compaction. In addition, L-type VGCCs of NG2 glia contribute to the regulation of long-term neuronal plasticity

Contribution of Intravital Neuroimaging to Study Animal Models of Multiple Sclerosis

Multiple sclerosis (MS) is a complex and long-lasting neurodegenerative disease of the central nervous system (CNS), characterized by the loss of myelin within the white matter and cortical fbers, axonopathy, and infammatory responses leading to consequent sensory-motor and cognitive defcits of patients. While complete resolution of the disease is not yet a reality, partial tissue repair has been observed in patients which ofers hope for therapeutic strategies. To address the molecular and cellular events of the pathomechanisms, a variety of animal models have been developed to investigate distinct aspects of MS disease. Recent advances of multiscale intravital imaging facilitated the direct in vivo analysis of MS in the animal models with perspective of clinical transfer to patients. This review gives an overview of MS animal models, focusing on the current imaging modalities at the microscopic and macroscopic levels and emphasizing the importance of multimodal approaches to improve our understanding of the disease and minimize the use of animals

Prednisolone as preservation additive prevents from ischemia reperfusion injury in a rat model of orthotopic lung transplantation

The lung is, more than other solid organs, susceptible for ischemia reperfusion injury after orthotopic transplantation. Corticosteroids are known to potently suppress pro-inflammatory processes when given in the post-operative setting or during rejection episodes. Whereas their use has been approved for these clinical indications, there is no study investigating its potential as a preservation additive in preventing vascular damage already in the phase of ischemia. To investigate these effects we performed orthotopic lung transplantations (LTX) in the rat. Prednisolone was either added to the perfusion solution for lung preservation or omitted and rats were followed for 48 hours after LTX. Prednisolone preconditioning significantly increased survival and diminished reperfusion edema. Hypoxia induced vasoactive cytokines such as VEGF were reduced. Markers of leukocyte invasiveness like matrix metalloprotease (MMP)-2, or common pro-inflammatory molecules like the CXCR4 receptor or the chemokine (C-C motif) ligand (CCL)-2 were downregulated by prednisolone. Neutrophil recruitment to the grafts was only increased in Perfadex treated lungs. Together with this, prednisolone treated animals displayed significantly reduced lung protein levels of neutrophil chemoattractants like CINC-1, CINC-2α/β and LIX and upregulated tissue inhibitor of matrix metalloproteinase (TIMP)-1. Interestingly, lung macrophage invasion was increased in both, Perfadex and prednisolone treated grafts, as measured by MMP-12 or RM4. Markers of anti-inflammatory macrophage transdifferentiation like MRC-1, IL-13, IL-4 and CD163, significantly correlated with prednisolone treatment. These observations lead to the conclusion that prednisolone as an additive to the perfusion solution protects from hypoxia triggered danger signals already in the phase of ischemia and thus reduces graft edema in the phase of reperfusion. Additionally, prednisolone preconditioning might also lead to macrophage polarization as a beneficial long-term effect

From Physiology to Pathology of Cortico-Thalamo-Cortical Oscillations: Astroglia as a Target for Further Research

The electrographic hallmark of childhood absence epilepsy (CAE) and other idiopathic forms of epilepsy are 2.5–4 Hz spike and wave discharges (SWDs) originating from abnormal electrical oscillations of the cortico-thalamo-cortical network. SWDs are generally associated with sudden and brief non-convulsive epileptic events mostly generating impairment of consciousness and correlating with attention and learning as well as cognitive deficits. To date, SWDs are known to arise from locally restricted imbalances of excitation and inhibition in the deep layers of the primary somatosensory cortex. SWDs propagate to the mostly GABAergic nucleus reticularis thalami (NRT) and the somatosensory thalamic nuclei that project back to the cortex, leading to the typical generalized spike and wave oscillations. Given their shared anatomical basis, SWDs have been originally considered the pathological transition of 11–16 Hz bursts of neural oscillatory activity (the so-called sleep spindles) occurring during Non-Rapid Eye Movement (NREM) sleep, but more recent research revealed fundamental functional differences between sleep spindles and SWDs, suggesting the latter could be more closely related to the slow (<1 Hz) oscillations alternating active (Up) and silent (Down) cortical activity and concomitantly occurring during NREM. Indeed, several lines of evidence support the fact that SWDs impair sleep architecture as well as sleep/wake cycles and sleep pressure, which, in turn, affect seizure circadian frequency and distribution. Given the accumulating evidence on the role of astroglia in the field of epilepsy in the modulation of excitation and inhibition in the brain as well as on the development of aberrant synchronous network activity, we aim at pointing at putative contributions of astrocytes to the physiology of slow-wave sleep and to the pathology of SWDs. Particularly, we will address the astroglial functions known to be involved in the control of network excitability and synchronicity and so far mainly addressed in the context of convulsive seizures, namely (i) interstitial fluid homeostasis, (ii) K+ clearance and neurotransmitter uptake from the extracellular space and the synaptic cleft, (iii) gap junction mechanical and functional coupling as well as hemichannel function, (iv) gliotransmission, (v) astroglial Ca2+ signaling and downstream effectors, (vi) reactive astrogliosis and cytokine release

