Reactive microglia are the major source of tumor necrosis factor alpha and contribute to astrocyte dysfunction and acute seizures in experimental temporal lobe epilepsy.

Extensive microglia reactivity has been well described in human and experimental temporal lobe epilepsy (TLE). To date, however, it is not clear whether and based on which molecular mechanisms microglia contribute to the development and progression of focal epilepsy. Astroglial gap junction coupled networks play an important role in regulating neuronal activity and loss of interastrocytic coupling causally contributes to TLE. Here, we show in the unilateral intracortical kainate (KA) mouse model of TLE that reactive microglia are primary producers of tumor necrosis factor (TNF)α and contribute to astrocyte dysfunction and severity of status epilepticus (SE). Immunohistochemical analyses revealed pronounced and persistent microglia reactivity, which already started 4 h after KA-induced SE. Partial depletion of microglia using a colony stimulating factor 1 receptor inhibitor prevented early astrocyte uncoupling and attenuated the severity of SE, but increased the mortality of epileptic mice following surgery. Using microglia-specific inducible TNFα knockout mice we identified microglia as the major source of TNFα during early epileptogenesis. Importantly, microglia-specific TNFα knockout prevented SE-induced gap junction uncoupling in astrocytes. Continuous telemetric EEG recordings revealed that during the first 4 weeks after SE induction, microglial TNFα did not significantly contribute to spontaneous generalized seizure activity. Moreover, the absence of microglial TNFα did not affect the development of hippocampal sclerosis but attenuated gliosis. Taken together, these data implicate reactive microglia in astrocyte dysfunction and network hyperexcitability after an epileptogenic insult.

PIEZO1 expression at the glio-vascular unit adjusts to neuroinflammation in seizure conditions.

Mechanosensors are emerging players responding to hemodynamic and physical inputs. Their significance in the central nervous system remains relatively uncharted. Using human-derived brain specimens or cells and a pre-clinical model of mesio-temporal lobe epilepsy (MTLE), we examined how the mRNA levels of the mechanosensitive channel PIEZO1 adjust to disease-associated pro-inflammatory trajectories. In brain tissue micro-punches obtained from 18 drug-resistant MTLE patients, PIEZO1 expression positively correlated with pro-inflammatory biomarkers TNFα, IL-1ß, and NF-kB in the epileptogenic hippocampus compared to the adjacent amygdala and temporal cortex tissues. In an experimental MTLE model, hippocampal Piezo1 and cytokine expression levels were increased post-status epilepticus (SE) and during epileptogenesis. Piezo1 expression positively correlated with Tnfα, Il1ß, and Nf-kb in the hippocampal foci. Next, by combining RNAscope with immunohistochemistry, we identified Piezo1 in glio-vascular cells. Post-SE and during epileptogenesis, ameboid IBA1 microglia, hypertrophic GFAP astrocytes, and damaged NG2DsRed pericytes exhibited time-dependent patterns of increased Piezo1 expression. Digital droplet PCR analysis confirmed the Piezo1 trajectory in isolated hippocampal microvessels in the ipsi and contralateral hippocampi. The combined examinations performed in this model showed Piezo1 expression returning towards basal levels after the epileptogenesis-associated peak inflammation. From these associations, we next asked whether pro-inflammatory players directly regulate PIEZO1 expression. We used human-derived brain cells and confirmed that endothelium, astrocytes, and pericytes expressed PIEZO1. Exposure to human recombinant TNFα or IL1ß upregulated NF-kB in all cells. Furthermore, TNFα induced PIEZO1 expression in a dose and time-dependent manner, primarily in astrocytes. This exploratory study describes a spatiotemporal dialogue between PIEZO1 brain cell-mechanobiology and neuro-inflammatory cell remodeling. The precise functional mechanisms regulating this interplay in disease conditions warrant further investigation.

Restoring glutamate receptosome dynamics at synapses rescues autism-like deficits in Shank3-deficient mice.

