Specialized astrocytes mediate glutamatergic gliotransmission in the CNS.

Multimodal astrocyte-neuron communications govern brain circuitry assembly and function1. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions4-7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca2+-dependent exocytosis similar to neurons8-10. However, the existence of this mechanism has been questioned11-13 owing to inconsistent data14-17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes18-21 and using molecular, bioinformatic and imaging approaches, together with cell-specific genetic tools that interfere with glutamate exocytosis in vivo. By analysing existing single-cell RNA-sequencing databases and our patch-seq data, we identified nine molecularly distinct clusters of hippocampal astrocytes, among which we found a notable subpopulation that selectively expressed synaptic-like glutamate-release machinery and localized to discrete hippocampal sites. Using GluSnFR-based glutamate imaging22 in situ and in vivo, we identified a corresponding astrocyte subgroup that responds reliably to astrocyte-selective stimulations with subsecond glutamate release events at spatially precise hotspots, which were suppressed by astrocyte-targeted deletion of vesicular glutamate transporter 1 (VGLUT1). Furthermore, deletion of this transporter or its isoform VGLUT2 revealed specific contributions of glutamatergic astrocytes in cortico-hippocampal and nigrostriatal circuits during normal behaviour and pathological processes. By uncovering this atypical subpopulation of specialized astrocytes in the adult brain, we provide insights into the complex roles of astrocytes in central nervous system (CNS) physiology and diseases, and identify a potential therapeutic target.

Vesicular Glutamate Transporters in Astrocytes as Potential New Therapeutic Targets: Astrocyte-targeted Viral Vectors Expressing Inhibitory Nanobodies.

Astrocytes, the main Central Nervous System (CNS) glial cell type, actively release transmitters, including glutamate, and thereby participate in physiological brain information processing. However, dysregulated transmitter release from astrocytes can contribute to CNS disease pathogenesis and progression. Therefore, targeting astrocyte glutamate release is a promising new therapeutic strategy in hyper-glutamatergic brain conditions, as it does not directly block glutamatergic neurotransmission. Basing on the evidence that astrocytes express Vesicular Glutamate Transporters (VGLUT), in collaboration with other NCCR TransCure partners, we developed an innovative approach for astrocyte-selective delivery of nanobodies inhibiting VGLUT. We inserted the anti-VGLUT nanobody constructs in astrocyte-directed viral vectors that were administered peripherally, crossed the blood-brain-barrier and led to successful cell-specific CNS-wide expression of the nanobodies.

Astrocyte control of the entorhinal cortex-dentate gyrus circuit: Relevance to cognitive processing and impairment in pathology.

The entorhinal cortex-dentate gyrus circuit is centrally involved in memory processing conveying to the hippocampus spatial and nonspatial context information via, respectively, medial and lateral perforant path (MPP and LPP) excitatory projections onto dentate granule cells (GCs). Here, we review work of several years from our group showing that astrocytes sense local synaptic transmission and exert in turn a presynaptic control at PP-GC synapses. Modulation of neurotransmitter release probability by astrocytes sets basal synaptic strength and dynamic range for long-term potentiation of PP-GC synapses. Intriguingly, this astrocyte control is circuit-specific, being present only at MPP-GC (not LPP-GC) synapses, which selectively express atypical presynaptic N-methyl-D-aspartate receptors (NMDAR) suitable to activation by astrocyte-released glutamate. Moreover, the astrocytic control is peculiarly dependent on the cytokine TNFα, which at constitutive levels acts as a gating factor for the astrocyte signaling. During inflammation/infection processes, increased levels of TNFα lead to uncontrolled astrocyte glutamate release, altered PP-GC circuit processing and, ultimately, impaired contextual memory performance. The TNFα-dependent pathological switch of the synaptic control from astrocytes and its deleterious consequences are observed in animal models of HIV brain infection and multiple sclerosis, conditions both known to cause cognitive disturbances in up to 50% of patients. The review also discusses open issues related to the identified astrocytic pathway: its role in contextual memory processing, potential damaging role in Alzheimer's disease, the existence of vesicular glutamate release from DG astrocytes, and the possible synaptic-like connectivity between astrocytic output sites and PP receptive sites.

Reactive astrocyte nomenclature, definitions, and future directions.

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.

Circuit-specific control of the medial entorhinal inputs to the dentate gyrus by atypical presynaptic NMDARs activated by astrocytes.

