Search

Astrocytes are morphologically complex cells that serve essential roles. They are widely implicated in central nervous system (CNS) disorders, with changes in astrocyte morphology and gene expression accompanying disease. In the Sapap3 knockout (KO) mouse model of compulsive and anxiety-related behaviors related to obsessive-compulsive disorder (OCD), striatal astrocytes display reduced morphology and altered actin cytoskeleton and Gi-G-protein-coupled receptor (Gi-GPCR) signaling proteins. Here, we show that normalizing striatal astrocyte morphology, actin cytoskeleton, and essential homeostatic support functions by targeting the astrocyte Gi-GPCR pathway using chemogenetics corrected phenotypes in Sapap3 KO mice, including anxiety-related and compulsive behaviors. Our data portend an astrocytic pharmacological strategy for rescuing phenotypes in brain disorders that include compromised astrocyte morphology and tissue support., Competing Interests: Declaration of interests UCLA filed a US provisional patent (no. 63/658,760) based on this work. B.S.K. is on the editorial advisory board of Neuron., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Astrocytes are predominant glial cells that tile the central nervous system and participate in well-established functional and morphological interactions with neurons, blood vessels, and other glia. These ubiquitous cells display rich intracellular Ca 2+ signaling, which has now been studied for over 30 years. In this review, we provide a summary and perspective of recent progress concerning the study of astrocyte intracellular Ca 2+ signaling as well as discussion of its potential functions. Progress has occurred in the areas of imaging, silencing, activating, and analyzing astrocyte Ca 2+ signals. These insights have collectively permitted exploration of the relationships of astrocyte Ca 2+ signals to neural circuit function and behavior in a variety of species. We summarize these aspects along with a framework for mechanistically interpreting behavioral studies to identify directly causal effects. We finish by providing a perspective on new avenues of research concerning astrocyte Ca 2+ signaling., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Region-specific synapse loss is an early pathological hallmark in Alzheimer's disease (AD). Emerging data in mice and humans highlight microglia, the brain-resident macrophages, as cellular mediators of synapse loss; however, the upstream modulators of microglia-synapse engulfment remain elusive. Here, we report a distinct subset of astrocytes, which are glial cells essential for maintaining synapse homeostasis, appearing in a region-specific manner with age and amyloidosis at onset of synapse loss. These astrocytes are distinguished by their peri-synaptic processes which are 'bulbous' in morphology, contain accumulated p62-immunoreactive bodies, and have reduced territorial domains, resulting in a decrease of astrocyte-synapse coverage. Using integrated in vitro and in vivo approaches, we show that astrocytes upregulate and secrete phagocytic modulator, milk fat globule-EGF factor 8 (MFG-E8), which is sufficient and necessary for promoting microglia-synapse engulfment in their local milieu. Finally, we show that knocking down Mfge8 specifically from astrocytes using a viral CRISPR-saCas9 system prevents microglia-synapse engulfment and ameliorates synapse loss in two independent amyloidosis mouse models of AD. Altogether, our findings highlight astrocyte-microglia crosstalk in determining synapse fate in amyloid models and nominate astrocytic MFGE8 as a potential target to ameliorate synapse loss during the earliest stages of AD., Competing Interests: Declaration of interests SH has acted as a paid consultant to Eisai Ltd, Novo Nordisk, and Alnylam; receives research funding from AstraZeneca and Eisai Ltd; and has a collaborative project with Ionis Ltd. During this research, OJF was employed by AstraZeneca; OJF is now employed by MSD. The following patents have been granted or applied for: PCT/2015/010288, US14/988387 and EP14822330 (SH). All the other authors declare that they have no competing interests.

Astrocytes intricately weave within the neuropil, giving rise to characteristic bushy morphologies. Pioneering studies suggested that primate astrocytes are more complex due to increased branch numbers and territory size compared to rodent counterparts. However, there has been no comprehensive comparison of astrocyte morphology across species. We employed several techniques to investigate astrocyte morphology and directly compared them between mice and rhesus macaques in cortical and subcortical regions. We assessed astrocyte density, territory size, branching structure, fine morphological complexity, and interactions with neuronal synapses using a combination of techniques, including immunohistochemistry, adeno-associated virus-mediated transduction of astrocytes, diOlistics, confocal imaging, and electron microscopy. We found significant morphological similarities between primate and rodent astrocytes, suggesting that astrocyte structure has scaled with evolution. Our findings show that primate astrocytes are larger and more numerous than those in rodents but contest the view that primate astrocytes are morphologically far more complex., (© 2024 Wiley Periodicals LLC.)

