The Shh receptor Boc is important for myelin formation and repair.

Myelination leads to the formation of myelin sheaths surrounding neuronal axons and is crucial for function, plasticity and repair of the central nervous system (CNS). It relies on the interaction of the axons and the oligodendrocytes: the glial cells producing CNS myelin. Here, we have investigated the role of a crucial component of the Sonic hedgehog (Shh) signalling pathway, the co-receptor Boc, in developmental and repairing myelination. During development, Boc mutant mice display a transient decrease in oligodendroglial cell density together with delayed myelination. Despite recovery of oligodendroglial cells at later stages, adult mutants still exhibit a lower production of myelin basic protein correlated with a significant decrease in the calibre of callosal axons and a reduced amount of the neurofilament NF-M. During myelin repair, the altered OPC differentiation observed in the mutant is reminiscent of the phenotype observed after blockade of Shh signalling. In addition, Boc mutant microglia/macrophages unexpectedly exhibit the apparent inability to transition from a highly to a faintly ramified morphology in vivo Altogether, these results identify Boc as an important component of myelin formation and repair.

Microglia modulate gliotransmission through the regulation of VAMP2 proteins in astrocytes.

Vesicular release is one of the release mechanisms of various signaling molecules. In neurons, the molecular machinery involved in vesicular release has been designed through evolution to trigger fast and synchronous release of neurotransmitters. Similar machinery with a slower kinetic and a slightly different molecular assembly allows astrocytes to release various transmitters such as adenosine triphosphate (ATP), glutamate, and D-serine. Astrocytes are important modulators of neurotransmission through gliotransmitter release. We recently demonstrated that microglia, another type of glia, release ATP to modulate synaptic transmission using astrocytes as intermediate. We now report that microglia regulate astrocytic gliotransmission through the regulation of SNARE proteins in astrocytes. Indeed, we found that gliotransmission triggered by P2Y1 agonist is impaired in slices from transgenic mice devoid of microglia. Using total internal reflection fluorescence imaging, we found that the vesicular release of gliotransmitter by astrocytes was different in cultures lacking microglia compared to vesicular release in astrocytes cocultured with microglia. Quantification of the kinetic of vesicular release indicates that the overall release appears to be faster in pure astrocyte cultures with more vesicles close to the membrane when compared to astrocytes cocultured with microglia. Finally, biochemical investigation of SNARE protein expression indicates an upregulation of VAMP2 in absence of microglia. Altogether, these results indicate that microglia seems to be involved in the regulation of an astrocytic phenotype compatible with proper gliotransmission. The mechanisms described in this study could be of importance for central nervous system diseases where microglia are activated.

Role of LGI1 protein in synaptic transmission: From physiology to pathology.

Leucine-Rich Glioma Inactivated protein 1 (LGI1) is a secreted neuronal protein highly expressed in the central nervous system and high amount are found in the hippocampus. An alteration of its function has been described in few families of patients with autosomal dominant temporal lobe epilepsy (ADLTE) or with autoimmune limbic encephalitis (LE), both characterized by epileptic seizures. Studies have shown that LGI1 plays an essential role during development, but also in neuronal excitability through an action on voltage-gated potassium Kv1.1 channels, and in synaptic transmission by regulating the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-R). Over the last decade, a growing number of studies investigating LGI1 functions have been published. They aimed to improve the understanding of LGI1 function in the regulation of neuronal networks using different animal and cellular models. LGI1 appears to be a major actor of synaptic regulation by modulating trans-synaptically pre- and post-synaptic proteins. In this review, we will focus on LGI1 binding partners, "A Disintegrin And Metalloprotease (ADAM) 22 and 23", the complex they form at the synapse, and will discuss the effects of LGI1 on neuronal excitability and synaptic transmission in physiological and pathological conditions. Finally, we will highlight new insights regarding N-terminal Leucine-Rich Repeat (LRR) domain and C-terminal Epitempin repeat (EPTP) domain and their potentially distinct role in LGI1 function.

Impact of anti-CASPR2 autoantibodies from patients with autoimmune encephalitis on CASPR2/TAG-1 interaction and Kv1 expression.

