Astrocytes are endogenous regulators of basal transmission at central synapses.

Basal synaptic transmission involves the release of neurotransmitters at individual synapses in response to a single action potential. Recent discoveries show that astrocytes modulate the activity of neuronal networks upon sustained and intense synaptic activity. However, their ability to regulate basal synaptic transmission remains ill defined and controversial. Here, we show that astrocytes in the hippocampal CA1 region detect synaptic activity induced by single-synaptic stimulation. Astrocyte activation occurs at functional compartments found along astrocytic processes and involves metabotropic glutamate subtype 5 receptors. In response, astrocytes increase basal synaptic transmission, as revealed by the blockade of their activity with a Ca(2+) chelator. Astrocytic modulation of basal synaptic transmission is mediated by the release of purines and the activation of presynaptic A(2A) receptors by adenosine. Our work uncovers an essential role for astrocytes in the regulation of elementary synaptic communication and provides insight into fundamental aspects of brain function.

Interpericyte tunnelling nanotubes regulate neurovascular coupling.

Signalling between cells of the neurovascular unit, or neurovascular coupling, is essential to match local blood flow with neuronal activity. Pericytes interact with endothelial cells and extend processes that wrap capillaries, covering up to 90% of their surface area1,2. Pericytes are candidates to regulate microcirculatory blood flow because they are strategically positioned along capillaries, contain contractile proteins and respond rapidly to neuronal stimulation3,4, but whether they synchronize microvascular dynamics and neurovascular coupling within a capillary network was unknown. Here we identify nanotube-like processes that connect two bona fide pericytes on separate capillary systems, forming a functional network in the mouse retina, which we named interpericyte tunnelling nanotubes (IP-TNTs). We provide evidence that these (i) have an open-ended proximal side and a closed-ended terminal (end-foot) that connects with distal pericyte processes via gap junctions, (ii) carry organelles including mitochondria, which can travel along these processes, and (iii) serve as a conduit for intercellular Ca2+ waves, thus mediating communication between pericytes. Using two-photon microscope live imaging, we demonstrate that retinal pericytes rely on IP-TNTs to control local neurovascular coupling and coordinate light-evoked responses between adjacent capillaries. IP-TNT damage following ablation or ischaemia disrupts intercellular Ca2+ waves, impairing blood flow regulation and neurovascular coupling. Notably, pharmacological blockade of Ca2+ influx preserves IP-TNTs, rescues light-evoked capillary responses and restores blood flow after reperfusion. Our study thus defines IP-TNTs and characterizes their critical role in regulating neurovascular coupling in the living retina under both physiological and pathological conditions.

Astrocytes Transplanted during Early Postnatal Development Integrate, Mature, and Survive Long Term in Mouse Cortex.

Astrocytes have complex structural, molecular, and physiological properties and form specialized microenvironments that support circuit-specific functions in the CNS. To better understand how astrocytes acquire their unique features, we transplanted immature mouse cortical astrocytes into the developing cortex of male and female mice and assessed their integration, maturation, and survival. Within days, transplanted astrocytes developed morphologies and acquired territories and tiling behavior typical of cortical astrocytes. At 35-47 d post-transplantation, astrocytes appeared morphologically mature and expressed levels of EAAT2/GLT1 similar to nontransplanted astrocytes. Transplanted astrocytes also supported excitatory/inhibitory (E/I) presynaptic terminals within their territories, and displayed normal Ca2+ events. Transplanted astrocytes showed initially reduced expression of aquaporin 4 (AQP4) at endfeet and elevated expression of EAAT1/GLAST, with both proteins showing normalized expression by 110 d and one year post-transplantation, respectively. To understand how specific brain regions support astrocytic integration and maturation, we transplanted cortical astrocytes into the developing cerebellum. Cortical astrocytes interlaced with Bergmann glia (BG) in the cerebellar molecular layer to establish discrete territories. However, transplanted astrocytes retained many cortical astrocytic features including higher levels of EAAT2/GLT1, lower levels of EAAT1/GLAST, and the absence of expression of the AMPAR subunit GluA1. Collectively, our findings demonstrate that immature cortical astrocytes integrate, mature, and survive (more than one year) following transplantation and retain cortical astrocytic properties. Astrocytic transplantation can be useful for investigating cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms contributing to astrocytic development/diversity, and for determining the optimal timing for transplanting astrocytes for cellular delivery or replacement in regenerative medicine.SIGNIFICANCE STATEMENT The mechanisms that enable astrocytes to acquire diverse molecular and structural properties remain to be better understood. In this study, we systematically analyzed the properties of cortical astrocytes following their transplantation to the early postnatal brain. We found that immature cortical astrocytes transplanted into cerebral cortex during early postnatal mouse development integrate and establish normal astrocytic properties, and show long-term survival in vivo (more than one year). In contrast, transplanted cortical astrocytes display reduced or altered ability to integrate into the more mature cerebral cortex or developing cerebellum, respectively. This study demonstrates the developmental potential of transplanted cortical astrocytes and provides an approach to tease apart cell-autonomous (intrinsic) and non-cell-autonomous (environmental) mechanisms that determine the structural, molecular, and physiological phenotype of astrocytes.