Astrocytes are predominant glial cells that tile the central nervous system (CNS). A cardinal feature of astrocytes is their complex and visually enchanting morphology, referred to as bushy, spongy, and star-like. A central precept of this review is that such complex morphological shapes evolved to allow astrocytes to contact and signal with diverse cells at a range of distances in order to sample, regulate, and contribute to the extracellular milieu, and thus participate widely in cell-cell signaling during physiology and disease. The recent use of improved imaging methods and cell-specific molecular evaluations has revealed new information on the structural organization and molecular underpinnings of astrocyte morphology, the mechanisms of astrocyte morphogenesis, and the contributions to disease states of reduced morphology. These insights have reignited interest in astrocyte morphological complexity as a cornerstone of fundamental glial biology and as a critical substrate for multicellular spatial and physiological interactions in the CNS., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Investigative exploration and foraging leading to food consumption have vital importance, but are not well-understood. Since GABAergic inputs to the lateral and ventrolateral periaqueductal gray (l/vlPAG) control such behaviors, we dissected the role of vgat-expressing GABAergic l/vlPAG cells in exploration, foraging and hunting. Here, we show that in mice vgat l/vlPAG cells encode approach to food and consumption of both live prey and non-prey foods. The activity of these cells is necessary and sufficient for inducing food-seeking leading to subsequent consumption. Activation of vgat l/vlPAG cells produces exploratory foraging and compulsive eating without altering defensive behaviors. Moreover, l/vlPAG vgat cells are bidirectionally interconnected to several feeding, exploration and investigation nodes, including the zona incerta. Remarkably, the vgat l/vlPAG projection to the zona incerta bidirectionally controls approach towards food leading to consumption. These data indicate the PAG is not only a final downstream target of top-down exploration and foraging-related inputs, but that it also influences these behaviors through a bottom-up pathway., (© 2024. The Author(s).)

The central nervous system (CNS) comprises diverse and morphologically complex cells. To understand the molecular basis of their physiology, it is crucial to assess proteins expressed within intact cells. Commonly used methods utilize cell dissociation and sorting to isolate specific cell types such as neurons and astrocytes, the major CNS cells. Proteins purified from isolated cells are identified by mass spectrometry-based proteomics. However, dissociation and cell-sorting methods lead to near total loss of cellular morphology, thereby losing proteins from key relevant subcompartments such as processes, end feet, dendrites and axons. Here we provide a systematic protocol for cell- and subcompartment-specific labeling and identification of proteins found within intact astrocytes and neurons in vivo. This protocol utilizes the proximity-dependent biotinylation system BioID2, selectively expressed in either astrocytes or neurons, to label proximal proteins in a cell-specific manner. BioID2 is targeted genetically to assess the subproteomes of subcellular compartments such as the plasma membrane and sites of cell-cell contacts. We describe in detail the expression methods (variable timing), stereotaxic surgeries for expression (1-2 d and then 3 weeks), in vivo protein labeling (7 d), protein isolation (2-3 d), protein identification methods (2-3 d) and data analysis (1 week). The protocol can be applied to any area of the CNS in mouse models of physiological processes and for disease-related research., (© 2023. Springer Nature Limited.)

Astrocytes are heterogeneous glial cells of the central nervous system 1-3 . However, the physiological relevance of astrocyte diversity for neural circuits and behaviour remains unclear. Here we show that a specific population of astrocytes in the central striatum expresses μ-crystallin (encoded by Crym in mice and CRYM in humans) that is associated with several human diseases, including neuropsychiatric disorders 4-7 . In adult mice, reducing the levels of μ-crystallin in striatal astrocytes through CRISPR-Cas9-mediated knockout of Crym resulted in perseverative behaviours, increased fast synaptic excitation in medium spiny neurons and dysfunctional excitatory-inhibitory synaptic balance. Increased perseveration stemmed from the loss of astrocyte-gated control of neurotransmitter release from presynaptic terminals of orbitofrontal cortex-striatum projections. We found that perseveration could be remedied using presynaptic inhibitory chemogenetics 8 , and that this treatment also corrected the synaptic deficits. Together, our findings reveal converging molecular, synaptic, circuit and behavioural mechanisms by which a molecularly defined and allocated population of striatal astrocytes gates perseveration phenotypes that accompany neuropsychiatric disorders 9-12 . Our data show that Crym-positive striatal astrocytes have key biological functions within the central nervous system, and uncover astrocyte-neuron interaction mechanisms that could be targeted in treatments for perseveration., (© 2024. The Author(s).)

Myelin is essential for rapid nerve signaling and is increasingly found to play important roles in learning and in diverse diseases of the CNS. Morphological parameters of myelin such as sheath length are thought to precisely tune conduction velocity, but the mechanisms controlling sheath morphology are poorly understood. Local calcium signaling has been observed in nascent myelin sheaths and can be modulated by neuronal activity. However, the role of calcium signaling in sheath formation remains incompletely understood. Here, we use genetic tools to attenuate oligodendrocyte calcium signaling during myelination in the developing mouse CNS. Surprisingly, genetic calcium attenuation does not grossly affect the number of myelinated axons or myelin thickness. Instead, calcium attenuation causes myelination defects resulting in shorter, dysmorphic sheaths. Mechanistically, calcium attenuation reduces actin filaments in oligodendrocytes, and an intact actin cytoskeleton is necessary and sufficient to achieve accurate myelin morphology. Together, our work reveals a cellular mechanism required for accurate CNS myelin formation and may provide mechanistic insight into how oligodendrocytes respond to neuronal activity to sculpt and refine myelin sheaths., (© 2024. The Author(s).)