Autoantibodies against CASPR2 (contactin-associated protein-like 2) have been linked to autoimmune limbic encephalitis that manifests with memory disorders and temporal lobe seizures. According to the growing number of data supporting a role for CASPR2 in neuronal excitability, CASPR2 forms a molecular complex with transient axonal glycoprotein-1 (TAG-1) and shaker-type voltage-gated potassium channels (Kv1.1 and Kv1.2) in compartments critical for neuronal activity and is required for Kv1 proper positioning. Whereas the perturbation of these functions could explain the symptoms observed in patients, the pathogenic role of anti-CASPR2 antibodies has been poorly studied. In the present study, we find that patient autoantibodies alter Caspr2 distribution at the cell membrane promoting cluster formation. We confirm in a HEK cellular model that the anti-CASPR2 antibodies impede CASPR2/TAG-1 interaction and we identify the domains of CASPR2 and TAG-1 taking part in this interaction. Moreover, introduction of CASPR2 into HEK cells induces a marked increase of the level of Kv1.2 surface expression and in cultures of hippocampal neurons Caspr2-positive inhibitory neurons appear to specifically express high levels of Kv1.2. Importantly, in both cellular models, anti-CASPR2 patient autoAb increase Kv1.2 expression. These results provide new insights into the pathogenic role of autoAb in the disease.

Contactin-associated protein-like 2, a protein of the neurexin family involved in several human diseases.

Contactin-associated protein-like 2 (CASPR2) is a cell adhesion protein of the neurexin family. Proteins of this family have been shown to play a role in the development of the nervous system, in synaptic functions, and in neurological diseases. Over recent years, CASPR2 function has gained an increasing interest as demonstrated by the growing number of publications. Here, we gather published data to comprehensively review CASPR2 functions within the nervous system in relation to CASPR2-related diseases in humans. On the one hand, studies on Cntnap2 (coding for CASPR2) knockout mice revealed its role during development, especially, in setting-up the inhibitory network. Consistent with this result, mutations in the CNTNAP2 gene coding for CASPR2 in human have been identified in neurodevelopmental disorders such as autism, intellectual disability, and epilepsy. On the other hand, CASPR2 was shown to play a role beyond development, in the localization of voltage-gated potassium channel (VGKC) complex that is composed of TAG-1, Kv1.1, and Kv1.2. This complex was found in several subcellular compartments essential for action potential propagation: the node of Ranvier, the axon initial segment, and the synapse. In line with a role of CASPR2 in the mature nervous system, neurological autoimmune diseases have been described in patients without neurodevelopmental disorders but with antibodies directed against CASPR2. These autoimmune diseases were of two types: central with memory disorders and temporal lobe seizures, or peripheral with muscular hyperactivity. Overall, we review the up-to-date knowledge on CASPR2 function and pinpoint confused or lacking information that will need further investigation.

Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C.

Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the ß-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor-gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR-gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the ß-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity.

Microglia activation triggers astrocyte-mediated modulation of excitatory neurotransmission.

Fine control of neuronal activity is crucial to rapidly adjust to subtle changes of the environment. This fine tuning was thought to be purely neuronal until the discovery that astrocytes are active players of synaptic transmission. In the adult hippocampus, microglia are the other major glial cell type. Microglia are highly dynamic and closely associated with neurons and astrocytes. They react rapidly to modifications of their environment and are able to release molecules known to control neuronal function and synaptic transmission. Therefore, microglia display functional features of synaptic partners, but their involvement in the regulation of synaptic transmission has not yet been addressed. We have used a combination of pharmacological approaches with electrophysiological analysis on acute hippocampal slices and ATP assays in purified cell cultures to show that activation of microglia induces a rapid increase of spontaneous excitatory postsynaptic currents. We found that this modulation is mediated by binding of ATP to P2Y1R located on astrocytes and is independent of TNFα or NOS2. Our data indicate that, on activation, microglia cells rapidly release small amounts of ATP, and astrocytes, in turn, amplified this release. Finally, P2Y1 stimulation of astrocytes increased excitatory postsynaptic current frequency through a metabotropic glutamate receptor 5-dependent mechanism. These results indicate that microglia are genuine regulators of neurotransmission and place microglia as upstream partners of astrocytes. Because pathological activation of microglia and alteration of neurotransmission are two early symptoms of most brain diseases, our work also provides a basis for understanding synaptic dysfunction in neuronal diseases.

