Brain stars take the lead during critical periods of early postnatal brain development: relevance of astrocytes in health and mental disorders.

In the brain, astrocytes regulate shape and functions of the synaptic and vascular compartments through a variety of released factors and membrane-bound proteins. An imbalanced astrocyte activity can therefore have drastic negative impacts on brain development, leading to the onset of severe pathologies. Clinical and pre-clinical studies show alterations in astrocyte cell number, morphology, molecular makeup and astrocyte-dependent processes in different affected brain regions in neurodevelopmental (ND) and neuropsychiatric (NP) disorders. Astrocytes proliferate, differentiate and mature during the critical period of early postnatal brain development, a time window of elevated glia-dependent regulation of a proper balance between synapse formation/elimination, which is pivotal in refining synaptic connectivity. Therefore, any intrinsic and/or extrinsic factors altering these processes during the critical period may result in an aberrant synaptic remodeling and onset of mental disorders. The peculiar bridging position of astrocytes between synaptic and vascular compartments further allows them to "compute" the brain state and consequently secrete factors in the bloodstream, which may serve as diagnostic biomarkers of distinct healthy or disease conditions. Here, we collect recent advancements regarding astrogenesis and astrocyte-mediated regulation of neuronal network remodeling during early postnatal critical periods of brain development, focusing on synapse elimination. We then propose alternative hypotheses for an involvement of aberrancies in these processes in the onset of ND and NP disorders. In light of the well-known differential prevalence of certain brain disorders between males and females, we also discuss putative sex-dependent influences on these neurodevelopmental events. From a translational perspective, understanding age- and sex-dependent astrocyte-specific molecular and functional changes may help to identify biomarkers of distinct cellular (dys)functions in health and disease, favouring the development of diagnostic tools or the selection of tailored treatment options for male/female patients.

Early Age- and Sex-Dependent Regulation of Astrocyte-Mediated Glutamatergic Synapse Elimination in the Rat Prefrontal Cortex: Establishing an Organotypic Brain Slice Culture Investigating Tool.

Clinical and pre-clinical studies of neuropsychiatric (NP) disorders show altered astrocyte properties and synaptic networks. These are refined during early postnatal developmental (PND) stages. Thus, investigating early brain maturational trajectories is essential to understand NP disorders. However, animal experiments are highly time-/resource-consuming, thereby calling for alternative methodological approaches. The function of MEGF10 in astrocyte-mediated synapse elimination (pruning) is crucial to refine neuronal networks during development and adulthood. To investigate the impact of MEGF10 during PND in the rat prefrontal cortex (PFC) and its putative role in brain disorders, we established and validated an organotypic brain slice culture (OBSC) system. Using Western blot, we characterized the expression of MEGF10 and the synaptic markers synaptophysin and PSD95 in the cortex of developing pups. We then combined immunofluorescent-immunohistochemistry with Imaris-supported 3D analysis to compare age- and sex-dependent astrocyte-mediated pruning within the PFC in pups and OBSCs. We thereby validated this system to investigate age-dependent astrocyte-mediated changes in pruning during PND. However, further optimizations are required to use OBSCs for revealing sex-dependent differences. In conclusion, OBSCs offer a valid alternative to study physiological astrocyte-mediated synaptic remodeling during PND and might be exploited to investigate the pathomechanisms of brain disorders with aberrant synaptic development.

Neuroinflammation and psychiatric disorders: Relevance of C1q, translocator protein (18 kDa) (TSPO), and neurosteroids.