Background: The formation and retrieval of fear memories depends on orchestrated synaptic activity of neuronal ensembles within the hippocampus, and it is becoming increasingly evident that astrocytes residing in the environment of these synapses play a central role in shaping cellular memory representations. Astrocyte distal processes, known as leaflets, fine-tune synaptic activity by clearing neurotransmitters and limiting glutamate diffusion. However, how astroglial synaptic coverage contributes to mnemonic processing of fearful experiences remains largely unknown., Methods: We used electron microscopy to observe changes in astroglial coverage of hippocampal synapses during consolidation of fear memory in mice. To manipulate astroglial synaptic coverage, we depleted ezrin, an integral leaflet-structural protein, from hippocampal astrocytes using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing. Next, a combination of Föster resonance energy transfer analysis, genetically encoded glutamate sensors, and whole-cell patch-clamp recordings was used to determine whether the proximity of astrocyte leaflets to the synapse is critical for synaptic integrity and function., Results: We found that consolidation of a recent fear memory is accompanied by a transient retraction of astrocyte leaflets from hippocampal synapses and increased activation of NMDA receptors. Accordingly, astrocyte-specific depletion of ezrin resulted in shorter astrocyte leaflets and reduced astrocyte contact with the synaptic cleft, which consequently boosted extrasynaptic glutamate diffusion and NMDA receptor activation. Importantly, after fear conditioning, these cellular phenotypes translated to increased retrieval-evoked activation of CA1 pyramidal neurons and enhanced fear memory expression., Conclusions: Together, our data show that withdrawal of astrocyte leaflets from the synaptic cleft is an experience-induced, temporally regulated process that gates the strength of fear memories., (Copyright © 2022 Society of Biological Psychiatry. All rights reserved.)

Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation 1 to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive-compulsive disorder (OCD) and repetitive behaviours 2-8 , was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD 4 lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders., (© 2023. The Author(s).)

Huntington's disease (HD) is a fatal, monogenic, autosomal dominant neurodegenerative disease caused by a polyglutamine-encoding CAG expansion in the huntingtin (HTT) gene that results in mutant huntingtin proteins (mHTT) in cells throughout the body. Although large parts of the central nervous system (CNS) are affected, the striatum is especially vulnerable and undergoes marked atrophy. Astrocytes are abundant within the striatum and contain mHTT in HD, as well as in mouse models of the disease. We focus on striatal astrocytes and summarize how they participate in, and contribute to, molecular pathophysiology and disease-related phenotypes in HD model mice. Where possible, reference is made to pertinent astrocyte alterations in human HD. Astrocytic dysfunctions related to cellular morphology, extracellular ion and neurotransmitter homeostasis, and metabolic support all accompany the development and progression of HD, in both transgenic mouse and human cellular and chimeric models of HD. These findings reveal the potential for the therapeutic targeting of astrocytes so as to restore synaptic as well as tissue homeostasis in HD. Elucidation of the mechanisms by which astrocytes contribute to HD pathogenesis may inform a broader understanding of the role of glial pathology in neurodegenerative disorders and, by so doing, enable new strategies of glial-directed therapeutics., (© 2023 New York Academy of Sciences.)

Astrocytes, the most abundant glial cells in the central nervous system, play vital roles in maintaining neuronal function. A new study using focused ion-beam scanning electron microscopy reveals the architecture of astrocytes at the nanoscale and provides new insights on how astrocytes perform their diverse activities., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Huntington's disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) resulting in expression of mutant HTT proteins (mHTT) with extended polyglutamine tracts, including in striatal neurons and astrocytes. It is unknown whether pathophysiology in vivo can be attenuated by lowering mHTT in either cell type throughout the brain, and the relative contributions of neurons and astrocytes to HD remain undefined. We use zinc finger protein (ZFP) transcriptional repressors to cell-selectively lower mHTT in vivo. Astrocytes display loss of essential functions such as cholesterol metabolism that are partly driven by greater neuronal dysfunctions, which encompass neuromodulation, synaptic, and intracellular signaling pathways. Using transcriptomics, proteomics, electrophysiology, and behavior, we dissect neuronal and astrocytic contributions to HD pathophysiology. Remarkably, brain-wide delivery of neuronal ZFPs results in strong mHTT lowering, rescue of HD-associated behavioral and molecular phenotypes, and significant extension of lifespan, findings that support translational development., Competing Interests: Declaration of interests B.S.K. is on the editorial advisory board of Neuron., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Functional hyperemia occurs when enhanced neuronal activity signals to increase local cerebral blood flow (CBF) to satisfy regional energy demand. Ca 2+ elevation in astrocytes can drive arteriole dilation to increase CBF, yet affirmative evidence for the necessity of astrocytes in functional hyperemia in vivo is lacking. In awake mice, we discovered that functional hyperemia is bimodal with a distinct early and late component whereby arteriole dilation progresses as sensory stimulation is sustained. Clamping astrocyte Ca 2+ signaling in vivo by expressing a plasma membrane Ca 2+ ATPase (CalEx) reduces sustained but not brief sensory-evoked arteriole dilation. Elevating astrocyte free Ca 2+ using chemogenetics selectively augments sustained hyperemia. Antagonizing NMDA-receptors or epoxyeicosatrienoic acid production reduces only the late component of functional hyperemia, leaving brief increases in CBF to sensory stimulation intact. We propose that a fundamental role of astrocyte Ca 2+ is to amplify functional hyperemia when neuronal activation is prolonged., (© 2022. The Author(s).)