Adaptive and non-adaptive changes in activity-deprived presynaptic terminals.

How the number of docked vesicles is regulated is still unclear. Following chronic activity blockade the number of docked vesicles increases, providing a model through which to address this issue. We tested the hypotheses that the number of docked vesicles is regulated with the size of the terminal, and by the level of Rab3-interacting molecule 1/2 (RIM1/2). We immobilized mouse hippocampal slice cultures by high-pressure freezing after 3 days of tetrodotoxin treatment and analysed them by electron microscopy. The number of docked vesicles, the size of the active zones and the amount of GluA2 were increased after activity blockade. However, there was no modification of either the total number of synaptic vesicles or the area of presynaptic profiles. Surprisingly, immunocytochemistry showed no change in the mean level of RIM1/2 per terminal but its distribution was modified. Additionally, there was no modification of the mean frequency or amplitude of miniature excitatory postsynaptic currents, but the distribution of amplitudes was modified. These results indicate a specific homeostatic regulation of the synaptic junction. The number of docked vesicles does not seem to be regulated by the amount of RIM1/2. The modification of the distribution, but not the amount, of RIM1/2 may explain the contradiction between the morphological and electrophysiological findings.

Use of metal-based contrast agents for in vivo MR and CT imaging of phagocytic cells in neurological pathologies.

The activation of phagocytic cells is a hallmark of many neurological diseases. Imaging them in their 3-dimensional cerebral environment over time is crucial to better understand their role in disease pathogenesis and to monitor their potential therapeutic effects. Phagocytic cells have the ability to internalize metal-based contrast agents both in vitro and in vivo and can thus be tracked by magnetic resonance imaging (MRI) or computed tomography (CT). In this review article, we summarize the different labelling strategies, contrast agents, and in vivo imaging modalities that can be used to monitor cells with phagocytic activity in the central nervous system using MRI and CT, with a focus on clinical applications. Metal-based nanoparticle contrast agents such as gadolinium, gold and iron are ideal candidates for these applications as they have favourable magnetic and/or radiopaque properties and can be fine-tuned for optimal uptake by phagocytic cells. However, they also come with downsides due to their potential toxicity, especially in the brain where they might accumulate. We therefore conclude our review by discussing the pitfalls, safety and potential for clinical translation of these metal-based neuroimaging techniques. Early results in patients with neuropathologies such as multiple sclerosis, stroke, trauma, cerebral aneurysm and glioblastoma are promising. If the challenges represented by safety issues are overcome, phagocytic cells imaging will be a very valuable tool for studying and understanding the inflammatory response and evaluating treatments that aim at mitigating this response in patients with neurological diseases.

Astrocyte-neuron communication: functional consequences.

Astrocyte-neuron communication has recently been proposed as a potential mechanism participating to synaptic transmission. With the development of this concept and accumulating evidences in favor of a modulation of synaptic transmission by astrocytes, has emerged the term gliotransmission. It refers to the capacity of astrocytes to release various transmitters, such as ATP, glutamate, D-serine, and GABA in the vicinity of synapses. While the cellular mechanisms involved in gliotransmission still need to be better described and, for some, identified, the aim of more and more studies is to determine the role of astrocytes from a functional point of view. This review will summarize the principal studies that have investigated a potential role of astrocytes in the various functions regulated by the brain (sleep, breathing, perception, learning and memory…). This will allow us to highlight the similarities and discrepancies in the signaling pathways involved in the different areas of the brain related to these functions.

Nitric oxide regulates astrocyte maturation in the hippocampus: involvement of NOS2.