Cooperative and competitive regulation of the astrocytic transcriptome by neurons and endothelial cells: Impact on astrocyte maturation.

Astrocytes have essential roles in central nervous system (CNS) health and disease. During development, immature astrocytes show complex interactions with neurons, endothelial cells, and other glial cell types. Our work and that of others have shown that these interactions are important for astrocytic maturation. However, whether and how these cells work together to control this process remains poorly understood. Here, we test the hypothesis that cooperative interactions of astrocytes with neurons and endothelial cells promote astrocytic maturation. Astrocytes were cultured alone, with neurons, endothelial cells, or a combination of both. This was followed by astrocyte sorting, RNA sequencing, and bioinformatic analysis to detect transcriptional changes. Across culture configurations, 7302 genes were differentially expressed by 4 or more fold and organized into 8 groups that demonstrate cooperative and antagonist effects of neurons and endothelia on astrocytes. We also discovered that neurons and endothelial cells caused splicing of 200 and 781 mRNAs, respectively. Changes in gene expression were validated using quantitative PCR, western blot (WB), and immunofluorescence analysis. We found that the transcriptomic data from the three-culture configurations correlated with protein expression of three representative targets (FAM107A, GAT3, and GLT1) in vivo. Alternative splicing results also correlated with cortical tissue isoform representation of a target (Fibronectin 1) at different developmental stages. By comparing our results to published transcriptomes of immature and mature astrocytes, we found that neurons or endothelia shift the astrocytic transcriptome toward a mature state and that the presence of both cell types has a greater effect on maturation than either cell alone. These results increase our understanding of cellular interactions/pathways that contribute to astrocytic maturation. They also provide insight into how alterations to neurons and/or endothelial cells may alter astrocytes with implications for astrocytic changes in CNS disorders and diseases.

Human iPSC-derived Down syndrome astrocytes display genome-wide perturbations in gene expression, an altered adhesion profile, and increased cellular dynamics.

Down syndrome (DS), caused by the triplication of human chromosome 21, leads to significant alterations in brain development and is a major genetic cause of intellectual disability. While much is known about changes to neurons in DS, the effects of trisomy 21 on non-neuronal cells such as astrocytes are poorly understood. Astrocytes are critical for brain development and function, and their alteration may contribute to DS pathophysiology. To better understand the impact of trisomy 21 on astrocytes, we performed RNA-sequencing on astrocytes from newly produced DS human induced pluripotent stem cells (hiPSCs). While chromosome 21 genes were upregulated in DS astrocytes, we found consistent up- and down-regulation of genes across the genome with a strong dysregulation of neurodevelopmental, cell adhesion and extracellular matrix molecules. ATAC (assay for transposase-accessible chromatin)-seq also revealed a global alteration in chromatin state in DS astrocytes, showing modified chromatin accessibility at promoters of cell adhesion and extracellular matrix genes. Along with these transcriptomic and epigenomic changes, DS astrocytes displayed perturbations in cell size and cell spreading as well as modifications to cell-cell and cell-substrate recognition/adhesion, and increases in cellular motility and dynamics. Thus, triplication of chromosome 21 is associated with genome-wide transcriptional, epigenomic and functional alterations in astrocytes that may contribute to altered brain development and function in DS.

Light microscopic and heterogeneity analysis of astrocytes in the common marmoset brain.