Here, we investigated the properties of presynaptic N-methyl-d-aspartate receptors (pre-NMDARs) at corticohippocampal excitatory connections between perforant path (PP) afferents and dentate granule cells (GCs), a circuit involved in memory encoding and centrally affected in Alzheimer's disease and temporal lobe epilepsy. These receptors were previously reported to increase PP release probability in response to gliotransmitters released from astrocytes. Their activation occurred even under conditions of elevated Mg2+ and lack of action potential firing in the axons, although how this could be accomplished was unclear. We now report that these pre-NMDARs contain the GluN3a subunit conferring them low Mg2+ sensitivity. GluN3a-containing NMDARs at PP-GC synapses are preponderantly presynaptic vs. postsynaptic and persist beyond the developmental period. Moreover, they are expressed selectively at medial-not lateral-PP axons and act to functionally enhance release probability specifically of the medial perforant path (MPP) input to GC dendrites. By controlling release probability, GluN3a-containing pre-NMDARs also control the dynamic range for long-term potentiation (LTP) at MPP-GC synapses, an effect requiring Ca2+ signaling in astrocytes. Consistent with the functional observations, GluN3a subunits in MPP terminals are localized at sites away from the presynaptic release sites, often facing astrocytes, in line with a primary role for astrocytic inputs in their activation. Overall, GluN3A-containing pre-NMDARs emerge as atypical modulators of dendritic computations in the MPP-GC memory circuit.

Morphine withdrawal recruits lateral habenula cytokine signaling to reduce synaptic excitation and sociability.

The lateral habenula encodes aversive stimuli contributing to negative emotional states during drug withdrawal. Here we report that morphine withdrawal in mice leads to microglia adaptations and diminishes glutamatergic transmission onto raphe-projecting lateral habenula neurons. Chemogenetic inhibition of this circuit promotes morphine withdrawal-like social deficits. Morphine withdrawal-driven synaptic plasticity and reduced sociability require tumor necrosis factor-α (TNF-α) release and neuronal TNF receptor 1 activation. Hence, habenular cytokines control synaptic and behavioral adaptations during drug withdrawal.

Astrocyte function from information processing to cognition and cognitive impairment.

Astrocytes serve important roles that affect recruitment and function of neurons at the local and network levels. Here we review the contributions of astrocyte signaling to synaptic plasticity, neuronal network oscillations, and memory function. The roles played by astrocytes are not fully understood, but astrocytes seem to contribute to memory consolidation and seem to mediate the effects of vigilance and arousal on memory performance. Understanding the role of astrocytes in cognitive processes may also advance our understanding of how these processes go awry in pathological conditions. Indeed, abnormal astrocytic signaling can cause or contribute to synaptic and network imbalances, leading to cognitive impairment. We discuss evidence for this from animal models of Alzheimer's disease and multiple sclerosis and from animal studies of sleep deprivation and drug abuse and addiction. Understanding the emerging roles of astrocytes in cognitive function and dysfunction will open up a large array of new therapeutic opportunities.

Studying Axon-Astrocyte Functional Interactions by 3D Two-Photon Ca2+ Imaging: A Practical Guide to Experiments and "Big Data" Analysis.

Recent advances in fast volumetric imaging have enabled rapid generation of large amounts of multi-dimensional functional data. While many computer frameworks exist for data storage and analysis of the multi-gigabyte Ca2+ imaging experiments in neurons, they are less useful for analyzing Ca2+ dynamics in astrocytes, where transients do not follow a predictable spatio-temporal distribution pattern. In this manuscript, we provide a detailed protocol and commentary for recording and analyzing three-dimensional (3D) Ca2+ transients through time in GCaMP6f-expressing astrocytes of adult brain slices in response to axonal stimulation, using our recently developed tools to perform interactive exploration, filtering, and time-correlation analysis of the transients. In addition to the protocol, we release our in-house software tools and discuss parameters pertinent to conducting axonal stimulation/response experiments across various brain regions and conditions. Our software tools are available from the Volterra Lab webpage at https://wwwfbm.unil.ch/dnf/group/glia-an-active-synaptic-partner/member/volterra-andrea-volterra in the form of software plugins for Image J (NIH)-a de facto standard in scientific image analysis. Three programs are available: MultiROI_TZ_profiler for interactive graphing of several movable ROIs simultaneously, Gaussian_Filter5D for Gaussian filtering in several dimensions, and Correlation_Calculator for computing various cross-correlation parameters on voxel collections through time.

Gliotransmission: Beyond Black-and-White.