Neurons and astrocytes are generated sequentially from radial glia. Once neurogenesis is completed, radial glia starts to differentiate into immature astrocytes. Astrocytes then maturate and change their morphology and electrophysiological properties. Neurotrophic cytokines or bone morphogenetic proteins have been identified as inducers of the developmental switch from neurogenesis to astrogenesis. However, the factors and mechanisms regulating the late differentiation of radial glia and the subsequent astrocyte maturation are poorly described. We used two independent approaches to examine the role of nitric oxide (NO) in the process of astrogenesis and maturation of astrocytes. First using a pharmacological approach, we depleted NO from developing hippocampus by intraventricular injection of a specific scavenger. Then by a genetic approach, we analyzed a nitric oxide synthase-2 (NOS2) knockout mouse. In both models, we found that differentiation of RC2-positive radial glia into late GFAP-positive radial glia was impaired. The cell-fate analysis after incorporation of BrdU demonstrated that astrogenesis was not altered upon NOS2 deficiency. Maturation of astrocytes was assessed by electrophysiological recordings at P7 and functional analysis. In wild type, 20% of astrocytes were immature as shown by their non-linear I/V relationship and high membrane resistance, whereas in NOS2-/- hippocampus, 51% of the astrocytes displayed an immature profile. The reduced branching of astrocytes upon NOS2 deficiency and their low content in connexin-43 further confirmed their immature profile. Our results highlight a novel developmental role of NO and NOS2 in the differentiation of radial glia and the maturation of astrocytes.

Dysregulation of the hippocampal neuronal network by LGI1 auto-antibodies.

LGI1 is a neuronal secreted protein highly expressed in the hippocampus. Epileptic seizures and LGI1 hypo-functions have been found in both ADLTE, a genetic epileptogenic syndrome and LGI1 limbic encephalitis (LE), an autoimmune disease. Studies, based mainly on transgenic mouse models, investigated the function of LGI1 in the CNS and strangely showed that LGI1 loss of function, led to a decreased AMPA-receptors (AMPA-R) expression. Our project intends at better understanding how an altered function of LGI1 leads to epileptic seizures. To reach our goal, we infused mice with LGI1 IgG purified from the serum of patients diagnozed with LGI1 LE. Super resolution imaging revealed that LGI1 IgG reduced AMPA-R expression at the surface of inhibitory and excitatory neurons only in the dentate gyrus of the hippocampus. Complementary electrophysiological approaches indicated that despite reduced AMPA-R expression, LGI1 IgG increased the global hyperexcitability in the hippocampal neuronal network. Decreased AMPA-R expression at inhibitory neurons and the lack of LGI1 IgG effect in presence of GABA antagonist on excitability, led us to conclude that LGI1 function might be essential for the proper functioning of the overall network and orchestrate the imbalance between inhibition and excitation. Our work suggests that LGI1 IgG reduced the inhibitory network activity more significantly than the excitatory network shedding lights on the essential role of the inhibitory network to trigger epileptic seizures in patients with LGI1 LE.

Microglia shape the embryonic development of mammalian respiratory networks.

Microglia, brain-resident macrophages, play key roles during prenatal development in defining neural circuitry function, including ensuring proper synaptic wiring and maintaining homeostasis. Mammalian breathing rhythmogenesis arises from interacting brainstem neural networks that are assembled during embryonic development, but the specific role of microglia in this process remains unknown. Here, we investigated the anatomical and functional consequences of respiratory circuit formation in the absence of microglia. We first established the normal distribution of microglia within the wild-type (WT, Spi1+/+ (Pu.1 WT)) mouse (Mus musculus) brainstem at embryonic ages when the respiratory networks are known to emerge (embryonic day (E) 14.5 for the parafacial respiratory group (epF) and E16.5 for the preBötzinger complex (preBötC)). In transgenic mice depleted of microglia (Spi1-/- (Pu.1 KO) mutant), we performed anatomical staining, calcium imaging, and electrophysiological recordings of neuronal activities in vitro to assess the status of these circuits at their respective times of functional emergence. Spontaneous respiratory-related activity recorded from reduced in vitro preparations showed an abnormally slow rhythm frequency expressed by the epF at E14.5, the preBötC at E16.5, and in the phrenic motor nerves from E16.5 onwards. These deficits were associated with a reduced number of active epF neurons, defects in commissural projections that couple the bilateral preBötC half-centers, and an accompanying decrease in their functional coordination. These abnormalities probably contribute to eventual neonatal death, since plethysmography revealed that E18.5 Spi1-/- embryos are unable to sustain breathing activity ex utero. Our results thus point to a crucial contribution of microglia in the proper establishment of the central respiratory command during embryonic development.