Astrocytes are abundant cells of the central nervous system (CNS) and are involved in processes including synapse formation/function, ion homeostasis, neurotransmitter uptake, and neurovascular coupling. Recent evidence indicates that astrocytes show diverse molecular, structural, and physiological properties within the CNS. This heterogeneity is reflected in differences in astrocyte structure, gene expression, functional properties, and responsiveness to injury/pathological conditions. Deeper investigation of astrocytic heterogeneity is needed to understand how astrocytes are configured to enable diverse roles in the CNS. While much has been learned about astrocytic heterogeneity in rodents, much less is known about astrocytic heterogeneity in the primate brain where astrocytes have greater size and complexity. The common marmoset (Callithrix jacchus) is a promising non-human primate model because of similarities between marmosets and humans with respect to genetics, brain anatomy, and cognition/behavior. Here, we investigated the molecular and structural heterogeneity of marmoset astrocytes using an array of astrocytic markers, multi-label confocal microscopy, and quantitative analysis. We used male and female marmosets and found that marmoset astrocytes show differences in expression of astrocytic markers in cortex, hippocampus, and cerebellum. These differences were accompanied by intra-regional variation in expression of markers for glutamate/GABA transporters, and potassium and water channels. Differences in astrocyte structure were also found, along with complex interactions with blood vessels, microglia, and neurons. This study contributes to our knowledge of the cellular and molecular features of marmoset astrocytes and is useful for understanding the complex properties of astrocytes in the primate CNS.

MIC-MAC: An automated pipeline for high-throughput characterization and classification of three-dimensional microglia morphologies in mouse and human postmortem brain samples.

The phenotypic changes of microglia in brain diseases are particularly diverse and their role in disease progression, beneficial, or detrimental, is still elusive. High-throughput molecular approaches such as single-cell RNA-sequencing can now resolve the high heterogeneity in microglia population for a specific physiological condition, however, the relation between the different microglial signatures and their surrounding brain microenvironment is barely understood. Thus, better tools to characterize the phenotypic variations of microglia in situ are needed, particularly for human brain postmortem samples analysis. To address this challenge, we developed MIC-MAC, a Microglia and Immune Cells Morphologies Analyser and Classifier pipeline that semiautomatically segments, extracts, and classifies all microglia and immune cells labeled in large three-dimensional (3D) confocal image stacks of mouse and human brain samples. Our imaging-based approach enables automatic 3D-morphology characterization and classification of thousands of individual microglia in situ and revealed species- and disease-specific morphological phenotypes in mouse aging, human Alzheimer's disease, and dementia with Lewy Bodie's samples. MIC-MAC is a precision diagnostic tool that allows a rapid, unbiased, and large-scale analysis of microglia morphological states in mouse models and patient brain samples.

Detection of activity-dependent vasopressin release from neuronal dendrites and axon terminals using sniffer cells.

Our understanding of neuropeptide function within neural networks would be improved by methods allowing dynamic detection of peptide release in living tissue. We examined the usefulness of sniffer cells as biosensors to detect endogenous vasopressin (VP) release in rat hypothalamic slices and from isolated neurohypophyses. Human embryonic kidney cells were transfected to express the human V1a VP receptor (V1aR) and the genetically encoded calcium indicator GCaMP6m. The V1aR couples to Gq11, thus VP binding to this receptor causes an increase in intracellular [Ca2+] that can be detected by a rise in GCaMP6 fluorescence. Dose-response analysis showed that VP sniffer cells report ambient VP levels >10 pM (EC50 = 2.6 nM), and this effect could be inhibited by the V1aR antagonist SR 49059. When placed over a coverslip coated with sniffer cells, electrical stimulation of the neurohypophysis provoked a reversible, reproducible, and dose-dependent increase in VP release using as few as 60 pulses delivered at 3 Hz. Suspended sniffer cells gently plated over a slice adhered to the preparation and allowed visualization of VP release in discrete regions. Electrical stimulation of VP neurons in the suprachiasmatic nucleus caused significant local release as well as VP secretion in distant target sites. Finally, action potentials evoked in a single magnocellular neurosecretory cell in the supraoptic nucleus provoked significant VP release from the somatodendritic compartment of the neuron. These results indicate that sniffer cells can be used for the study of VP secretion from various compartments of neurons in living tissue. NEW & NOTEWORTHY The specific functional roles of neuropeptides in neuronal networks are poorly understood due to the absence of methods allowing their real-time detection in living tissue. Here, we show that cultured "sniffer cells" can be engineered to detect endogenous release of vasopressin as an increase in fluorescence.

