Perisynaptic Astrocytic Processes Research Articles

Ultrastructure of neuron-glia interaction in the norm and experimental pathology

Long-term experience was gained in analyzing synapses and their glial surroundings in normal, natural, and experimental models of functional plasticity and brain pathology development. However, most studies in this area rely on electrophysiological techniques combined with fluorescent imaging. Notably, fine synapse structure and astrocytic processes cannot be resolved using light or some electron microscopy techniques. Studies on experimental brain pathology using volume electron microscopy methods [1] were previously restricted due to their high labor intensity. However, automated methods for sample preparation and analysis, using machine vision and artificial intelligence, significantly simplified this task. Using transmission electron microscopy methods and 3D reconstructions, this study examined the Str. radiatum CA1 hippocampal region of rat brains in a chronic lithium-pilocarpine model of epilepsy. The results indicate a decrease in synaptic number along with an increase in their size and a reduction in astrocytic isolation of the active zones. A decrease in glial ensheathment of enlarged active zones and facilitation of neurotransmitter diffusion to active synapses may have a multiplicative effect on epileptiform activity growth and excitotoxicity. The simplification of the astrocytes meshwork in the somatosensory cortex’s layer 2/3 is comparable to that of layer 1. This decrease in layer 1’s inhibitory action enables pyramidal neurons in layer 2/3 to potentially exhibit epileptiform activity. Thus, the superficial cortical layers’ structural-functional aspects can be used as a natural cellular model in epilepsy development studies. Reduction of Ca2+ events in astrocyte processes in lithium-pilocarpine induced epilepsy may result from the low buffer capacity of Ca2+ ions in the smooth endoplasmic reticulum (sER) and/or impaired Ca2+ wave transmission through the gap junctions between astrocytic processes. High resolution is necessary to analyze the gap junctions, and special methods are required for sER visualization in perisynaptic astrocytic processes. We developed original sER staining methods [2] to quantitatively evaluate gap junctions and sER cisternae within astrocytic meshworks in layers 1 and 2/3 of the somatosensory cortex. In layer 1, the area of gap junctions in relation to the volume of an astrocyte was twice as high as in layer 2/3. The proportion of sER volume differed between layer 1 and layer 2/3 tissues. Specifically, the total sER cisternae volume in layer 2/3 was twice as high as in layer 1, relative to the volume of astrocytic processes [3]. Additionally, a doubling of single astrocytic gap junction area concomitant with decreased calcium events was observed. The results suggest a normal balance between Ca2+ stores (sER) and gap junctions, whose disruption may contribute to the development of seizures.

Heroin Self-Administration and Extinction Increase Prelimbic Cortical Astrocyte-Synapse Proximity and Alter Dendritic Spine Morphometrics That Are Reversed by N-Acetylcysteine.

Clinical and preclinical studies indicate that adaptations in corticostriatal neurotransmission significantly contribute to heroin relapse vulnerability. In animal models, heroin self-administration and extinction produce cellular adaptations in both neurons and astrocytes within the nucleus accumbens (NA) core that are required for cue-induced heroin seeking. Specifically, decreased glutamate clearance and reduced association of perisynaptic astrocytic processes with NAcore synapses allow glutamate release from prelimbic (PrL) cortical terminals to engage synaptic and structural plasticity in NAcore medium spiny neurons. Normalizing astrocyte glutamate homeostasis with drugs like the antioxidant N-acetylcysteine (NAC) prevents cue-induced heroin seeking. Surprisingly, little is known about heroin-induced alterations in astrocytes or pyramidal neurons projecting to the NAcore in the PrL cortex (PrL-NAcore). Here, we observe functional adaptations in the PrL cortical astrocyte following heroin self-administration (SA) and extinction as measured by the electrophysiologically evoked plasmalemmal glutamate transporter 1 (GLT-1)-dependent current. We likewise observed the increased complexity of the glial fibrillary acidic protein (GFAP) cytoskeletal arbor and increased association of the astrocytic plasma membrane with synaptic markers following heroin SA and extinction training in the PrL cortex. Repeated treatment with NAC during extinction reversed both the enhanced astrocytic complexity and synaptic association. In PrL-NAcore neurons, heroin SA and extinction decreased the apical tuft dendritic spine density and enlarged dendritic spine head diameter in male Sprague-Dawley rats. Repeated NAC treatment during extinction prevented decreases in spine density but not dendritic spine head expansion. Moreover, heroin SA and extinction increased the co-registry of the GluA1 subunit of AMPA receptors in both the dendrite shaft and spine heads of PrL-NAcore neurons. Interestingly, the accumulation of GluA1 immunoreactivity in spine heads was further potentiated by NAC treatment during extinction. Finally, we show that the NAC treatment and elimination of thrombospondin 2 (TSP-2) block cue-induced heroin relapse. Taken together, our data reveal circuit-level adaptations in cortical dendritic spine morphology potentially linked to heroin-induced alterations in astrocyte complexity and association at the synapses. Additionally, these data demonstrate that NAC reverses PrL cortical heroin SA-and-extinction-induced adaptations in both astrocytes and corticostriatal neurons.

