Andrea Volterra

Astrocytes participate in local inflammation and cognitive decline in Alzheimer’s disease (AD). Aberrant cytokine TNFα signaling via astrocyte type-1 receptor (aTNFR1) could causally link the two AD pathology aspects. To verify this hypothesis, we crossed transgenic AD mice with mice enabling astrocyte-specific conditional TNFR1 deletion (aTNFR1KO). Induction of aTNFR1KO at early AD stages, preserved memory and reduced β-amyloid load and astrogliosis in the aged mice. Induction of aTNFR1KO at late AD stages, in mice already memory-impaired, surprisingly produced rapid memory rescue, without affecting β-amyloid load and astrogliosis. Single nucleus-RNA-seq analysis of all hippocampal cell populations revealed that late-stage aTNFR1KO rapidly modifies gene expression mainly in neurons, primarily targeting synaptic pathways, causing combined glutamatergic downregulation and GABAergic up-regulation. Consistently, hippocampal EEGs showed a pro-inhibitory effect of aTNFR1KO, which thus restores memory by “rebalancing” hippocampal circuitry excitability. This pro-memory effect identifies a new mechanism and astrocyte target against cognitive decline in AD.

This chapter reviews studies in the last two decades that led to identifying the rapid bidirectional communication existing between astrocytes and neurons, and its roles in the control of synaptic transmission at local level as well as in the multiscale coordination of large synaptic networks. This chapter first describes the most relevant observations that demonstrated how astrocyte signaling modulates neuronal activity and plasticity, focusing on the role of astrocytic metabotropic receptors, intracellular Ca²⁺ elevations, and release of glutamate as neuroactive molecule. It then examines evidence of how astrocytes contribute to synaptic networks oscillations and to their behavioral consequences, notably in learning and memory processes. This chapter also discusses recent discoveries about a heterogeneity of the astrocytes, with molecular, morphological and functional differences at both inter- and intra-regional levels, implying a new level of complexity in the modalities by which astrocytes communicate with neurons and participate to brain functions.

Multimodal astrocyte–neuron communications govern brain circuitry assembly and function¹. For example, through rapid glutamate release, astrocytes can control excitability, plasticity and synchronous activity^2,3 of synaptic networks, while also contributing to their dysregulation in neuropsychiatric conditions^4–7. For astrocytes to communicate through fast focal glutamate release, they should possess an apparatus for Ca²⁺-dependent exocytosis similar to neurons^8–10. However, the existence of this mechanism has been questioned^11–13 owing to inconsistent data^14–17 and a lack of direct supporting evidence. Here we revisited the astrocyte glutamate exocytosis hypothesis by considering the emerging molecular heterogeneity of astrocytes^18–21 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.

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 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.

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.

Significance: We previously identified a mechanism controlling transmitter release probability at perforant path (PP)–granule cell excitatory synapses, a memory-related circuit that goes awry in Alzheimer’s disease and temporal lobe epilepsy. We found that the mechanism involves activation of presynaptic N -methyl- d -aspartate receptors (pre-NMDARs) by astrocytes but could not explain its specificities, notably why pre-NMDARs were activated in conditions in which classical NMDARs are not. We show that pre-NMDARs ( i ) contain an atypical subunit, GluN3a, responsible for their properties and ( ii ) are localized in PP terminals facing astrocytes, restricted to a subset of PP afferents and controlling only the synapses made by those afferents. This circuit-specific modulatory mechanism by astrocytes may be important for memory processing and its alterations in pathological conditions.

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.

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.