In vivo regulation of steroid hormones by the Chst10 sulfotransferase in mouse.

Chst10 adds sulfate to glucuronic acid to form a carbohydrate antigen, HNK-1, in glycoproteins and glycolipids. To determine the role of Chst10 in vivo, we generated systemic Chst10-deficient mutant mice. Although Chst10(-/-) mice were born and grew to adulthood with no gross defects, they were subfertile. Uteri from Chst10(-/-) females at the pro-estrus stage were larger than those from wild-type females and exhibited a thick uterine endometrium. Serum estrogen levels in Chst10(-/-) females were higher than those from wild-type females, suggesting impaired down-regulation of estrogen. Because steroid hormones are often conjugated to glucuronic acid, we hypothesized that Chst10 sulfates glucuronidated steroid hormone to regulate steroid hormone in vivo. Enzymatic activity assays and structural analysis of Chst10 products by HPLC and mass spectrometry revealed that Chst10 indeed sulfates glucuronidated estrogen, testosterone, and other steroid hormones. We also identified an HPLC peak corresponding to sulfated and glucuronidated estradiol in serum from wild-type but not from Chst10 null female mice. Estrogen-response element reporter assays revealed that Chst10-modified estrogen likely did not bind to its receptor. These results suggest that subfertility exhibited by female mice following Chst10 loss results from dysregulation of estrogen. Given that Chst10 transfers sulfates to several steroid hormones, Chst10 likely functions in widespread regulation of steroid hormones in vivo.

Comparative analyses of the Smith-Magenis syndrome protein RAI1 in mice and common marmoset monkeys.

Retinoic acid-induced 1 (RAI1) encodes a transcriptional regulator critical for brain development and function. RAI1 haploinsufficiency in humans causes a syndromic autism spectrum disorder known as Smith-Magenis syndrome (SMS). The neuroanatomical distribution of RAI1 has not been quantitatively analyzed during the development of the prefrontal cortex, a brain region critical for cognitive function and social behaviors and commonly implicated in autism spectrum disorders, including SMS. Here, we performed comparative analyses to uncover the evolutionarily convergent and divergent expression profiles of RAI1 in major cell types during prefrontal cortex maturation in common marmoset monkeys (Callithrix jacchus) and mice (Mus musculus). We found that while RAI1 in both species is enriched in neurons, the percentage of excitatory neurons that express RAI1 is higher in newborn mice than in newborn marmosets. By contrast, RAI1 shows similar neural distribution in adult marmosets and adult mice. In marmosets, RAI1 is expressed in several primate-specific cell types, including intralaminar astrocytes and MEIS2-expressing prefrontal GABAergic neurons. At the molecular level, we discovered that RAI1 forms a protein complex with transcription factor 20 (TCF20), PHD finger protein 14 (PHF14), and high mobility group 20A (HMG20A) in the marmoset brain. In vitro assays in human cells revealed that TCF20 regulates RAI1 protein abundance. This work demonstrates that RAI1 expression and protein interactions are largely conserved but with some unique expression in primate-specific cells. The results also suggest that altered RAI1 abundance could contribute to disease features in disorders caused by TCF20 dosage imbalance.

EphA signaling promotes actin-based dendritic spine remodeling through slingshot phosphatase.

Actin cytoskeletal remodeling plays a critical role in transforming the morphology of subcellular structures across various cell types. In the brain, restructuring of dendritic spines through actin cytoskeleletal reorganization is implicated in the regulation of synaptic efficacy and the storage of information in neural circuits. However, the upstream pathways that provoke actin-based spine changes remain only partly understood. Here we show that EphA receptor signaling remodels spines by triggering a sequence of events involving actin filament rearrangement and synapse/spine reorganization. Rapid EphA signaling over minutes activates the actin filament depolymerizing/severing factor cofilin, alters F-actin distribution in spines, and causes transient spine elongation through the phosphatases slingshot 1 (SSH1) and calcineurin/protein phosphatase 2B (PP2B). This early phase of spine extension is followed by synaptic reorganization events that take place over minutes to hours and involve the relocation of pre/postsynaptic components and ultimately spine retraction. Thus, EphA receptors utilize discrete cellular and molecular pathways to promote actin-based structural plasticity of excitatory synapses.