Physiological roles of microglia during development.

In all the species examined thus far, the behavior of microglia during development appears to be highly stereotyped. This reproducibility supports the notion that these cells have a physiological role in development. Microglia are macrophages that migrate from the yolk sac and colonize the central nervous system early during development. The first invading yolk-sac macrophages are highly proliferative and their role has not yet been addressed. At later developmental stages, microglia can be found throughout the brain and tend to preferentially reside at specific locations that are often associated with known developmental processes. Thus, it appears that microglia concentrate in areas of cell death, in proximity of developing blood vessels, in the marginal layer, which contains developing axon fascicles, and in close association with radial glial cells. This review describes the main features of brain colonization by microglia and discusses the possible physiological roles of these cells during development.

Multimodal Imaging with NanoGd Reveals Spatiotemporal Features of Neuroinflammation after Experimental Stroke.

The purpose of this study is to propose and validate a preclinical in vivo magnetic resonance imaging (MRI) tool to monitor neuroinflammation following ischemic stroke, based on injection of a novel multimodal nanoprobe, NanoGd, specifically designed for internalization by phagocytic cells. First, it is verified that NanoGd is efficiently internalized by microglia in vitro. In vivo MRI coupled with intravenous injection of NanoGd in a permanent middle cerebral artery occlusion mouse model results in hypointense signals in the ischemic lesion. In these mice, longitudinal two-photon intravital microscopy shows NanoGd internalization by activated CX3CR1-GFP/+ cells. Ex vivo analysis, including phase contrast imaging with synchrotron X-ray, histochemistry, and transmission electron microscopy corroborate NanoGd accumulation within the ischemic lesion and uptake by immune phagocytic cells. Taken together, these results confirm the potential of NanoGd-enhanced MRI as an imaging biomarker of neuroinflammation at the subacute stage of ischemic stroke. As far as it is known, this work is the first to decipher the working mechanism of MR signals induced by a nanoparticle passively targeted at phagocytic cells by performing intravital microscopy back-to-back with MRI. Furthermore, using a gadolinium-based rather than an iron-based contrast agent raises future perspectives for the development of molecular imaging with emerging computed tomography technologies.

Hybrid multimodal contrast agent for multiscale in vivo investigation of neuroinflammation.

Neuroinflammation is a process common to several brain pathologies. Despites its medical relevance, it still remains poorly understood; there is therefore a need to develop new in vivo preclinical imaging strategies to monitor inflammatory processes longitudinally. We here present the development of a hybrid imaging nanoprobe named NP3, that was specifically designed to get internalized by phagocytic cells and imaged in vivo with MRI and bi-photon microscopy. NP3 is composed of a 16 nm core of gadolinium fluoride (GdF3), coated with bisphosphonate polyethylene glycol (PEG) and functionalized with a Lemke-type fluorophore. It has a hydrodynamic diameter of 28 ± 8 nm and a zeta potential of -42 ± 6 mV. The MR relaxivity ratio at 7 T is r1/r2 = 20; therefore, NP3 is well suited as a T2/T2* contrast agent. In vitro cytotoxicity assessments performed on four human cell lines revealed no toxic effects of NP3. In addition, NP3 is internalized by macrophages in vitro without inducing inflammation or cytotoxicity. In vivo, uptake of NP3 has been observed in the spleen and the liver. NP3 has a prolonged vascular remanence, which is an advantage for macrophage uptake in vivo. The proof-of-concept that NP3 may be used as a contrast agent targeting phagocytic cells is provided in an animal model of ischemic stroke in transgenic CX3CR1-GFP/+ mice using three complementary imaging modalities: MRI, intravital two-photon microscopy and phase contrast imaging with synchrotron X-rays. In summary, NP3 is a promising preclinical tool for the multiscale and multimodal investigation of neuroinflammation.