Sublamina-specific organization of the blood brain barrier in the mouse olfactory nerve layer

Astrocytes constitute the main glial component of the mammalian blood brain barrier (BBB). However, in the olfactory bulb (OB), the olfactory nerve layer (ONL) is almost devoid of astrocytes, raising the question which glial cells are part of the BBB. We used mice expressing EGFP in astrocytes and tdTomato in olfactory ensheathing cells (OECs), a specialized type of glial cells in the ONL, to unequivocally identify both glial cell types and investigate their contribution to the BBB in the olfactory bulb. OECs were located exclusively in the ONL, while somata of astrocytes were located in deeper layers and extended processes in the inner sublamina of the ONL. These processes surrounded blood vessels and contained aquaporin-4, an astrocytic protein enriched at the BBB. In the outer sublamina of the ONL, in contrast, blood vessels were surrounded by aquaporin-4-negative processes of OECs. Transcardial perfusion of blood vessels with lanthanum and subsequent visualization by electron microscopy showed that blood vessels enwrapped by OECs possessed intact tight junctions. In acute olfactory bulb preparations, injection of fluorescent glucose 6-NBDG into blood vessels resulted in labeling of OECs, indicating glucose transport from the perivascular space into OECs. In addition, Ca2+ transients in OECs in the outer sublamina evoked vasoconstriction, whereas Ca2+ signaling in OECs of the inner sublamina had no effect on adjacent blood vessels. Our results demonstrate that the BBB in the inner sublamina of the ONL contains astrocytes, while in the outer ONL OECs are part of the BBB

Conditional Deletion of LRP1 Leads to Progressive Loss of Recombined NG2-Expressing Oligodendrocyte Precursor Cells in a Novel Mouse Model

The low-density lipoprotein receptor-related protein 1 (LRP1) is a transmembrane receptor, mediating endocytosis and activating intracellular signaling cascades. LRP1 is highly expressed in the central nervous system (CNS), especially in oligodendrocyte precursor cells (OPCs). Previous studies have suggested LRP1 as a regulator in early oligodendrocyte development, repair of chemically induced white matter lesions, and cholesterol homeostasis. To circumvent embryonic lethality observed in the case of global LRP1 deletion, we generated a new inducible conditional knockout (KO) mouse model, which enabled an NG2-restricted LRP1 deficiency (NG2-CreERT2ct2/wtxR26eGFPflox/floxxLRP1flox/flox). When characterizing our triple transgenic mouse model, we noticed a substantial and progressive loss of recombined LRP1-deficient cells in the oligodendrocyte lineage. On the other hand, we found comparable distributions and fractions of oligodendroglia within the Corpus callosum of the KO and control animals, indicating a compensation of these deficits. An initial study on experimental autoimmune encephalomyelitis (EAE) was performed in triple transgenic and control mice and the cell biology of oligodendrocytes obtained from the animals was studied in an in vitro myelination assay. Differences could be observed in these assays, which, however, did not achieve statistical significance, presumably because the majority of recombined LRP1-deficient cells has been replaced by non-recombined cells. Thus, the analysis of the role of LRP1 in EAE will require the induction of acute recombination in the context of the disease process. As LRP1 is necessary for the survival of OPCs in vivo, we assume that it will play an important role in myelin repair