There is increasing evidence that neuroinflammatory processes may play a role in the pathophysiology of psychiatric disorders. Recently, the complement protein C1q and the translocator protein (18 kDa) (TSPO) have attracted considerable interest in this context. C1q is a small molecule which is involved into synaptic pruning mechanisms, increases during ageing and may contribute to neurodegenerative disorders. TSPO is a transmembrane channel protein and mediates numerous biological functions such as bioenergetics and steroid synthesis. Meanwhile, there is evidence that both C1q and TSPO may be elevated in psychiatric disorders, e.g. major depression. Moreover, preclinical and first clinical studies suggest that TSPO ligands may exert antidepressant and anxiolytic properties by promoting endogenous neurosteroid synthesis. In addition, certain neurosteroids, e.g. allopregnanolone, are potent positive allosteric modulators of GABAA receptors and their composition is altered in depression and anxiety disorders. Recently, neurosteroid compounds such as brexanolone or zuranolone have been reported to reduce depressive and anxiety symptoms in postpartum depression and major depressive disorder. In conclusion, compounds enhancing GABAergic neurotransmission such as neurosteroids and TSPO ligands, which also may exert anti-inflammatory properties in concert with immunomodulators such as C1q may open new avenues for the treatment of psychiatric disorders.

Morphological Features of Astrocytes in Health and Neuropsychiatric Disorders.

Astroglial cells actively partner with several cell types to regulate the arrangement of neuronal circuits both in the developing and adult brain. Morphological features of astroglial cells strongly impact their functional interactions, thereby supporting the hypothesis that aberrancies in glial morphology may trigger the onset of neuropsychiatric disorders. Thus, understanding the factors which modulate astroglial shapes and the development of tools to examine them may help to gain valuable insights about the role of astroglia in physiological and pathological brain states.Here, we present a collection of representative review and original articles describing the major morphological features which define different subtypes of glial cells and emphasize a high degree of heterogeneity typical of these cell types, besides neurons. Furthermore, we offer an overview about first in vitro and in vivo evidences, which highlight an altered morphology of glial cells in brains of psychiatric patients and animal models of neuropsychiatric disorders.

Microglia react to partner loss in a sex- and brain site-specific manner in prairie voles.

Positive social relationships are paramount for the survival of mammals and beneficial for mental and physical health, buffer against stressors, and even promote appropriate immune system functioning. By contrast, impaired social relationships, social isolation, or the loss of a bonded partner lead to aggravated physical and mental health. For example, in humans partner loss is detrimental for the functioning of the immune system and heightens the susceptibility for the development of post-traumatic stress disorders, anxiety disorders, and major depressive disorders. To understand potential underlying mechanisms, the monogamous prairie vole can provide important insights. In the present study, we separated pair bonded male and female prairie voles after five days of co-housing, subjected them to the forced swim test on the fourth day following separation, and studied their microglia morphology and activation in specific brain regions. Partner loss increased passive stress-coping in male, but not female, prairie voles. Moreover, partner loss was associated with microglial priming within the parvocellular region of the paraventricular nucleus of the hypothalamus (PVN) in male prairie voles, whereas in female prairie voles the morphological activation within the whole PVN and the prelimbic cortex (PrL) was decreased, marked by a shift towards ramified microglial morphology. Expression of the immediate early protein c-Fos following partner loss was changed within the PrL of male, but not female, prairie voles. However, the loss of a partner did not affect the investigated aspects of the peripheral immune response. These data suggest a potential sex-dependent mechanism for the regulation of microglial activity following the loss of a partner, which might contribute to the observed differences in passive stress-coping. This study furthers our understanding of the effects of partner loss and its short-term impact on the CNS as well as the CNS immune system and the peripheral innate immune system in both male and female prairie voles.

Astrocytic-neuronal crosstalk gets jammed: Alternative perspectives on the onset of neuropsychiatric disorders.