Shank3 monogenic mutations lead to autism spectrum disorders (ASD). Shank3 is part of the glutamate receptosome that physically links ionotropic NMDA receptors to metabotropic mGlu5 receptors through interactions with scaffolding proteins PSD95-GKAP-Shank3-Homer. A main physiological function of the glutamate receptosome is to control NMDA synaptic function that is required for plasticity induction. Intact glutamate receptosome supports glutamate receptors activation and plasticity induction, while glutamate receptosome disruption blocks receptors activity, preventing the induction of subsequent plasticity. Despite possible impact on metaplasticity and cognitive behaviors, scaffold interaction dynamics and their consequences are poorly defined. Here, we used mGlu5-Homer interaction as a biosensor of glutamate receptosome integrity to report changes in synapse availability for plasticity induction. Combining BRET imaging and electrophysiology, we show that a transient neuronal depolarization inducing NMDA-dependent plasticity disrupts glutamate receptosome in a long-lasting manner at synapses and activates signaling pathways required for the expression of the initiated neuronal plasticity, such as ERK and mTOR pathways. Glutamate receptosome disruption also decreases the NMDA/AMPA ratio, freezing the sensitivity of the synapse to subsequent changes of neuronal activity. These data show the importance of a fine-tuning of protein-protein interactions within glutamate receptosome, driven by changes of neuronal activity, to control plasticity. In a mouse model of ASD, a truncated mutant form of Shank3 prevents the integrity of the glutamate receptosome. These mice display altered plasticity, anxiety-like, and stereotyped behaviors. Interestingly, repairing the integrity of glutamate receptosome and its sensitivity to the neuronal activity rescued synaptic transmission, plasticity, and some behavioral traits of Shank3∆C mice. Altogether, our findings characterize mechanisms by which Shank3 mutations cause ASD and highlight scaffold dynamics as new therapeutic target.

Seizure activity triggers tau hyperphosphorylation and amyloidogenic pathways.

OBJECTIVE: Although epilepsies and neurodegenerative disorders show pathophysiological similarities, their direct functional associations are unclear. Here, we tested the hypothesis that experimental seizures can induce tau hyperphosphorylation and amyloidogenic modifications over time, with intersections with neuroinflammation. METHODS: We used a model of mesial temporal lobe epilepsy (MTLE) where unilateral intrahippocampal injection of kainic acid (KA) in C57BL/6 mice elicits epileptogenesis and spontaneous focal seizures. We used a model of generalized status epilepticus (SE) obtained by intraperitoneal KA injection in C57BL/6 mice. We performed analyses and cross-comparisons according to a schedule of 72 h, 1 week, and 8 weeks after KA injection. RESULTS: In experimental MTLE, we show AT100, PHF1, and CP13 tau hyperphosphorylation during epileptogenesis (72 h-1 week) and long-term (8 weeks) during spontaneous seizures in the ipsilateral hippocampi, the epileptogenic zone. These pathological modifications extended to the contralateral hippocampus, a seizure propagating zone with no histological lesion or sclerosis. Two kinases, Cdk5 and GSK3ß, implicated in the pathological phosphorylation of tau, were activated. In this MTLE model, the induction of the amyloidogenic pathway (APP, C99, BACE1) was prominent and long-lasting in the epileptogenic zone. These Alzheimer's disease (AD)-relevant markers, established during seizure progression and recurrence, reciprocated an enduring glial (GFAP, Iba1) inflammation and the inadequate activation of the endogenous, anti-inflammatory, glucocorticoid receptor system. By contrast, a generalized SE episode provoked a predominantly transient induction of tau hyperphosphorylation and amyloidogenic markers in the hippocampus, along with resolving inflammation. Finally, we identified overlapping profiles of long-term hippocampal tau hyperphosphorylation by comparing MTLE to J20 mice, the latter a model relevant to AD. SIGNIFICANCE: MTLE and a generalized SE prompt persistent and varying tau hyperphosphorylation or amyloidogenic modifications in the hippocampus. In MTLE, an AD-relevant molecular trajectory intertwines with neuroinflammation, spatiotemporally involving epileptogenic and nonlesional seizure propagating zones.

Dietary fat exacerbates postprandial hypothalamic inflammation involving glial fibrillary acidic protein-positive cells and microglia in male mice.