Astrocytes, a type of glia, are abundant and morphologically complex cells. Here, we report astrocyte molecular profiles, diversity, and morphology across the mouse central nervous system (CNS). We identified shared and region-specific astrocytic genes and functions and explored the cellular origins of their regional diversity. We identified gene networks correlated with astrocyte morphology, several of which unexpectedly contained Alzheimer's disease (AD) risk genes. CRISPR/Cas9-mediated reduction of candidate genes reduced astrocyte morphological complexity and resulted in cognitive deficits. The same genes were down-regulated in human AD, in an AD mouse model that displayed reduced astrocyte morphology, and in other human brain disorders. We thus provide comprehensive molecular data on astrocyte diversity and mechanisms across the CNS and on the molecular basis of astrocyte morphology in health and disease.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Cell-specific RNA sequencing has revolutionized the study of cell biology. Here, we present a protocol to assess cell-specific translatomes of genetically targeted cell types. We focus on astrocytes and describe RNA purification using RiboTag tools. Unlike single-cell RNA sequencing, this approach allows high sequencing depth to detect low expression genes, and the exploration of RNAs translated in subcellular compartments. Furthermore, it avoids underestimation of transcripts from cells susceptible to cell isolation procedures. The protocol can be applied to a variety of cell types. For complete details on the use and execution of this protocol, please refer to Chai et al. (2017), Díaz-Castro et al. (2021), Díaz-Castro et al. (2019), Srinivasan et al. (2016), and Yu et al. (2018)., Competing Interests: B.S.K. is a consultant for Third Rock Ventures., (© 2022 The Authors.)

Astrocytic contributions to neuroinflammation are widely implicated in disease, but they remain incompletely explored. We assess medial prefrontal cortex (PFC) and visual cortex (VCX) astrocyte and whole-tissue gene expression changes in mice following peripherally induced neuroinflammation triggered by a systemic bacterial endotoxin, lipopolysaccharide, which produces sickness-related behaviors, including anhedonia. Neuroinflammation-mediated behavioral changes and astrocyte-specific gene expression alterations peak when anhedonia is greatest and then reverse to normal. Notably, region-specific molecular identities of PFC and VCX astrocytes are largely maintained during reactivity changes. Gene pathway analyses reveal alterations of diverse cell signaling pathways, including changes in cell-cell interactions of multiple cell types that may underlie the central effects of neuroinflammation. Certain astrocyte molecular signatures accompanying neuroinflammation are shared with changes reported in Alzheimer's disease and mouse models. However, we find no evidence of altered neuronal survival or function in the PFC even when neuroinflammation-induced astrocyte reactivity and behavioral changes are significant., Competing Interests: Declaration of interests B.S.K. is a consultant for Third Rock Ventures. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Astrocytes respond to neurotransmitters and neuromodulators using G-protein-coupled receptors (GPCRs) to mediate physiological responses. Despite their importance, there has been no method to genetically, specifically, and effectively attenuate astrocyte G q GPCR pathways to explore consequences of this prevalent signaling mechanism in vivo. We report a 122-residue inhibitory peptide from β-adrenergic receptor kinase 1 (iβARK; and inactive D110A control) to attenuate astrocyte G q GPCR signaling. iβARK significantly attenuated G q GPCR Ca 2+ signaling in brain slices and, in vivo, altered behavioral responses, spared other GPCR responses, and did not alter astrocyte spontaneous Ca 2+ signals, morphology, electrophysiological properties, or gene expression in the striatum. Furthermore, brain-wide attenuation of astrocyte G q GPCR signaling with iβARK using PHP.eB adeno-associated viruses (AAVs), when combined with c-Fos mapping, suggested nuclei-specific contributions to behavioral adaptation and spatial memory. iβARK extends the toolkit needed to explore functions of astrocyte G q GPCR signaling within neural circuits in vivo., Competing Interests: Declaration of interests The authors declare no competing interests relevant to this study. However, B.S.K. is a consultant for Third Rock Ventures., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Astrocytes are indispensable for proper neuronal functioning. Given the diverse needs of neuronal circuits and the variety of tasks astrocytes perform, the perceived homogeneous nature of astrocytes has been questioned. In the spinal dorsal horn, complex neuronal circuitries regulate the integration of sensory information of different modalities. The dorsal horn is organized in a distinct laminar manner based on termination patterns of high- and low-threshold afferent fibers and neuronal properties. Neurons in laminae I (L1) and II (L2) integrate potentially painful, nociceptive information, whereas neurons in lamina III (L3) and deeper laminae integrate innocuous, tactile information from the periphery. Sensory information is also integrated by an uncharacterized network of astrocytes. How these lamina-specific characteristics of neuronal circuits of the dorsal horn are of functional importance for properties of astrocytes is currently unknown. We addressed if astrocytes in L1, L2, and L3 of the upper dorsal horn of mice are differentially equipped for the needs of neuronal circuits that process sensory information of different modalities. We found that astrocytes in L1 and L2 were characterized by a higher density, higher expression of GFAP, Cx43, and GLAST and a faster coupling speed than astrocytes located in L3. L1 astrocytes were more responsive to Kir4.1 blockade and had higher levels of AQP4 compared to L3 astrocytes. In contrast, basic membrane properties, network formation, and somatic intracellular calcium signaling were similar in L1-L3 astrocytes. Our data indicate that the properties of spinal astrocytes are fine-tuned for the integration of nociceptive versus tactile information., (© 2021 The Authors. GLIA published by Wiley Periodicals LLC.)

Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is necessary for context-specific escape induced by moderate threats but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Astrocytes exist throughout the CNS and affect neural circuits and behavior through intracellular Ca 2+ signaling. Studying the function(s) of astrocyte Ca 2+ signaling has proven difficult because of the paucity of tools to achieve selective attenuation. Based on recent studies, we generated and used male and female knock-in mice for Cre-dependent expression of mCherry-tagged hPMCA2w/b to attenuate astrocyte Ca 2+ signaling in genetically defined cells in vivo (CalEx flox mice for Ca lcium Ex trusion). We characterized CalEx flox mice following local AAV-Cre microinjections into the striatum and found reduced astrocyte Ca 2+ signaling (∼90%) accompanied with repetitive self-grooming behavior. We also crossed CalEx flox mice to astrocyte-specific Aldh1l1 -Cre/ERT2 mice to achieve inducible global CNS-wide Ca 2+ signaling attenuation. Within 6 d of induction in the bigenic mice, we observed significantly altered ambulation in the open field, disrupted motor coordination and gait, and premature lethality. Furthermore, with histologic, imaging, and transcriptomic analyses, we identified cellular and molecular alterations in the cerebellum following mCherry-tagged hPMCA2w/b expression. Our data show that expression of mCherry-tagged hPMCA2w/b with CalEx flox mice throughout the CNS resulted in substantial attenuation of astrocyte Ca 2+ signaling and significant behavioral alterations in adult mice. We interpreted these findings candidly in relation to the ability of CalEx to attenuate astrocyte Ca 2+ signaling, with regards to additional mechanistic interpretations of the data, and their relation to past studies that reduced astrocyte Ca 2+ signaling throughout the CNS. The data and resources provide complementary ways to interrogate the function(s) of astrocytes in multiple experimental scenarios. SIGNIFICANCE STATEMENT Astrocytes represent a significant fraction of all brain cells and tile the entire central nervous system. Unlike neurons, astrocytes lack propagated electrical signals. Instead, astrocytes are proposed to use diverse and dynamic intracellular Ca 2+ signals to communicate with other cells. An open question concerns if and how astrocyte Ca 2+ signaling regulates behavior in adult mice. We approached this problem by generating a new transgenic mouse line to achieve inducible astrocyte Ca 2+ signaling attenuation in vivo We report our data with this mouse line and we interpret the findings candidly in relation to past studies and within the framework of different mechanistic interpretations., (Copyright © 2021 the authors.)

Trauma can cause dysfunctional fear regulation leading some people to develop disorders, such as post-traumatic stress disorder (PTSD). The amygdala regulates fear, whereas PACAP (pituitary adenylate activating peptide) and PAC1 receptors are linked to PTSD symptom severity at genetic/epigenetic levels, with a strong link in females with PTSD. We discovered a PACAPergic projection from the basomedial amygdala (BMA) to the medial intercalated cells (mICCs) in adult mice. In vivo optogenetic stimulation of this pathway increased CFOS expression in mICCs, decreased fear recall, and increased fear extinction. Selective deletion of PAC1 receptors from the mICCs in females reduced fear acquisition, but enhanced fear generalization and reduced fear extinction in males. Optogenetic stimulation of the BMA-mICC PACAPergic pathway produced EPSCs in mICC neurons, which were enhanced by the PAC1 receptor antagonist, PACAP 6-38. Our findings show that mICCs modulate contextual fear in a dynamic and sex-dependent manner via a microcircuit containing the BMA and mICCs, and in a manner that was dependent on behavioral state. SIGNIFICANCE STATEMENT Traumatic stress can affect different aspects of fear behaviors, including fear learning, generalization of learned fear to novel contexts, how the fear of the original context is recalled, and how fear is reduced over time. While the amygdala has been studied for its role in regulation of different aspects of fear, the molecular circuitry of this structure is quite complex. In addition, aspects of fear can be modulated differently in males and females. Our findings show that a specific circuitry containing the neuropeptide PACAP and its receptor, PAC1, regulates various aspects of fear, including acquisition, generalization, recall, and extinction in a sexually dimorphic manner, characterizing a novel pathway that modulates traumatic fear., (Copyright © 2021 Rajbhandari et al.)

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.

Astrocytes are a large and diverse population of morphologically complex cells that exist throughout nervous systems of multiple species. Progress over the last two decades has shown that astrocytes mediate developmental, physiological, and pathological processes. However, a long-standing open question is how astrocytes regulate neural circuits in ways that are behaviorally consequential. In this regard, we summarize recent studies using Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and Mus musculus. The data reveal diverse astrocyte mechanisms operating in seconds or much longer timescales within neural circuits and shaping multiple behavioral outputs. We also refer to human diseases that have a known primary astrocytic basis. We suggest that including astrocytes in mechanistic, theoretical, and computational studies of neural circuits provides new perspectives to understand behavior, its regulation, and its disease-related manifestations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Astrocytes tile the central nervous system and are widely implicated in brain diseases, but the molecular mechanisms by which astrocytes contribute to brain disorders remain incompletely explored. By performing astrocyte gene expression analyses following 14 experimental perturbations of relevance to the striatum, we discovered that striatal astrocytes mount context-specific molecular responses at the level of genes, pathways, and upstream regulators. Through data mining, we also identified astrocyte pathways in Huntington's disease (HD) that were reciprocally altered with respect to the activation of striatal astrocyte G protein-coupled receptor (GPCR) signaling. Furthermore, selective striatal astrocyte stimulation of the G i -GPCR pathway in vivo corrected several HD-associated astrocytic, synaptic, and behavioral phenotypes, with accompanying improvement of HD-associated astrocyte signaling pathways, including those related to synaptogenesis and neuroimmune functions. Overall, our data show that astrocytes are malleable, using context-specific responses that can be dissected molecularly and used for phenotypic benefit in brain disorders., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The first issue of Nature Reviews Neuroscience was published 20 years ago, in 2000. To mark this anniversary, in this Viewpoint article we asked a selection of researchers from across the field who have authored pieces published in the journal in recent years for their thoughts on notable and interesting developments in neuroscience, and particularly in their areas of the field, over the past two decades. They also provide some thoughts on current lines of research and questions that excite them.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.