Investigating interactions of glia cells and synapses during development and in adulthood is the focus of several research programmes which aim at understanding the neurobiology of brain physiological and pathological processes. Both glia-specific released and membrane-bound proteins play essential roles in the development, maintenance and functionality of synaptic connections. Alterations in synaptic contacts in specific brain areas are hallmarks of several brain diseases, such as major depressive disorder, autism spectrum disorder and schizophrenia. Thus, a deeper knowledge about putative astrocyte dysfunctions which might affect the synaptic compartment is warranted to improve treatment options. Here, we present the latest advances about the role of glia cells in orchestrating the arrangement of synapses and neuronal networks in physiological and pathological states. We specifically focus on the role of astrocytes in the phagocytosis of neuronal synapses as a novel mechanism which drives the refinement of neuronal circuits and might be affected in pathological conditions. Finally, we propose this astrocyte-dependent mechanism as a putative alternative target of pharmacological interventions for the treatment of brain disorders.

GDF15 promotes simultaneous astrocyte remodeling and tight junction strengthening at the blood-brain barrier.

Perivascular astrocyte processes (PAP) surround cerebral endothelial cells (ECs) and modulate the strengthening of tight junctions to influence blood-brain barrier (BBB) permeability. Morphologically altered astrocytes may affect barrier properties and trigger the onset of brain pathologies. However, astrocyte-dependent mediators of these events remain poorly studied. Here, we show a pharmacologically driven elevated expression and release of growth/differentiation factor 15 (GDF15) in rat primary astrocytes and cerebral PAP. GDF15 has been shown to possess trophic properties for motor neurons, prompting us to hypothesize similar effects on astrocytes. Indeed, its increased expression and release occurred simultaneously to morphological changes of astrocytes in vitro and PAP, suggesting modulatory effects of GDF15 on these cells, but also neighboring EC. Administration of recombinant GDF15 was sufficient to promote astrocyte remodeling and enhance barrier properties between ECs in vitro, whereas its pharmacogenetic abrogation prevented these effects. We validated our findings in male high anxiety-related behavior rats, an animal model of depressive-like behavior, with shrunk PAP associated with reduced expression of the junctional protein claudin-5, which were both restored by a pharmacologically induced increase in GDF15 expression. Thus, we identified GDF15 as an astrocyte-derived trigger of astrocyte process remodeling linked to enhanced tight junction strengthening at the BBB.

Minocycline alters behavior, microglia and the gut microbiome in a trait-anxiety-dependent manner.

Major depressive disorder is the main cause of disability worldwide with imperfect treatment options. However, novel therapeutic approaches are currently discussed, from augmentation strategies to novel treatments targeting the immune system or the microbiome-gut-brain axis. Therefore, we examined the potential beneficial effects of minocycline, a tetracycline antibiotic with pleiotropic, immunomodulatory action, alone or as augmentation of escitalopram on behavior, prefrontal microglial density, and the gut microbiome in rats selectively bred for high anxiety-like behavior (HAB). We show that concomitant with their high innate anxiety and depression, HABs have lower microglial numbers in the infralimbic and prelimbic prefrontal cortex and an altered gut microbiota composition compared with controls. Three weeks of minocycline treatment alleviated the depressive-like phenotype, further reduced microglial density, exclusively in male HAB rats, and reduced plasma concentrations of pro-inflammatory cytokines. However, coadministration of escitalopram, which had no effect alone, prevented these minocycline-induced effects. Moreover, minocycline led to a robust shift in cecal microbial composition in both HABs and rats non-selected for anxiety-like behavior. Minocycline markedly increased relative abundance of Lachnospiraceae and Clostridiales Family XIII, families known for their butyrate production, with a corresponding increase and positive correlation in plasma 3-OH-butyrate levels in a trait-dependent manner. Thus, our data suggest that the antidepressant effect of minocycline is sex- and trait-dependent, associated with a reduced microglial number in the prefrontal cortex, and with changes in microbial composition and their metabolites. These results further support the microbiome-gut-brain axis as potential target in the treatment of depression.

Native Chromatin Immunoprecipitation (N-ChIP) in Primary Cortical Rat Astrocytes.