In humans, obesity is associated with brain inflammation, glial reactivity, and immune cells infiltration. Studies in rodents have shown that glial reactivity occurs within 24 hr of high-fat diet (HFD) consumption, long before obesity development, and takes place mainly in the hypothalamus (HT), a crucial brain structure for controlling body weight. Here, we sought to characterize the postprandial HT inflammatory response to 1, 3, and 6 hr of exposure to either a standard diet (SD) or HFD. HFD exposure increased gene expression of astrocyte and microglial markers (glial fibrillary acidic protein [GFAP] and Iba1, respectively) compared to SD-treated mice and induced morphological modifications of microglial cells in HT. This remodeling was associated with higher expression of inflammatory genes and differential regulation of hypothalamic neuropeptides involved in energy balance regulation. DREADD and PLX5622 technologies, used to modulate GFAP-positive or microglial cells activity, respectively, showed that both glial cell types are involved in hypothalamic postprandial inflammation, with their own specific kinetics and reactiveness to ingested foods. Thus, recurrent exacerbated postprandial inflammation in the brain might promote obesity and needs to be characterized to address this worldwide crisis.

Microglia proliferation plays distinct roles in acquired epilepsy depending on disease stages.

OBJECTIVE: Microgliosis occurs in animal models of acquired epilepsy and in patients. It includes cell proliferation that is associated with seizure frequency and decreased neuronal cells in human epilepsy. The role of microglia proliferation in the development of acquired epilepsy is unknown; thus, we examined its contribution to spontaneous seizure, neurodegeneration, and cognitive deficits in different disease phases. METHODS: We used a model of acquired epilepsy triggered by intra-amygdala kainic acid in C57BL6N adult male mice. Mice were electroencephalographically (EEG) monitored (24/7) during status epilepticus and in early and chronic disease. Microglia proliferation was blocked by GW2580, a selective CSF1 receptor inhibitor, supplemented in the diet for 21 days from status epilepticus onset. Then, mice were returned to placebo diet until experiment completion. Control mice were exposed to status epilepticus and fed with placebo diet. Experimental mice were tested in the novel object recognition test (NORT) and in Barnes maze, and compared to control and sham mice. At the end of the behavioral test, mice were killed for brain histopathological analysis. Additionally, seizure baseline was monitored in chronic epileptic mice, then mice were fed for 14 days with GW2580 or placebo diet under 24/7 EEG recording. RESULTS: GW2580 prevented microglia proliferation in mice undergoing epilepsy, whereas it did not affect microglia or basal excitatory neurotransmission in the hippocampus of naive mice. Mice with occluded microglia proliferation during early disease development underwent status epilepticus and subsequent epilepsy similar to placebo diet mice, and were similarly impaired in NORT, with improvement in Barnes maze. GW2580-treated mice displayed neuroprotection in the hippocampus. In contrast, blockade of microglia proliferation in chronic epileptic mice resulted in spontaneous seizure reduction versus placebo mice. SIGNIFICANCE: Microglia proliferation during early disease contributes to neurodegeneration, whereas in late chronic disease it contributes to seizures. Timely pharmacological interference with microglia proliferation may offer a potential target for improving disease outcomes.

Role of astrocyte purinergic signaling in epilepsy.

Epilepsy is characterized by unpredictable recurrent seizures resulting from hypersynchronous discharges from neuron assemblies. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability. Purinergic signaling is central in neuron-glia and glia-glia interactions and dysfunctions in communication pathways involving purinergic receptors have been reported in various CNS pathologies, such as Alzheimer disease, stroke, major depression, schizophrenia, and epilepsy. In the present review we will first discuss the mechanisms by which astrocytes influence neuronal activity. We will then review in more details recent evidence indicating that dysregulation of astrocyte purinergic signaling actively contributes to the appearance of abnormal neuronal activity in epilepsy.