Astrocytes are a type of glial cell that tile the CNS. They interact with multiple cell types, including neurons, glial cells and blood vessels, and are involved or implicated in brain disorders. Progress has been made in understanding astrocytes, but the field lacks detailed information concerning how they perform their multifarious functions, and how and when they influence the operations of the neural circuits with which they interact. One recognized bottleneck to progress has been the paucity of reliable tools with which to explore astrocytes within the adult vertebrate CNS in vivo. However, improved tools for molecular, genetic, morphological and physiological assessments have been developed recently or have been adapted from their original purposes to study neurons and are now being used to systematically document and interrogate astrocyte biology in vivo. These tools, their uses and limitations, and the insights that they afford are summarized in this Review.

Astrocytes are morphologically complex cells with numerous close contacts with neurons at the level of their somata, branches, and branchlets. The smallest astrocyte processes make discrete contacts with synapses at scales that cannot be observed by standard light microscopy. At such contact points, astrocytes are thought to perform both homeostatic and neuromodulatory roles-functions that are proposed to be determined by their close spatial apposition. To study such spatial interactions, we previously developed a Förster resonance energy transfer (FRET)-based approach, which enables observation and tracking of the static and dynamic proximity of astrocyte processes with synapses. The approach is compatible with standard imaging techniques such as confocal microscopy and permits assessment of the most proximate contacts between astrocytes and neurons in live tissues. In this protocol article we describe the approach to analyze the contacts between striatal astrocyte processes and corticostriatal neuronal projection terminals onto medium spiny neurons. We report the required protocols in detail, including adeno-associated virus microinjections, acute brain slice preparation, imaging, and post hoc FRET quantification. The article provides a detailed description that can be used to characterize and study astrocyte process proximity to synapses in living tissue. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Förster resonance energy transfer imaging in cultured cells Basic Protocol 2: Förster resonance energy transfer imaging with the neuron-astrocyte proximity assay in acute brain slices., (© 2020 John Wiley & Sons, Inc.)

Temporal lobe epilepsy causes severe cognitive deficits, but the circuit mechanisms remain unknown. Interneuron death and reorganization during epileptogenesis may disrupt the synchrony of hippocampal inhibition. To test this, we simultaneously recorded from the CA1 and dentate gyrus in pilocarpine-treated epileptic mice with silicon probes during head-fixed virtual navigation. We found desynchronized interneuron firing between the CA1 and dentate gyrus in epileptic mice. Since hippocampal interneurons control information processing, we tested whether CA1 spatial coding was altered in this desynchronized circuit, using a novel wire-free miniscope. We found that CA1 place cells in epileptic mice were unstable and completely remapped across a week. This spatial instability emerged around 6 weeks after status epilepticus, well after the onset of chronic seizures and interneuron death. Finally, CA1 network modeling showed that desynchronized inputs can impair the precision and stability of CA1 place cells. Together, these results demonstrate that temporally precise intrahippocampal communication is critical for spatial processing.

Astrocytes are implicated in neurodegenerative disorders and may contribute to striatal neuron loss or dysfunction in Huntington's disease (HD). Here, we assessed striatal astrocyte gene and protein signatures in two HD mouse models at three stages and compared our results to human HD data at four clinical grades and to mice exhibiting polyglutamine length-dependent pathology. We found disease-model and stage-specific alterations and discovered a core disease-associated astrocyte molecular signature comprising 62 genes that were conserved between mice and humans. Our results show little evidence of neurotoxic A1 astrocytes that have been proposed to be causal for neuronal death in neurodegenerative disorders such as HD. Furthermore, 61 of the 62-core gene expression changes within astrocytes were reversed in a HD mouse model by lowering astrocyte mutant huntingtin protein (mHTT) expression using zinc finger protein (ZFP) transcriptional repressors. Our findings indicate that HD astrocytes progressively lose essential normal functions, some of which can be remedied by lowering mHTT. The data have implications for neurodegenerative disease rescue and repair strategies as well as specific therapeutic relevance for mHTT reduction and contribute to a better understanding of fundamental astrocyte biology and its contributions to disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Astrocytes are essential components of neural circuits. They tile the entire central nervous system (CNS) and are involved in a variety of functions, which include neurotransmitter clearance, ion regulation, synaptic modulation, metabolic support to neurons, and blood flow regulation. Astrocytes are complex cells that have a soma, several major branches, and numerous fine processes that contact diverse cellular elements within the neuropil. In order to assess the morphology of astrocytes, it is necessary to have a reliable and reproducible method to visualize their structure. We report a reliable protocol to perform intracellular iontophoresis of astrocytes using fluorescent Lucifer yellow (LY) dye in lightly fixed brain tissue from adult mice. This method has several features that are useful to characterize astrocyte morphology. It allows for three-dimensional reconstruction of individual astrocytes, which is useful to perform morphological analyses on different aspects of their structure. Immunohistochemistry together with LY iontophoresis can also be utilized to understand the interaction of astrocytes with different components of nervous system and to evaluate the expression of proteins within the labelled astrocytes. This protocol can be implemented in a variety of mouse models of CNS disorders to rigorously examine astrocyte morphology with light microscopy. LY iontophoresis provides an experimental approach to evaluate astrocyte structure, especially in the context of injury or disease where these cells are proposed to undergo significant morphological changes.