FABP7: a glial integrator of sleep, circadian rhythms, plasticity, and metabolic function.

Sleep and circadian rhythms are observed broadly throughout animal phyla and influence neural plasticity and cognitive function. However, the few phylogenetically conserved cellular and molecular pathways that are implicated in these processes are largely focused on neuronal cells. Research on these topics has traditionally segregated sleep homeostatic behavior from circadian rest-activity rhythms. Here we posit an alternative perspective, whereby mechanisms underlying the integration of sleep and circadian rhythms that affect behavioral state, plasticity, and cognition reside within glial cells. The brain-type fatty acid binding protein, FABP7, is part of a larger family of lipid chaperone proteins that regulate the subcellular trafficking of fatty acids for a wide range of cellular functions, including gene expression, growth, survival, inflammation, and metabolism. FABP7 is enriched in glial cells of the central nervous system and has been shown to be a clock-controlled gene implicated in sleep/wake regulation and cognitive processing. FABP7 is known to affect gene transcription, cellular outgrowth, and its subcellular localization in the fine perisynaptic astrocytic processes (PAPs) varies based on time-of-day. Future studies determining the effects of FABP7 on behavioral state- and circadian-dependent plasticity and cognitive processes, in addition to functional consequences on cellular and molecular mechanisms related to neural-glial interactions, lipid storage, and blood brain barrier integrity will be important for our knowledge of basic sleep function. Given the comorbidity of sleep disturbance with neurological disorders, these studies will also be important for our understanding of the etiology and pathophysiology of how these diseases affect or are affected by sleep.

The ribosome-associated protein RACK1 represses Kir4.1 translation in astrocytes and influences neuronal activity

The regulation of translation in astrocytes, the main glial cells in the brain, remains poorly characterized. We developed a high-throughput proteomics screen for polysome-associated proteins in astrocytes and focused on ribosomal protein receptor of activated protein C kinase 1 (RACK1), a critical factor in translational regulation. In astrocyte somata and perisynaptic astrocytic processes (PAPs), RACK1 preferentially binds to a number of mRNAs, including Kcnj10, encoding the inward-rectifying potassium (K+) channel Kir4.1. By developing an astrocyte-specific, conditional RACK1 knockout mouse model, we show that RACK1 represses production of Kir4.1 in hippocampal astrocytes and PAPs. Upregulation of Kir4.1 in the absence of RACK1 increases astrocytic Kir4.1-mediated K+ currents and volume. It also modifies neuronal activity attenuating burst frequency and duration. Reporter-based assays reveal that RACK1 controls Kcnj10 translation through the transcript's 5' untranslated region. Hence, translational regulation by RACK1 in astrocytes represses Kir4.1 expression and influences neuronal activity.