The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

Immune changes occur in experimental and clinical epilepsy. Here, we tested the hypothesis that during epileptogenesis and spontaneous recurrent seizures (SRS) an impairment of the endogenous anti-inflammatory pathway glucocorticoid receptor (GR)-annexin A1 (ANXA1) occurs. By administrating exogenous ANXA1, we studied whether pharmacological potentiation of the anti-inflammatory response modifies seizure activity and pathophysiology. We used an in vivo model of temporal lobe epilepsy based on intrahippocampal kainic acid (KA) injection. Video-electroencephalography, molecular biology analyses on brain and peripheral blood samples, and pharmacological investigations were performed in this model. Human epileptic cortices presenting type II focal cortical dysplasia (IIa and b), hippocampi with or without hippocampal sclerosis (HS), and available controls were used to study ANXA1 expression. A decrease of phosphorylated (phospho-) GR and phospho-GR/tot-GR protein expression occurred in the hippocampus during epileptogenesis. Downstream to GR, the anti-inflammatory protein ANXA1 remained at baseline levels while inflammation installed and endured. In peripheral blood, ANXA1 and corticosterone levels showed no significant modifications during disease progression except for an early and transient increase poststatus epilepticus. These results indicate inadequate ANXA1 engagement over time and in these experimental conditions. By analyzing human brain specimens, we found that where significant inflammation exists, the pattern of ANXA1 immunoreactivity was abnormal because the typical perivascular ANXA1 immunoreactivity was reduced. We next asked whether potentiation of the endogenous anti-inflammatory mechanism by ANXA1 administration modifies the disease pathophysiology. Although with varying efficacy, administration of exogenous ANXA1 somewhat reduced the time spent in seizure activity as compared to saline. These results indicate that the anti-inflammatory GR-ANXA1 pathway is defective during experimental seizure progression. The prospect of pharmacologically restoring or potentiating this endogenous anti-inflammatory mechanism as an add-on therapeutic strategy for specific forms of epilepsy is proposed.-Zub, E., Canet, G., Garbelli, R., Blaquiere, M., Rossini, L., Pastori, C., Sheikh, M., Reutelingsperger, C., Klement, W., de Bock, F., Audinat, E., Givalois, L., Solito, E., Marchi, N. The GR-ANXA1 pathway is a pathological player and a candidate target in epilepsy.

A pericyte-glia scarring develops at the leaky capillaries in the hippocampus during seizure activity.

OBJECTIVE: Inflammatory cerebrovascular damage occurs in epilepsy. Here, we tested the hypothesis that a pericyte-glia scar forms around the outer wall of hippocampal capillaries in a model of temporal lobe epilepsy associated with hippocampal sclerosis. We studied the participation of stromal cells expressing platelet-derived growth factor receptor beta (PDGFRß) and extracellular matrix modifications to the perivascular scar during epileptogenesis. METHODS: We used NG2DsRed/C57BL6 mice and induced status epilepticus (SE) followed by epileptogenesis and spontaneous recurrent seizures (SRS) by means of unilateral intrahippocampal injection of kainic acid (KA). For pharmacological assessment, we used organotypic hippocampal cultures (OHCs) where ictal electrographic activity was elicited by KA or bicuculline. RESULTS: NG2DsRed pericytes, GFAP astroglia, and IBA1 microglia are reactive and converge to form a pericapillary multicellular scar in the CA hippocampal regions during epileptogenesis and at SRS. The capillaries are leaky as indicated by fluorescein entering the parenchyma from the peripheral blood. Concomitantly, PDGFRß transcript and protein levels were significantly increased. Within the regional scar, a fibrotic-like PDGFRß mesh developed around the capillaries, peaking at 1 week post-SE and regressing, but not resolving, at SRS. Abnormal distribution or accumulation of extracellular matrix collagens III/IV occurred in the CA regions during seizure progression. PDGFRß/DAPI cells were in direct contact with or adjacent to the damaged NG2DsRed pericytes at the capillary interface, consistent with the notion of stromal cell reactivity or fibroblast formation. Inducing electrographic activity in OHCs was sufficient to augment PDGFRß reactivity around the capillaries. The latter effect was pharmacologically mimicked by treating OHCs with the PDGFRß agonist PDGF-BB and it was diminished by the PDGFRß inhibitor imatinib. SIGNIFICANCE: The reported multicellular activation and scar are traits of perivascular inflammation and hippocampal sclerosis in experimental epilepsy, with an implication for neurovascular dysfunction. Modulation of PDGFRß could be exploited to target inflammation in this chronic disease setting.

Blocking TNFα-driven astrocyte purinergic signaling restores normal synaptic activity during epileptogenesis.

Epilepsy is characterized by unpredictable recurrent seizures resulting from abnormal neuronal excitability. Increasing evidence indicates that aberrant astrocyte signaling to neurons plays an important role in driving the network hyperexcitability, but the underlying mechanism that alters glial signaling in epilepsy remains unknown. Increase in glutamate release by astrocytes participates in the onset and progression of seizures. Epileptic seizures are also accompanied by increase of tumor necrosis factor alpha (TNFα), a cytokine involved in the regulation of astrocyte glutamate release. Here we tested whether TNFα controls abnormal astrocyte glutamate signaling in epilepsy and through which mechanism. Combining Ca2+ imaging, optogenetics, and electrophysiology, we report that TNFα triggers a Ca2+ -dependent glutamate release from astrocytes that boosts excitatory synaptic activity in the hippocampus through a mechanism involving autocrine activation of P2Y1 receptors by astrocyte-derived ATP/ADP. In a mouse model of temporal lobe epilepsy, such TNFα-driven astrocytic purinergic signaling is permanently active, promotes glial glutamate release, and drives abnormal synaptic activity in the hippocampus. Blocking this pathway by inhibiting P2Y1 receptors restores normal excitatory synaptic activity in the inflamed hippocampus. Our findings indicate that targeting the coupling of TNFα with astrocyte purinergic signaling may be a therapeutic strategy for reducing glial glutamate release and normalizing synaptic activity in epilepsy.