Chromatin immunoprecipitation (ChIP) in conjunction with qPCR or next generation sequencing (ChIP-seq) is used to detect protein-DNA interaction. Typically, DNA bound to a protein of interest is captured with an antibody against this protein, and DNA is then purified from DNA-protein complexes. Here, we describe a native Chromatin immunoprecipitation (N-ChIP) approach which is an efficient ChIP method with high resolution for histone modifications and a number of transcription factors. This protocol has been tailored for cultured primary rat astrocytes, and we included the preparation of astrocytic cell cultures in this protocol.

Examining the Coverage of Blood Vessels by Astrocyte Endfeet in an Animal Model of Major Depressive Disorder.

The use of immunofluorescent immunohistochemical methods has been instrumental to characterize the distribution and extents of colocalization of molecules located in various cellular compartments. Such information has been pivotal to formulate hypothesis about their roles in physiological and pathological conditions.In the brain, astrocytes interact with endothelial cells to form the blood-brain barrier (BBB). Astrocytes regulate features of the BBB through the tips of their processes, called astrocyte endfeet, which surround and contact brain microvessels. Any disruption in astrocyte endfeet can have dramatic consequences on the integrity of the BBB, with consequent development of brain disorders.Here we describe a method to characterize the integrity of astrocyte endfeet around blood vessels in an animal model of major depressive disorder (MDD) using confocal microscopy, Adobe Photoshop, and the AutoQuantX3 software.

The Blood-Brain Barrier and the EphR/Ephrin System: Perspectives on a Link Between Neurovascular and Neuropsychiatric Disorders.

Interactions among endothelial cells (EC) forming blood vessels and their surrounding cell types are essential to establish the blood-brain barrier (BBB), an integral part of the neurovascular unit (NVU). Research on the NVU has recently seen a renaissance to especially understand the neurobiology of vascular and brain pathologies and their frequently occurring comorbidities. Diverse signaling molecules activated in the near proximity of blood vessels trigger paracellular pathways which regulate the formation and stabilization of tight junctions (TJ) between EC and thereby influence BBB permeability. Among regulatory molecules, the erythropoietin-producing-hepatocellular carcinoma receptors (EphR) and their Eph receptor-interacting signals (ephrins) play a pivotal role in EC differentiation, angiogenesis and BBB integrity. Multiple EphR-ligand interactions between EC and other cell types influence different aspects of angiogenesis and BBB formation. Such interactions additionally control BBB sealing properties and thus the penetration of substances into the brain parenchyma. Thus, they play critical roles in the healthy brain and during the pathogenesis of brain disorders. In this mini-review article, we aim at integrating the constantly growing literature about the functional roles of the EphR/ephrin system for the development of the vascular system and the BBB and in the pathogenesis of neurovascular and neuropsychiatric disorders. We suggest the hypothesis that a disrupted EphR/ephrin signaling at the BBB might represent an underappreciated molecular hub of disease comorbidity. Finally, we propose the possibility that the EphR/ephrin system bears the potential of becoming a novel target for the development of alternative therapeutic treatments, focusing on such comorbidities.

Fluoxetine Requires the Endfeet Protein Aquaporin-4 to Enhance Plasticity of Astrocyte Processes.