Two subcompartments of the smooth endoplasmic reticulum in perisynaptic astrocytic processes: ultrastructure and distribution in hippocampal and neocortical synapses

Perisynaptic astrocytic processes involved in the tripartite synapse functioning respond to its activation by local depolarization with calcium release from the intracellular stores inside nodes of astrocytic processes and develop local and generalized calcium events. However, based on the first electron microscopy studies a point of view was formed that terminal astrocytic lamellae are devoid of any organelles, including the main astrocytic calcium store - the cisternae of the smooth endoplasmic reticulum. Indeed, analysis of smooth endoplasmic reticulum cisternae could be limited by their weak electron contrast, the studying of astrocytic processes on single sections, and insufficient optical resolution of the equipment used. Here, by using serial section transmission electron microscopy and 3D reconstructions, we analyzed astrocytic processes in murine hippocampal and cortical synapses. As a result of unit membranes contrast enhancement, it was shown for the first time that perisynaptic processes of astrocytes with a morphology of thin branchlets contain two types of smooth endoplasmic reticulum cisternae and microvesicles. Unlike branchlets, membrane organelles inside terminal lamellae were comprised by only short fragments of thin smooth endoplasmic reticulum cister-nae and microvesicles, whose groups tend to be located in close proximity to active zones of the most active synapses. We speculate both on reliability of the alternative methods in electron microscopy while studying astrocytic microenvironment of synapses and structure-function aspects of smooth endoplasmic reticulum cisternae compartmentalization inside the perisynaptic processes of astrocytes.

A novel role for MLC1 in regulating astrocyte-synapse interactions.

Loss of function of the astrocyte membrane protein MLC1 is the primary genetic cause of the rare white matter disease Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC), which is characterized by disrupted brain ion and water homeostasis. MLC1 is prominently present around fluid barriers in the brain, such as in astrocyte endfeet contacting blood vessels and in processes contacting the meninges. Whether the protein plays a role in other astrocyte domains is unknown. Here, we show that MLC1 is present in distal astrocyte processes, also known as perisynaptic astrocyte processes (PAPs) or astrocyte leaflets, which closely interact with excitatory synapses in the CA1 region of the hippocampus. We find that the PAP tip extending toward excitatory synapses is shortened in Mlc1-null mice. This affects glutamatergic synaptic transmission, resulting in a reduced rate of spontaneous release events and slower glutamate re-uptake under challenging conditions. Moreover, while PAPs in wildtype mice retract from the synapse upon fear conditioning, we reveal that this structural plasticity is disturbed in Mlc1-null mice, where PAPs are already shorter. Finally, Mlc1-null mice show reduced contextual fear memory. In conclusion, our study uncovers an unexpected role for the astrocyte protein MLC1 in regulating the structure of PAPs. Loss of MLC1 alters excitatory synaptic transmission, prevents normal PAP remodeling induced by fear conditioning and disrupts contextual fear memory expression. Thus, MLC1 is a new player in the regulation of astrocyte-synapse interactions.

Two Subcompartments of the Smooth Endoplasmic Reticulum in Perisynaptic Astrocytic Processes: Ultrastructure and Distribution in Hippocampal and Neocortical Synapses

Two Subcompartments of the Smooth Endoplasmic Reticulum in Perisynaptic Astrocytic Processes: Ultrastructure and Distribution in Hippocampal and Neocortical Synapses

Necl2/3-mediated mechanism for tripartite synapse formation.

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

Development of an optogenetic TrkB.T1 probe.

Astrocytes are the most abundant cell type in the human brain and are involved in regulating synaptic transmission and synapse formation. Astrocytes have a unique star-like morphology containing multi-branched processes that reach sub-diffraction limited scales. Fine processes protruding from astrocytes are known to interact directly with synapses to form the canonical tripartite synapse. These Perisynaptic Astrocytic Processes (PAPs) can release and uptake various neuroactive molecules to regulate synaptic function. Astrocyte morphology and PAP interactions are dynamic and vary throughout different brain regions and in diseased states, suggesting that astrocyte branching dynamics are important for regulating brain function. Despite this, no tools have been developed to directly probe astrocyte morphology for in vivo studies. Previously, signaling via a truncated receptor tyrosine kinase, TrkB.T1, has been found to regulate astrocyte morphology. TrkB.T1 is a transmembrane receptor highly expressed in astrocytes. Binding of Brain Derived Nerve Growth Factor (BDNF) to the extracellular receptor of TrkB.T1 results in the dimerization of its 23 amino acid intracellular domain. This dimerization activates Phospholipase C (PLC) calcium signaling and inhibits RhoGTPases via the release of the RhoGTPase inhibitor RhoGDI1 into the cytosol. Application of BDNF to astrocytes in culture and brain slices has been shown to increased astrocyte branching and promote synapse formation. In this work, we develop Cry2 and iLID based optogenetic TrkB.T1 probes (OptoT1s). OptoT1s allows for light induced dimerization of the intracellular TrkB.T1 domain at the cell membrane, which should subsequently activate downstream TrkB.T1 signaling pathways. We are currently characterizing the ability of these OptoT1 probes to induce PLC-dependent calcium transients and RhoGTPase inhibition. The development and optimization of this optogenetic tool will allow for in vivo spatiotemporal control of astrocyte branching that can help us investigate the role of astrocyte morphology on brain function and diseases.