Purinergic signaling in epilepsy.

Until recently, analysis of the mechanisms underlying epilepsy was centered on neuron dysfunctions. Accordingly, most of the available pharmacological treatments aim at reducing neuronal excitation or at potentiating neuronal inhibition. These therapeutic options can lead to obvious secondary effects, and, moreover, seizures cannot be controlled by any known medication in one-third of the patients. A purely neurocentric view of brain functions and dysfunctions has been seriously questioned during the past 2 decades because of the accumulation of experimental data showing the functional importance of reciprocal interactions between glial cells and neurons. In the case of epilepsy, our current knowledge of the human disease and analysis of animal models clearly favor the involvement of astrocytes and microglial cells during the progression of the disease, including at very early stages, opening the way to the identification of new therapeutic targets. Purinergic signaling is a fundamental feature of neuron-glia interactions, and increasing evidence indicates that modifications of this pathway contribute to the functional remodeling of the epileptic brain. This Review discusses the recent experimental results indicating the roles of astrocytic and microglial P2X and P2Y receptors in epilepsy. © 2016 Wiley Periodicals, Inc.

Postnatal down-regulation of the GABAA receptor γ2 subunit in neocortical NG2 cells accompanies synaptic-to-extrasynaptic switch in the GABAergic transmission mode.

NG2 cells, a main pool of glial progenitors, express γ-aminobutyric acid A (GABA(A)) receptors (GABA(A)Rs), the functional and molecular properties of which are largely unknown. We recently reported that transmission between GABAergic interneurons and NG2 cells drastically changes during development of the somatosensory cortex, switching from synaptic to extrasynaptic communication. Since synaptic and extrasynaptic GABA(A)Rs of neurons differ in their subunit composition, we hypothesize that GABA(A)Rs of NG2 cells undergo molecular changes during cortical development accompanying the switch of transmission modes. Single-cell RT-PCR and the effects of zolpidem and α5IA on evoked GABAergic currents reveal the predominance of functional α1- and α5-containing GABA(A)Rs at interneuron-NG2 cell synapses in the second postnatal week, while the α5 expression declines later in development when responses are exclusively extrasynaptic. Importantly, pharmacological and molecular analyses demonstrate that γ2, a subunit contributing to the clustering of GABA(A)Rs at postsynaptic sites in neurons, is down-regulated in NG2 cells in a cell type-specific manner in concomitance with the decline of synaptic activity and the switch of transmission mode. In keeping with the synaptic nature of γ2 in neurons, the down-regulation of this subunit is an important molecular hallmark of the change of transmission modes between interneurons and NG2 cells during development.

Fractalkine Signaling and Microglia Functions in the Developing Brain.

Microglial cells are the resident macrophages of the central nervous system (CNS). Besides their classical roles in pathological conditions, these immune cells also dynamically interact with neurons and influence their structure and function in physiological conditions. The neuronal chemokine fractalkine and its microglial receptor CX3CR1 are one important signaling pathway involved in these reciprocal interactions. In the present review, we will discuss recent evidence indicating that fractalkine signaling also determines several functions of microglial cells during normal CNS development. It has been known for a decade that microglial cells influence the neuronal death that normally occurs during CNS development. Surprisingly, recent evidence indicates that they can also support survival of developing neurons, control axon outgrowth, and laminar positioning of subsets of interneurons in the forebrain. Moreover, microglial cells influence the maturation of synaptic circuits at early postnatal stages: their phagocytic activity allows them to eliminate inappropriate synapses and they can also influence the functional expression of synaptic proteins by releasing mediators. Fractalkine signaling controls these functions of microglial cells in part by regulating their timely recruitment at sites of developing synapses. Finally, on-going research suggests that this signaling pathway is also a key player in neurodevelopmental disorders.