Morphological alterations in astrocytes are characteristic for post mortem brains of patients affected by major depressive disorder (MDD). Recently, a significant reduction in the coverage of blood vessels (BVs) by aquaporin-4 (AQP-4)-positive astrocyte endfeet has been shown in the prefrontal cortex (PFC) of MDD patients, suggesting that either alterations in the morphology of endfeet or in AQP-4 distribution might be responsible for the disease phenotype or constitute a consequence of its progress. Antidepressant drugs (ADs) regulate the expression of several proteins, including astrocyte-specific ones. Thus, they may target AQP-4 to induce morphological changes in astrocytes and restore their proper shape or relocate AQP-4 to endfeet. Using an animal model of depression, rats selectively bred for high anxiety-like behavior (HAB), we confirmed a reduced coverage of BVs in the adult PFC by AQP-4-immunoreactive (AQP-4-IR) astrocyte processes with respect to non-selected Wistar rats (NAB), thereby validating it for our study. A further evaluation of the morphology of astrocyte in brain slices (ex vivo) and in vitro using an antibody against the astrocyte-specific cytoskeletal protein glial fibrillary acidic protein (GFAP) revealed that HAB astrocytes extended less processes than NAB cells. Furthermore, short-term drug treatment in vitro with the AD fluoxetine (FLX) was sufficient to increase the plasticity of astrocyte processes, enhancing their number in NAB-derived cells and recovering their basal number in HAB-derived cells. This enhanced FLX-dependent plasticity occurred, however, only in the presence of intact AQP-4, as demonstrated by the lack of effect after the downregulation of AQP-4 with RNAi in both NAB and HAB cells. Nonetheless, a similar short-term treatment did neither modulate the coverage of BVs with AQP-4-positive astrocyte endfeet in NAB nor in HAB rats, although dosage and time of treatment were sufficient to fully recover GFAP expression in HAB brains. Thus, we suggest that longer treatment regimes may be needed to properly restore the coverage of BVs or to relocate AQP-4 to astrocyte endfeet. In conclusion, FLX requires AQP-4 to modulate the plasticity of astrocyte processes and this effect might be essential to re-establish a functional glia-vasculature interface necessary for a physiological communication between bloodstream and brain parenchyma.

Desipramine targets astrocytes to attenuate synaptic plasticity via modulation of the ephrinA3/EphA4 signalling.

Long-term potentiation (LTP), a major cellular correlate of memory storage, depends on activation of the ERK/MAPK signalling pathway, but the cell type-specific localization of activated MAPKs remains unknown. We found that in the CA1 field of the hippocampus, shortly after LTP induction, an increase in the number of MAPK-positive cells occurred specifically among astrocytes of the stratum radiatum, suggesting a putative role of astrocytes for LTP. Desipramine (DMI) is an antidepressant which is used to treat major depressive disorder, but also other pathologies such as neuropathic pain or attention-deficit/hyperactivity disorder. Tricyclic antidepressants such as DMI may cause memory impairment as a side effect. However, biological underpinnings of this effect still remain unclear. Here, we show that DMI inhibited the astrocytic MAPK activation and thereby hindered synaptic potentiation. These effects correlated with a reduced neuronal activation in the stratum pyramidale, thereby prompting us to analyse a regulator of LTP located at the astrocyte-neuron interface in the stratum radiatum, namely the ephrinA3/EphA4 signalling pathway. DMI enhanced EphA4 clustering, which favoured an increased ephrinA3-mediated EphA4 phosphorylation and elevated EphA4 forward signalling. The co-administration of DMI with the Src inhibitor SU6656, which blocks EphA4 forward signalling, could partially reverse the LTP attenuation, further supporting the targeting of the ephrinA3/EphA4 pathway by DMI. Thus, our findings suggest a putative novel mechanism for DMI to modulate LTP through the regulation of the ephrinA3/EphA4 signalling pathway. A further exploration of the molecular and behavioral consequences of targeting ephrinA3/EphA4 might help to improve the clinical use of DMI.

Pharmacological modulation of astrocytes and the role of cell type-specific histone modifications for the treatment of mood disorders.

Astrocytes orchestrate arrangement and functions of neuronal circuits and of the blood-brain barrier. Dysfunctional astrocytes characterize mood disorders, here showcased by deregulation of the astrocyte end-feet protein Aquaporin-4 around blood vessels and, hypothetically, of the astrocyte-specific phagocytic protein MEGF10 to shape synapses. Development of mood disorders is often a result of 'gene × environment' interactions, regulated among others by histone modifications and related modulator enzymes, which rapidly promote adaptive responses. Thus, they represent ideal targets of drugs aimed at inducing stable effects with quick onsets. One of the prevalent features of histone modifications and their modulators is their cell-type specificity. Investigating cell type-specific epigenetic modulations upon drug administration might therefore help to implement therapeutic treatments.