Electron microscopy analysis of astrocyte-synapse interactions shows altered dynamics in an Alzheimer's disease mouse model.

Astrocyte-synapse bi-directional communication is required for neuronal development and synaptic plasticity. Astrocytes structurally interact with synapses using their distal processes also known as leaflets or perisynaptic astrocytic processes (PAPs). We recently showed that these PAPs are retracted from hippocampal synapses, and involved in the consolidation of fear memory. However, whether astrocytic synaptic coverage is affected when memory is impaired is unknown. Here, we describe in detail an electron microscopy method that makes use of a large number of 2D images to investigate structural astrocyte-synapse interaction in paraformaldehyde fixed brain tissue of mice. We show that fear memory-induced synaptic activation reduces the interaction between the PAPs and the presynapse, but not the postsynapse, accompanied by retraction of the PAP tip from the synaptic cleft. Interestingly, this retraction is absent in the APP/PS1 mouse model of Alzheimer's disease, supporting the concept that alterations in astrocyte-synapse coverage contribute to memory processing.

Physiopathological changes of ferritin mRNA density and distribution in hippocampal astrocytes in the mouse brain.

Astrocytes are thought to play a crucial role in brain iron homeostasis. How they accomplish this regulation in vivo is unclear. In a recent transcriptomic analysis, we showed that polysomal Ftl1 and Fth1 mRNAs, encoding the ferritin light (Ftl) and heavy (Fth) chains that assemble into ferritin, a critical complex for iron storage and reduction, are enriched in perisynaptic astrocytic processes as compared to astrocytic soma. These data suggested that ferritin translation plays a specific role at the perisynaptic astrocytic interface and is tighly regulated by local translation. Here, we used our recently described AstroDot 3D in situ methodology to study the density and localization of ferritin mRNAs in astrocytes in the hippocampus in three different contexts in which local or systemic iron overload has been documented: aging, the hepcidin knock-out mouse model of hemochromatosis and the APP/PS1dE9 mouse model of Alzheimer's disease (AD). Our results showed that in wild type mice, Fth1 mRNA density was higher than Ftl1 and that both mRNAs were mostly distributed in astrocyte fine processes. Aging and absence of hepcidin caused an increased Fth1/Ftl1 ratio in astrocytes and in the case of aging, led to a redistribution of Fth1 mRNAs in astrocytic fine processes. In contrast, in AD mice, we observed a lower Fth1/Ftl1 ratio. Fth1 mRNAs became more somatic and Ftl1 mRNAs redistributed in large processes of astrocytes proximal to Amyloid beta (Aß) deposits. Hence, we propose that regulation of ferritin mRNA density and distribution in astrocytes contribute to iron homeostasis in physiology and pathophysiology.

EFFECT OF GLYCOLYSIS SUPRESSION ON ULTRASTRUCTURE OF THE HIPPOCAMPAL GIANT SYNAPSES

Suppression of glycolysis in the rat hippocampal formations was induced by daily intracerebroventricular injection of 2-deoxy-D-glucose for 10 days. Using electron microscopy, a disturbance of morphological interactions of axonal terminals of granular neurons with perisynaptic astrocytic processes was found. At the same time, both the determinant ultrastructural characteristics of giant synapses and two types of functional contacts (asymmetric chemical active zones with dendritic spines and symmetric adhesive junctions with dendrites) were retained. However, they showed morphological signs of a weakening of synaptic processes, which were expressed in a decrease in the number of intraterminal active zones, clustering of synaptic vesicles, and their removal from neurotransmitter release sites. Compared to the norm, an increase in the number of small mitochondria with an electron-dense matrix and the evidence of initiation of glycogen synthesis in the form of glycosomes were found in the giant terminals. The data obtained indicate that the giant synapses of the hippocampal mossy fibers are a plastic, self-correcting system that is able to function by adapting its own bioenergetic metabolism, when brain glycolysis is suppressed.