Astrocytes are highly complex cells with many emerging putative roles in brain function. Of these, gliotransmission (active information transfer from glia to neurons) has probably the widest implications on our understanding of how the brain works: do astrocytes really contribute to information processing within the neural circuitry? "Positive evidence" for this stems from work of multiple laboratories reporting many examples of modulatory chemical signaling from astrocytes to neurons in the timeframe of hundreds of milliseconds to several minutes. This signaling involves, but is not limited to, Ca2+-dependent vesicular transmitter release, and results in a variety of regulatory effects at synapses in many circuits that are abolished by preventing Ca2+ elevations or blocking exocytosis selectively in astrocytes. In striking contradiction, methodologically advanced studies by a few laboratories produced "negative evidence," triggering a heated debate on the actual existence and properties of gliotransmission. In this context, a skeptics' camp arose, eager to dismiss the whole positive evidence based on a number of assumptions behind the negative data, such as the following: (1) deleting a single Ca2+ release pathway (IP3R2) removes all the sources for Ca2+-dependent gliotransmission; (2) stimulating a transgenically expressed Gq-GPCR (MrgA1) mimics the physiological Ca2+ signaling underlying gliotransmitter release; (3) age-dependent downregulation of an endogenous GPCR (mGluR5) questions gliotransmitter release in adulthood; and (4) failure by transcriptome analysis to detect vGluts or canonical synaptic SNAREs in astrocytes proves inexistence/functional irrelevance of vesicular gliotransmitter release. We here discuss how the above assumptions are likely wrong and oversimplistic. In light of the most recent literature, we argue that gliotransmission is a more complex phenomenon than originally thought, possibly consisting of multiple forms and signaling processes, whose correct study and understanding require more sophisticated tools and finer scientific experiments than done until today. Under this perspective, the opposing camps can be reconciled and the field moved forward. Along the path, a more cautious mindset and an attitude to open discussion and mutual respect between opponent laboratories will be good companions.Dual Perspectives Companion Paper: Multiple Lines of Evidence Indicate That Gliotransmission Does Not Occur under Physiological Conditions, by Todd A. Fiacco and Ken D. McCarthy.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

Three-dimensional Ca2+ imaging advances understanding of astrocyte biology.

Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) imaging, but this method has not yielded conclusive data on the role of astrocytes in synaptic and vascular function. We developed a three-dimensional two-photon imaging approach and studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented between regions, preponderantly local within regions, and heterogeneously distributed regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active. In awake mice, activity was higher than in brain slices, particularly in endfeet and processes, and displayed occasional multifocal cellwide events. Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots.

Synaptic Adhesion Molecules Regulate the Integration of New Granule Neurons in the Postnatal Mouse Hippocampus and their Impact on Spatial Memory.

Postnatal hippocampal neurogenesis induces network remodeling and may participate to mechanisms of learning. In turn, the maturation and survival of newborn neurons is regulated by their activity. Here, we tested the effect of a cell-autonomous overexpression of synaptic adhesion molecules on the maturation and survival of neurons born postnatally and on hippocampal-dependent memory performances. Families of adhesion molecules are known to induce pre- and post-synaptic assembly. Using viral targeting, we overexpressed three different synaptic adhesion molecules, SynCAM1, Neuroligin-1B and Neuroligin-2A in newborn neurons in the dentate gyrus of 7- to 9-week-old mice. We found that SynCAM1 increased the morphological maturation of dendritic spines and mossy fiber terminals while Neuroligin-1B increased spine density. In contrast, Neuroligin-2A increased both spine density and size as well as GABAergic innervation and resulted in a drastic increase of neuronal survival. Surprisingly, despite increased neurogenesis, mice overexpressing Neuroligin-2A in new neurons showed decreased memory performances in a Morris water maze task. These results indicate that the cell-autonomous overexpression of synaptic adhesion molecules can enhance different aspects of synapse formation on new neurons and increase their survival. Furthermore, they suggest that the mechanisms by which new neurons integrate in the postnatal hippocampus conditions their functional implication in learning and memory.

Neuroinflammatory TNFα Impairs Memory via Astrocyte Signaling.

The occurrence of cognitive disturbances upon CNS inflammation or infection has been correlated with increased levels of the cytokine tumor necrosis factor-α (TNFα). To date, however, no specific mechanism via which this cytokine could alter cognitive circuits has been demonstrated. Here, we show that local increase of TNFα in the hippocampal dentate gyrus activates astrocyte TNF receptor type 1 (TNFR1), which in turn triggers an astrocyte-neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses. Astrocytic TNFR1 signaling is necessary for the hippocampal synaptic alteration and contextual learning-memory impairment observed in experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disturbances in MS, as well as in other CNS conditions accompanied by inflammatory states or infections.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy.

What do we know about gliotransmitter release from astrocytes?