Talking to the synapse: how antidepressants can target glial cells to reshape brain circuits.

Functional alterations in synaptic contacts in specific brain areas are a hallmark of major depressive disorder (MDD). Antidepressant treatments not only readjust the aberrant concentrations of neurotransmitters in the synaptic clefts, but have the capacity to reshape neuronal circuits by affecting synaptogenesis and synaptic stabilization in specific regions of the brain. Nevertheless, the underlying molecular mechanisms are still unclear. Glial cells are active partners of neurons in orchestrating molecular signals that regulate the arrangement of neuronal circuits both in the developing and adult brain. Here, we present evidences indicating that glial cells might be substrates of antidepressant action for restructuring neuronal networks that has become miswired after the onset or progression of MDD. We aim to offer an alternative approach (a "gliocentric" view) to study this complex neuropsychiatric disorder and to identify alternative mechanisms of action for the currently available antidepressant therapies. Such knowledge may help to improve current treatment regimens or identify novel targets for the development of more efficacious antidepressant drugs.

Antidepressants act directly on astrocytes: evidences and functional consequences.

Post-mortem histopathological studies report on reduced glial cell numbers in various frontolimbic areas of depressed patients implying that glial loss together with abnormal functioning could contribute to the pathophysiology of mood disorders. Astrocytes are regarded as the most abundant cell type in the brain and known for their housekeeping functions, but as recent developments suggest, they are also dynamic regulators of synaptogenesis, synaptic strength and stability and they control adult hippocampal neurogenesis. The primary aim of this review was to summarize the abundant experimental evidences demonstrating that antidepressant therapies have profound effect on astrocytes. Antidepressants modify astroglial physiology, morphology and by affecting gliogenesis they probably even regulate glial cell numbers. Antidepressants affect intracellular signaling pathways and gene expression of astrocytes, as well as the expression of receptors and the release of various trophic factors. We also assess the potential functional consequences of these changes on glutamate and glucose homeostasis and on synaptic communication between the neurons. We propose here a hypothesis that antidepressant treatment not only affects neurons, but also activates astrocytes, triggering them to carry out specific functions that result in the reactivation of cortical plasticity and can lead to the readjustment of abnormal neuronal networks. We argue here that these astrocyte specific changes are likely to contribute to the therapeutic effectiveness of the currently available antidepressant treatments and the better understanding of these cellular and molecular processes could help us to identify novel targets for the development of antidepressant drugs.

Lipid raft integrity affects GABAA receptor, but not NMDA receptor modulation by psychopharmacological compounds.

Lipid rafts have been shown to play an important role for G-protein mediated signal transduction and the function of ligand-gated ion channels including their modulation by psychopharmacological compounds. In this study, we investigated the functional significance of the membrane distribution of NMDA and GABAA receptor subunits in relation to the accumulation of the tricyclic antidepressant desipramine (DMI) and the benzodiazepine diazepam (Diaz). In the presence of Triton X-100, which allowed proper separation of the lipid raft marker proteins caveolin-1 and flotillin-1 from the transferrin receptor, all receptor subunits were shifted to the non-raft fractions. In contrast, under detergent-free conditions, NMDA and GABAA receptor subunits were detected both in raft and non-raft fractions. Diaz was enriched in non-raft fractions without Triton X-100 in contrast to DMI, which preferentially accumulated in lipid rafts. Impairment of lipid raft integrity by methyl-ß-cyclodextrine (MßCD)-induced cholesterol depletion did not change the inhibitory effect of DMI at the NMDA receptor, whereas it enhanced the potentiating effect of Diaz at the GABAA receptor at non-saturating concentrations of GABA. These results support the hypothesis that the interaction of benzodiazepines with the GABAA receptor likely occurs outside of lipid rafts while the antidepressant DMI acts on ionotropic receptors both within and outside these membrane microdomains.