Activity maintains structural plasticity of mossy fiber terminals in the hippocampus.

Neural activity plays an important role in organizing and optimizing neural circuits during development and in the mature nervous system. However, the cellular events that underlie this process still remain to be fully understood. In this study, we investigated the role of neural activity in regulating the structural plasticity of presynaptic terminals in the hippocampal formation. We designed a virus to drive the Drosophila Allatostatin receptor in individual dentate granule neurons to suppress activity of complex mossy fiber terminals 'on-demand' in organotypic slices and used time-lapse confocal imaging to determine the impact on presynaptic remodeling. We found that activity played an important role in maintaining the structural plasticity of the core region of the mossy fiber terminal (MFT) that synapses onto CA3 pyramidal cell thorny excrescences but was not essential for the motility of terminal filopodial extensions that contact local inhibitory neurons. Short-term suppression of activity did not have an impact on the size of the MFT, however, longer-term suppression reduced the overall size of the MFT. Remarkably, global blockade of activity with tetrodotoxin (TTX) interfered with the ability of single cell activity deprivation to slow down terminal dynamics suggesting that differences in activity levels among neighboring synapses promote synaptic remodeling events. The results from our studies indicate that neural activity plays an important role in maintaining structural plasticity of presynaptic compartments in the central nervous system and provide new insight into the time-frame during which activity can affect the morphology of synaptic connections.

Astrocyte morphology.

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.

Organizing principles of astrocytic nanoarchitecture in the mouse cerebral cortex.

Astrocytes are increasingly understood to be important regulators of central nervous system (CNS) function in health and disease; yet, we have little quantitative understanding of their complex architecture. While broad categories of astrocytic structures are known, the discrete building blocks that compose them, along with their geometry and organizing principles, are poorly understood. Quantitative investigation of astrocytic complexity is impeded by the absence of high-resolution datasets and robust computational approaches to analyze these intricate cells. To address this, we produced four ultra-high-resolution datasets of mouse cerebral cortex using serial electron microscopy and developed astrocyte-tailored computer vision methods for accurate structural analysis. We unearthed specific anatomical building blocks, structural motifs, connectivity hubs, and hierarchical organizations of astrocytes. Furthermore, we found that astrocytes interact with discrete clusters of synapses and that astrocytic mitochondria are distributed to lie closer to larger clusters of synapses. Our findings provide a geometrically principled, quantitative understanding of astrocytic nanoarchitecture and point to an unexpected level of complexity in how astrocytes interact with CNS microanatomy.

Astrocytes display complex and localized calcium responses to single-neuron stimulation in the hippocampus.

Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron-glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manipulations known to alter calcium levels in astrocytes. Light-evoked neuronal depolarization was elicited and calcium events in surrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity.

Astrocytes control glutamate receptor levels at developing synapses through SPARC-beta-integrin interactions.

Neurons recruit numerous mechanisms to facilitate the development of synaptic connections. However, little is known about activity-dependent mechanisms that control the timing and fidelity of this process. Here we describe a novel pathway used by neurons to regulate glutamate receptors at maturing central synapses. This pathway relies on communication between neurons and astrocytes and the ability of astrocytes to release the factor SPARC (secreted protein, acidic and rich in cysteine). SPARC expression is dynamically regulated and plays a critical role in determining the level of synaptic AMPARs. SPARC ablation in mice increases excitatory synapse function, causes an abnormal accumulation of surface AMPARs at synapses, and impairs synaptic plasticity during development. We further demonstrate that SPARC inhibits the properties of neuronal ß3-integrin complexes, which are intimately coupled to AMPAR stabilization at synapses. Thus neuron-glial signals control glutamate receptor levels at developing synapses to enable activity-driven modifications of synaptic strength.

Glial glutamate transport modulates dendritic spine head protrusions in the hippocampus.