The physiological functions of astrocytes within neural circuits remain incompletely understood. There has been progress in this regard from recent work on striatal astrocytes, where detailed studies are emerging. In this review, findings on striatal astrocyte identity, form, and function, are summarized with a focus on how astrocytes regulate striatal neurons, circuits, and behavior. Specific features of striatal astrocytes are highlighted to illustrate how they may be specialized to regulate medium spiny neurons (MSNs) by responding to, and altering, excitation and inhibition. Further experiments should reveal additional mechanisms for astrocyte-neuron interactions in the striatum and potentially reveal insights into the functions of astrocytes in neural circuits more generally., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Astrocytes are morphologically complex, ubiquitous cells that are viewed as a homogeneous population tiling the entire central nervous system (CNS). However, this view has been challenged in the last few years with the availability of RNA sequencing, immunohistochemistry, electron microscopy, morphological reconstruction, and imaging data. These studies suggest that astrocytes represent a diverse population of cells and that they display brain area- and disease-specific properties and functions. In this review, we summarize these observations, emphasize areas where clear conclusions can be made, and discuss potential unifying themes. We also identify knowledge gaps that need to be addressed in order to exploit astrocyte diversity as a biological phenomenon of physiological relevance in the CNS. We thus provide a summary and a perspective on astrocyte diversity in the vertebrate CNS.

Channelrhodopsin2 (ChR2) optogenetic excitation is widely used to study neurons, astrocytes, and circuits. Using complementary approaches in situ and in vivo, we found that ChR2 stimulation leads to significant transient elevation of extracellular potassium ions by ∼5 mM. Such elevations were detected in ChR2-expressing mice, following local in vivo expression of ChR2(H134R) with adeno-associated viruses (AAVs), in different brain areas and when ChR2 was expressed in neurons or astrocytes. In particular, ChR2-mediated excitation of striatal astrocytes was sufficient to increase medium spiny neuron (MSN) excitability and immediate early gene expression. The effects on MSN excitability were recapitulated in silico with a computational MSN model and detected in vivo as increased action potential firing in awake, behaving mice. We show that transient, physiologically consequential increases in extracellular potassium ions accompany ChR2 optogenetic excitation. This coincidental effect may be important to consider during astrocyte studies employing ChR2 to interrogate neural circuits and animal behavior., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Hyperactivity and disturbances of attention are common behavioral disorders whose underlying cellular and neural circuit causes are not understood. We report the discovery that striatal astrocytes drive such phenotypes through a hitherto unknown synaptic mechanism. We found that striatal medium spiny neurons (MSNs) triggered astrocyte signaling via γ-aminobutyric acid B (GABA B ) receptors. Selective chemogenetic activation of this pathway in striatal astrocytes in vivo resulted in acute behavioral hyperactivity and disrupted attention. Such responses also resulted in upregulation of the synaptogenic cue thrombospondin-1 (TSP1) in astrocytes, increased excitatory synapses, enhanced corticostriatal synaptic transmission, and increased MSN action potential firing in vivo. All of these changes were reversed by blocking TSP1 effects. Our data identify a form of bidirectional neuron-astrocyte communication and demonstrate that acute reactivation of a single latent astrocyte synaptogenic cue alters striatal circuits controlling behavior, revealing astrocytes and the TSP1 pathway as therapeutic targets in hyperactivity, attention deficit, and related psychiatric disorders., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adenosine 5' triphosphate (ATP) is a universal intracellular energy source and an evolutionarily ancient, ubiquitous extracellular signal in diverse species. Here, we report the generation and characterization of single-wavelength genetically encoded fluorescent sensors (iATPSnFRs) for imaging extracellular and cytosolic ATP from insertion of circularly permuted superfolder GFP into the epsilon subunit of F 0 F 1 -ATPase from Bacillus PS3. On the cell surface and within the cytosol, iATPSnFR 1.0 responds to relevant ATP concentrations (30 μM to 3 mM) with fast increases in fluorescence. iATPSnFRs can be genetically targeted to specific cell types and sub-cellular compartments, imaged with standard light microscopes, do not respond to other nucleotides and nucleosides, and when fused with a red fluorescent protein function as ratiometric indicators. After careful consideration of their modest pH sensitivity, iATPSnFRs represent promising reagents for imaging ATP in the extracellular space and within cells during a variety of settings, and for further application-specific refinements.