Role of Glia in the Regulation of Sleep in Health and Disease.

Sleep is a naturally occurring physiological state that is required to sustain physical and mental health. Traditionally viewed as strictly regulated by top-down control mechanisms, sleep is now known to also originate locally. Glial cells are emerging as important contributors to the regulation of sleep-wake cycles, locally and among dedicated neural circuits. A few pioneering studies revealed that astrocytes and microglia may influence sleep pressure, duration as well as intensity, but the precise involvement of these two glial cells in the regulation of sleep remains to be fully addressed, across contexts of health and disease. In this overview article, we will first summarize the literature pertaining to the role of astrocytes and microglia in the regulation of sleep under normal physiological conditions. Afterward, we will discuss the beneficial and deleterious consequences of glia-mediated neuroinflammation, whether it is acute, or chronic and associated with brain diseases, on the regulation of sleep. Sleep disturbances are a main comorbidity in neurodegenerative diseases, and in several brain diseases that include pain, epilepsy, and cancer. Identifying the relationships between glia-mediated neuroinflammation, sleep-wake rhythm disruption and brain diseases may have important implications for the treatment of several disorders. © 2020 American Physiological Society. Compr Physiol 10:687-712, 2020.

Ketamine/xylazine and barbiturates modulate microglial morphology and motility differently in a mouse model.

Microglia, the resident immune cells of the brain, are highly ramified and motile and their morphology is strongly linked to their function. Microglia constantly monitor the brain parenchyma and are crucial for maintaining brain homeostasis and fine-tuning neuronal networks. Besides affecting neurons, anesthetics may have wide-ranging effects mediated by non-neuronal cells and in particular microglia. We thus examined the effect of two commonly used anesthetic agents, ketamine/xylazine and barbiturates, on microglial motility and morphology. A combination of two-photon in vivo imaging and electroencephalography (EEG) recordings in unanesthetized and anesthetized mice as well as automated analysis of ex vivo sections were used to assess morphology and dynamics of microglia. We found that administration of ketamine/xylazine and pentobarbital anesthesia resulted in quite distinct EEG profiles. Both anesthetics reduced microglial motility, but only ketamine/xylazine administration led to reduction of microglial complexity in vivo. The change of cellular dynamics in vivo was associated with a region-dependent reduction of several features of microglial cells ex vivo, such as the complexity index and the ramification length, whereas thiopental altered the size of the cytoplasm. Our results show that anesthetics have considerable effects on neuronal activity and microglial morphodynamics and that barbiturates may be a preferred anesthetic agent for the study of microglial morphology. These findings will undoubtedly raise compelling questions about the functional relevance of anesthetics on microglial cells in neuronal physiology and anesthesia-induced neurotoxicity.

A common molecular basis for membrane docking and functional priming of synaptic vesicles.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes execute synaptic vesicle (SV) fusion. Vesicle fusion is preceded by an obligatory Munc13-dependent priming process that conveys fusion competence to SVs by facilitating SNARE complex assembly. Ultrastructural studies after chemical fixation indicated that vesicle docking to the plasma membrane is independent of Munc13s but these results may be misleading because aldehyde fixatives modify the localization of SVs with respect to the plasma membrane. To reinvestigate the role of Munc13s in vesicle docking, cultured hippocampal slices were immobilized using high-pressure freezing, which circumvents aldehyde artifacts. High-pressure freezing was combined with electron tomography to reach a resolution that allows the characterization of details of SV docking in a close-to-native state. In control slices, docked vesicles are not hemifused with the plasma membrane but linked to it and to dense material at the active zone by small strands. In slice cultures from Munc13-deficient mice, vesicles are not docked to the active zone plasma membrane. These results indicate that SV docking at the plasma membrane and functional priming are respective morphological and physiological manifestations of the same molecular process mediated by SNARE complexes and Munc13s.