Paradoxical effects of minocycline in the developing mouse somatosensory cortex.

Minocycline, a tetracycline derivative, is known to exert neuroprotective effects unrelated to its antimicrobial action. In particular, minocycline prevents microglial activation in pathological conditions and consequently reduces the production of proinflammatory factors contributing to the propagation of diseases. Accumulative evidence indicates that microglial cells contribute to the maturation of neuronal and synaptic networks during the normal development of the central nervous system (CNS) and that perinatal inflammation is a known risk factor for brain lesions. Although minocycline has been used to infer microglia functions during development, mechanisms by which this tetracycline derivative affect the immature CNS have not been analyzed in detail. In this study, we demonstrate that minocycline administration during the first postnatal week of development has paradoxical effects on microglia phenotype and on neuronal survival in the mouse somatosensory cortex. Using a combination of immunohistochemistry and electrophysiology, we show that intraperitoneal injections of minocycline between postnatal days 6 and 8 affect distribution, morphology, and functional properties of microglia cells of the whisker-related barrel cortex, leading to the development of a phenotype resembling that of microglia activated in pathological conditions. Minocyline also induced a massive cell death that developed faster than changes in microglia phenotype, suggesting that the latter is a consequence of the former. Finally, cell death and microglial activation were not observed when minocycline treatment was postponed by only 2 days (i.e., between postnatal days 8 and 10). These observations call into question the use of tetracycline derivatives during CNS development to study microglia or to reduce perinatal inflammation.

[Microglia: immune cells sculpting and controlling neuronal synapses]. / La microglie : des cellules immunitaires qui sculptent et contrôlent les synapses neuronales.

The development of new animal models and functional analysis methods has dramatically changed our understanding of the physiology of microglial cells. These cells which are the resident macrophages of central nervous system have the ability to adapt rapidly to subtle changes of their environment. Recent findings indicate in particular that they can establish contacts with neuronal synapses that they can eliminate and modulate by releasing specific mediators. Here we review the experimental observations that have revealed the occurrence of these interactions not only in pathological conditions but also in the healthy brain and in particular during normal brain development. The discovery of bi-directional communications between synapses and microglia sheds a new light on our understanding of brain functioning and should allow a better understanding of brain functioning and of interactions between immune and nervous systems.

Continuous low-level dietary exposure to glyphosate elicits dose and sex-dependent synaptic and microglial adaptations in the rodent brain.

Prolonged exposure to low levels of dietary contaminants is a context in modern life that could alter organ physiology gradually. Here, we aimed to investigate the impact of continuous exposure to acceptable daily intake (ADI) and non-observable adverse effect level (NOAEL) of glyphosate from gestation to adulthood using C57BL/6J mice and incorporating these levels into their food pellets. From adulthood, we analyzed neurophysiological and neuro-glia cellular adaptations in male and female animals. Using ex-vivo hippocampal slice electrophysiology, we found a reduced efficacy of Schaffer collateral-to-CA1 excitatory synapses in glyphosate-exposed dietary conditions, with ADI and NOAEL dose-dependent effects. Short-term facilitation of excitatory synaptic transmission was specifically increased in NOAEL conditions, with a predominant influence in males, suggesting a reduced probability of neurotransmitter release. Long-term synaptic potentiation (LTP) was decreased in NOAEL-exposed mice. Next, we explore whether these neurophysiological modifications are associated with neuro-glia changes in the somatosensory cortex and hippocampus. High-resolution confocal microscopy analyses unveil a dose-dependent increased density of excitatory Vglut1+ Homer1+ synapses. Microglial Iba1+ cells displayed a shortening of their ramifications, a sign of cellular reactivity that was more pronounced in males at NOAEL levels. The morphology of GFAP+ astrocytes was generally not modified. Finally, we asked whether mouse-specific cross-correlations exist among all data sets generated. This examination included the novel object recognition (NOR) test performed before ex vivo functional and immunohistochemical examinations. We report a negative linear regression between the number of synapses and NOR or LTP maintenance when plotting ADI and NOAEL datasets. These results outline synaptic and microglial cell adaptations resulting from prenatal and continuous dietary low levels of glyphosate, discernible in, but not limited to, adult males exposed to the NOAEL. We discuss the potential significance of these findings to real-world consumer situations and long-term brain resilience.

Deficiency of the microglial receptor CX3CR1 impairs postnatal functional development of thalamocortical synapses in the barrel cortex.