Advances in fluorescence microscopy enable monitoring larger brain areas in-vivo with finer time resolution. The resulting data rates require reproducible analysis pipelines that are reliable, fully automated, and scalable to datasets generated over the course of months. We present CaImAn, an open-source library for calcium imaging data analysis. CaImAn provides automatic and scalable methods to address problems common to pre-processing, including motion correction, neural activity identification, and registration across different sessions of data collection. It does this while requiring minimal user intervention, with good scalability on computers ranging from laptops to high-performance computing clusters. CaImAn is suitable for two-photon and one-photon imaging, and also enables real-time analysis on streaming data. To benchmark the performance of CaImAn we collected and combined a corpus of manual annotations from multiple labelers on nine mouse two-photon datasets. We demonstrate that CaImAn achieves near-human performance in detecting locations of active neurons., Competing Interests: AG, JF, PG, JK, BB, SK, JT, FN, JG, PZ, BK, DT, DC, EP No competing interests declared, (© 2019, Giovannucci et al.)

Astrocytes tile the central nervous system, but their functions in neural microcircuits in vivo and their roles in mammalian behavior remain incompletely defined. We used two-photon laser scanning microscopy, electrophysiology, MINIscopes, RNA-seq, and a genetic approach to explore the effects of reduced striatal astrocyte Ca 2+ signaling in vivo. In wild-type mice, reducing striatal astrocyte Ca 2+ -dependent signaling increased repetitive self-grooming behaviors by altering medium spiny neuron (MSN) activity. The mechanism involved astrocyte-mediated neuromodulation facilitated by ambient GABA and was corrected by blocking astrocyte GABA transporter 3 (GAT-3). Furthermore, in a mouse model of Huntington's disease, dysregulation of GABA and astrocyte Ca 2+ signaling accompanied excessive self-grooming, which was relieved by blocking GAT-3. Assessments with RNA-seq revealed astrocyte genes and pathways regulated by Ca 2+ signaling in a cell-autonomous and non-cell-autonomous manner, including Rab11a, a regulator of GAT-3 functional expression. Thus, striatal astrocytes contribute to neuromodulation controlling mouse obsessive-compulsive-like behavior., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Spreading depolarization is assumed to be the mechanism of migraine with aura, which is accompanied by an initial predominant hyperaemic response followed by persistent vasoconstriction. Cerebral blood flow responses are impaired in patients and in experimental animals after spreading depolarization. Understanding the regulation of cortical blood vessels during and after spreading depolarization could help patients with migraine attacks, but our knowledge of these vascular mechanisms is still incomplete. Recent findings show that control of cerebral blood flow does not only occur at the arteriole level but also at capillaries. Pericytes are vascular mural cells that can constrict or relax around capillaries, mediating local cerebral blood flow control. They participate in the constriction observed during brain ischaemia and might be involved the disruption of the microcirculation during spreading depolarization. To further understand the regulation of cerebral blood flow in spreading depolarization, we examined penetrating arterioles and capillaries with respect to vascular branching order, pericyte location and pericyte calcium responses during somatosensory stimulation and spreading depolarization. Mice expressing a red fluorescent indicator and intravenous injections of FITC-dextran were used to visualize pericytes and vessels, respectively, under two-photon microscopy. By engineering a genetically encoded calcium indicator we could record calcium changes in both pericytes around capillaries and vascular smooth muscle cells around arterioles. We show that somatosensory stimulation evoked a decrease in cytosolic calcium in pericytes located on dilating capillaries, up to the second order capillaries. Furthermore, we show that prolonged vasoconstriction following spreading depolarization is strongest in first order capillaries, with a persistent increase in pericyte calcium. We suggest that the persistence of the 'spreading cortical oligaemia' in migraine could be caused by this constriction of cortical capillaries. After spreading depolarization, somatosensory stimulation no longer evoked changes in capillary diameter and pericyte calcium. Thus, calcium changes in pericytes located on first order capillaries may be a key determinant in local blood flow control and a novel vascular mechanism in migraine. We suggest that prevention or treatment of capillary constriction in migraine with aura, which is an independent risk factor for stroke, may be clinically useful.

Potassium ions significantly contribute to the resting membrane potential of cells and, therefore, extracellular K + concentration is a crucial regulator of cell excitability. Altered concentrations of extracellular K + affect the resting membrane potential and cellular excitability by shifting the equilibria between closed, open and inactivated states for voltage-dependent ion channels that underlie action potential initiation and conduction. Hence, it is valuable to directly measure extracellular K + dynamics in health and diseased states. Here, we describe how to make, calibrate and use monopolar K + -selective microelectrodes. We deployed them in adult hippocampal brain slices to measure electrically evoked K + concentration dynamics. The judicious use of such electrodes is an important part of the tool-kit needed to evaluate cellular and biophysical mechanisms that control extracellular K + concentrations in the nervous system.

Astrocytes are complex bushy cells that serve important functions through close contacts between their processes and synapses. However, the spatial interactions and dynamics of astrocyte processes relative to synapses have proven problematic to study in adult living brain tissue. Here, we report a genetically targeted neuron-astrocyte proximity assay (NAPA) to measure astrocyte-synapse spatial interactions within intact brain preparations and at synaptic distance scales. The method exploits resonance energy transfer between extracellularly displayed fluorescent proteins targeted to synapses and astrocyte processes. We validated the method in the striatal microcircuitry following in vivo expression. We determined the proximity of striatal astrocyte processes to distinct neuronal input pathways, to D1 and D2 medium spiny neuron synapses, and we evaluated how astrocyte-to-excitatory synapse proximity changed following cortical afferent stimulation, during ischemia and in a model of Huntington's disease. NAPA provides a simple approach to measure astrocyte-synapse spatial interactions in a variety of experimental scenarios. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)