Accumulating evidence supports the idea that synapses are tripartite, whereby perisynaptic astrocytes modulate both pre- and postsynaptic function. Although some of these features have been uncovered by using electrophysiological methods, less is known about the structural interplay between synapses and glial processes. Here, we investigated how astrocytes govern the plasticity of individual hippocampal dendritic spines. Recently, we uncovered that a subgroup of innervated dendritic spines is able to undergo remodeling by extending spine head protrusions (SHPs) toward neighboring functional presynaptic boutons, resulting in new synapses. Although glutamate serves as a trigger, how this behavior is regulated is unknown. As astrocytes control extracellular glutamate levels through their high-affinity uptake transporters, together with their privileged access to synapses, we investigated a role for astrocytes in SHP formation. Using time-lapse confocal microscopy, we found that the volume overlap between spines and astrocytic processes decreased during the formation of SHPs. Focal application of glutamate also reduced spine-astrocyte overlap and induced SHPs. Importantly, SHP formation was prevented by blocking glial glutamate transporters, suggesting that glial control of extracellular glutamate is important for SHP-mediated plasticity of spines. Hence, the dynamic changes of both spines and astrocytes can rapidly modify synaptic connectivity.

The EphA4 receptor regulates dendritic spine remodeling by affecting beta1-integrin signaling pathways.

Remodeling of dendritic spines is believed to modulate the function of excitatory synapses. We previously reported that the EphA4 receptor tyrosine kinase regulates spine morphology in hippocampal pyramidal neurons, but the signaling pathways involved were not characterized (Murai, K.K., L.N. Nguyen, F. Irie, Y. Yamaguchi, and E.B. Pasquale. 2003. Nat. Neurosci. 6:153-160). In this study, we show that EphA4 activation by ephrin-A3 in hippocampal slices inhibits integrin downstream signaling pathways. EphA4 activation decreases tyrosine phosphorylation of the scaffolding protein Crk-associated substrate (Cas) and the tyrosine kinases focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) and also reduces the association of Cas with the Src family kinase Fyn and the adaptor Crk. Consistent with this, EphA4 inhibits beta1-integrin activity in neuronal cells. Supporting a functional role for beta1 integrin and Cas inactivation downstream of EphA4, the inhibition of integrin or Cas function induces spine morphological changes similar to those associated with EphA4 activation. Furthermore, preventing beta1-integrin inactivation blocks the effects of EphA4 on spines. Our results support a model in which EphA4 interferes with integrin signaling pathways that stabilize dendritic spines, thus modulating synaptic interactions with the extracellular environment.

Glial ephrin-A3 regulates hippocampal dendritic spine morphology and glutamate transport.

Increasing evidence indicates the importance of neuron-glia communication for synaptic function, but the mechanisms involved are not fully understood. We reported that the EphA4 receptor tyrosine kinase is in dendritic spines of pyramidal neurons of the adult hippocampus and regulates spine morphology. We now show that the ephrin-A3 ligand, which is located in the perisynaptic processes of astrocytes, is essential for maintaining EphA4 activation and normal spine morphology in vivo. Ephrin-A3-knockout mice have spine irregularities similar to those observed in EphA4-knockout mice. Remarkably, loss of ephrin-A3 or EphA4 increases the expression of glial glutamate transporters. Consistent with this, glutamate transport is elevated in ephrin-A3-null hippocampal slices whereas Eph-dependent stimulation of ephrin-A3 signaling inhibits glutamate transport. Furthermore, some forms of hippocampus-dependent learning are impaired in the ephrin-A3-knockout mice. Our results suggest that the interaction between neuronal EphA4 and glial ephrin-A3 bidirectionally controls synapse morphology and glial glutamate transport, ultimately regulating hippocampal function.

Foundations and implications of astrocyte heterogeneity during brain development and disease.

Astrocytes play crucial roles in regulating brain circuit formation and physiology. Recent technological advances have revealed unprecedented levels of astrocyte diversity encompassing molecular, morphological, and functional differences. This diversification is initiated during embryonic specification events and (in rodents) continues into the early postnatal period where it overlaps with peak synapse development and circuit refinement. In fact, several lines of evidence suggest astrocyte diversity both influences and is a consequence of molecular crosstalk among developing astrocytes and other cell types, notably neurons and their synapses. Neurological disease states exhibit additional layers of astrocyte heterogeneity, which could help shed light on these cells' key pathological roles. This review highlights recent advances in clarifying astrocyte heterogeneity and molecular/cellular crosstalk and identifies key outstanding questions.