Accumulative evidence indicates that microglial cells influence the normal development of brain synapses. Yet, the mechanisms by which these immune cells target maturating synapses and influence their functional development at early postnatal stages remain poorly understood. Here, we analyzed the role of CX3CR1, a microglial receptor activated by the neuronal chemokine CX3CL1 (or fractalkine) which controls key functions of microglial cells. In the whisker-related barrel field of the mouse somatosensory cortex, we show that the recruitment of microglia to the sites where developing thalamocortical synapses are concentrated (i.e., the barrel centers) occurs only after postnatal day 5 and is controlled by the fractalkine/CX3CR1 signaling pathway. Indeed, at this developmental stage fractalkine is overexpressed within the barrels and CX3CR1 deficiency delays microglial cell recruitment into the barrel centers. Functional analysis of thalamocortical synapses shows that CX3CR1 deficiency also delays the functional maturation of postsynaptic glutamate receptors which normally occurs at these synapses between the first and second postnatal week. These results show that reciprocal interactions between neurons and microglial cells control the functional maturation of cortical synapses.

Adaptive phenotype of microglial cells during the normal postnatal development of the somatosensory "Barrel" cortex.

Accumulative evidence indicates that microglial cells influence the normal development of central nervous system (CNS) synapses. Yet, the functional properties of microglia in relation with synapse development remain unclear. We recently showed that in layer 4 of the whisker-related barrel field of the mouse somatosensory cortex, microglial cells are recruited only after postnatal day (P)5 in the center of the barrels where thalamo-cortical synapses are concentrated and begin their maturation. In the present study, we analyzed the phenotype of microglia during this developmental process. We show that between P5 and P7 microglial cells acquire a more ramified morphology with a smaller soma, they express classical markers of microglia (Iba1, CD11b, and CD68) but never markers of activation (Mac-2 and MHCII) and rarely the proliferation marker Ki67. Electrophysiological recordings in acute cortical slices showed that at P5 a proportion of layer 4 microglia transiently express voltage-dependant potassium currents of the delayed rectifier family, mostly mediated by Kv1.3 subunits, which are usually expressed by activated microglia under pathological conditions. This proportion of cells with rectifying properties doubles between P5 and P6, in concomitance with the beginning of microglia invasion of the barrel centers. Finally, analysis of the responses mediated by purinergic receptors indicated that a higher percentage of rectifying microglia expressed functional P2Y6 and P2Y12 receptors, as compared with nonrectifying cells, whereas all cells expressed functional P2X7 receptors. Our results indicate that during normal cortical development distinct microglia properties mature differentially, some of them being exquisitely influenced by the local environment of the maturating neuronal network.

Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus.

Within the central nervous system, functions of the ATP-gated receptor-channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)-induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R-deficient mice, behavioral responses during KA-induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage-dependent potassium channels were impaired in P2X4R-deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity-dependent degenerative processes, the CA1 area was partially protected from SE-induced neuronal death in P2X4R-deficient mice compared with wild-type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation.

Therapeutic Potential of Astrocyte Purinergic Signalling in Epilepsy and Multiple Sclerosis.

Epilepsy and multiple sclerosis (MS), two of the most common neurological diseases, are characterized by the establishment of inflammatory environment in the central nervous system that drives disease progression and impacts on neurodegeneration. Current therapeutic approaches in the treatments of epilepsy and MS are targeting neuronal activity and immune cell response, respectively. However, the lack of fully efficient responses to the available treatments obviously shows the need to search for novel therapeutic candidates that will not exclusively target neurons or immune cells. Accumulating knowledge on epilepsy and MS in humans and analysis of relevant animal models, reveals that astrocytes are promising therapeutic candidates to target as they participate in the modulation of the neuroinflammatory response in both diseases from the initial stages and may play an important role in their development. Indeed, astrocytes respond to reactive immune cells and contribute to the neuronal hyperactivity in the inflamed brain. Mechanistically, these astrocytic cell to cell interactions are fundamentally mediated by the purinergic signalling and involve metabotropic P2Y1 receptors in case of astrocyte interactions with neurons, while ionotropic P2X7 receptors are mainly involved in astrocyte interactions with autoreactive immune cells. Herein, we review the potential of targeting astrocytic purinergic signalling mediated by P2Y1 and P2X7 receptors to develop novel approaches for treatments of epilepsy and MS at very early stages.