Astrocytes participate in information processing by actively modulating synaptic properties via gliotransmitter release. Various mechanisms of astrocytic release have been reported, including release from storage organelles via exocytosis and release from the cytosol via plasma membrane ion channels and pumps. It is still not fully clear which mechanisms operate under which conditions, but some of them, being Ca(2+)-regulated, may be physiologically relevant. The properties of Ca(2+)-dependent transmitter release via exocytosis or via ion channels are different and expected to produce different extracellular transmitter concentrations over time and to have distinct functional consequences. The molecular aspects of these two release pathways are still under active investigation. Here, we discuss the existing morphological and functional evidence in support of either of them. Transgenic mouse models, specific antagonists and localization studies have provided insight into regulated exocytosis, albeit not in a systematic fashion. Even more remains to be uncovered about the details of channel-mediated release. Better functional tools and improved ultrastructural approaches are needed in order fully to define specific modalities and effects of astrocytic gliotransmitter release pathways.

Identification and staining of distinct populations of secretory organelles in astrocytes.

Increasing evidence indicates that astrocytes, the most abundant glial cell type in the brain, respond to an elevation in cytoplasmic calcium concentration ([Ca(2+)]i) by releasing chemical transmitters (also called gliotransmitters) via regulated exocytosis of heterogeneous classes of organelles. By this process, astrocytes exert modulatory influences on neighboring cells and are thought to participate in the control of synaptic circuits and cerebral blood flow. Studying the properties of exocytosis in astrocytes is a challenge, because the cell biological basis of this process is incompletely defined. Astrocytic exocytosis involves multiple populations of secretory vesicles, including synaptic-like microvesicles (SLMVs), dense-core granules (DCGs), and lysosomes. Here we summarize the available information for identifying individual populations of secretory organelles in astrocytes, including DCGs, SLMVs, and lysosomes, and present experimental procedures for specifically staining such populations.

Imaging exocytosis and recycling of synaptic-like microvesicles in astrocytes.

Optical imaging techniques are well suited for following the dynamics of physiological processes in living cells. Total internal reflection fluorescence (TIRF) microscopy based on evanescent wave illumination (EWi) allows spectacular, real-time visualization of individual vesicle movements, fusions, and retrievals at the cell surface (i.e., within 100 nm of the plasma membrane). TIRF microscopy is an ideal approach for studying the properties of exocytosis and recycling in cultured astrocytes, particularly because these cells have a rather flat surface and contain secretory vesicles with sparse distribution. Among all populations of secretory vesicles, we focus here on synaptic-like microvesicles (SLMVs). We illustrate how TIRF microscopy using EWi is useful to study exocytosis and recycling of SLMVs at the single-vesicle level and, when combined with epifluorescence illumination (EPIi), can provide detailed information on the kinetics of exocytosis, endocytosis, and re-acidification at the whole-cell level.

Astrocyte Ca²âº signalling: an unexpected complexity.

Astrocyte Ca(2+) signalling has been proposed to link neuronal information in different spatial-temporal dimensions to achieve a higher level of brain integration. However, some discrepancies in the results of recent studies challenge this view and highlight key insufficiencies in our current understanding. In parallel, new experimental approaches that enable the study of astrocyte physiology at higher spatial-temporal resolution in intact brain preparations are beginning to reveal an unexpected level of compartmentalization and sophistication in astrocytic Ca(2+) dynamics. This newly revealed complexity needs to be attentively considered in order to understand how astrocytes may contribute to brain information processing.

Gliotransmitters travel in time and space.

The identification of the presence of active signaling between astrocytes and neurons in a process termed gliotransmission has caused a paradigm shift in our thinking about brain function. However, we are still in the early days of the conceptualization of how astrocytes influence synapses, neurons, networks, and ultimately behavior. In this Perspective, our goal is to identify emerging principles governing gliotransmission and consider the specific properties of this process that endow the astrocyte with unique functions in brain signal integration. We develop and present hypotheses aimed at reconciling confounding reports and define open questions to provide a conceptual framework for future studies. We propose that astrocytes mainly signal through high-affinity slowly desensitizing receptors to modulate neurons and perform integration in spatiotemporal domains complementary to those of neurons.

Computational quest for understanding the role of astrocyte signaling in synaptic transmission and plasticity.

The complexity of the signaling network that underlies astrocyte-synapse interactions may seem discouraging when tackled from a theoretical perspective. Computational modeling is challenged by the fact that many details remain hitherto unknown and conventional approaches to describe synaptic function are unsuitable to explain experimental observations when astrocytic signaling is taken into account. Supported by experimental evidence is the possibility that astrocytes perform genuine information processing by means of their calcium signaling and are players in the physiological setting of the basal tone of synaptic transmission. Here we consider the plausibility of this scenario from a theoretical perspective, focusing on the modulation of synaptic release probability by the astrocyte and its implications on synaptic plasticity. The analysis of the signaling pathways underlying such modulation refines our notion of tripartite synapse and has profound implications on our understanding of brain function.