Astrocytic Glutamate Transporters and Migraine.

Glutamate levels and lifetime in the brain extracellular space are dinamically regulated by a family of Na+- and K+-dependent glutamate transporters, which thereby control numerous brain functions and play a role in numerous neurological and psychiatric diseases. Migraine is a neurological disorder characterized by recurrent attacks of typically throbbing and unilateral headache and by a global dysfunction in multisensory processing. Familial hemiplegic migraine type 2 (FHM2) is a rare monogenic form of migraine with aura caused by loss-of-function mutations in the α2 Na/K ATPase (α2NKA). In the adult brain, this pump is expressed almost exclusively in astrocytes where it is colocalized with glutamate transporters. Knockin mouse models of FHM2 (FHM2 mice) show a reduced density of glutamate transporters in perisynaptic astrocytic processes (mirroring the reduced expression of α2NKA) and a reduced rate of glutamate clearance at cortical synapses during neuronal activity and sensory stimulation. Here we review the migraine-relevant alterations produced by the astrocytic glutamate transport dysfunction in FHM2 mice and their underlying mechanisms, in particular regarding the enhanced brain susceptibility to cortical spreading depression (the phenomenon that underlies migraine aura and can also initiate the headache mechanisms) and the enhanced algesic response to a migraine trigger.

SNARE protein VAMP-2, but not syntaxin-1, SNAP-25 and synaptotagmin 1, expressed in perisynaptic astrocytic processes in the CA1 area of the rat hippocampus.

Perisynaptic astrocytic processes have been suggested as sites for the regulated release of neuroactive substances. However, very little is known about the molecular properties of regulated exocytosis in these processes. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins mediate synaptic vesicle exocytosis from neuronal cells and might be candidates for regulated exocytosis also from astrocytic processes. The expression of SNARE proteins in astrocytes, however, is not clarified. Thus, we aimed to investigate the localization and relative concentrations of neuronal SNARE proteins syntaxin-1, synaptosomal nerve-associated protein 25 (SNAP-25), vesicle-associated membrane protein 2 (VAMP-2) (synaptobrevin-2) and calcium sensor synaptotagmin 1 in perisynaptic astrocytic processes compared to nerve terminals and dendrites. We used quantitative immunogold electron microscopy of the rat hippocampus to investigate the localization and concentration of neuronal SNARE proteins. As expected, analysis of the immunogold data revealed a lower labeling density of SNARE proteins in the perisynaptic astrocytic processes than in presynaptic terminals. The same was also true when compared to dendrites. Contrary to VAMP-2, labeling intensities for syntaxin-1, SNAP-25 and synaptotagmin 1 were not distinguishable from background labeling in the processes. The relative concentration of VAMP-2 stands out, as the mean perisynaptic astrocytic process concentration of the protein was only 68 % lower than in presynaptic terminals and still 32 % higher than in dendrites. VAMP-2 was associated with small vesicles in the processes. Some gold particles were located over the astrocytic plasma membrane. VAMP-2 is expressed in perisynaptic astrocytic processes, with a concentration higher than in the dendrites. Our results are compatible with the role of VAMP-2 in exocytosis from perisynaptic astrocytic processes.

Multi-transcriptomic analysis points to early organelle dysfunction in human astrocytes in Alzheimer's disease

The phenotypic transformation of astrocytes in Alzheimer's disease (AD) is still not well understood. Recent analyses based on single-nucleus RNA sequencing of postmortem Alzheimer's disease (AD) samples are limited by the low number of sequenced astrocytes, small cohort sizes, and low number of differentially expressed genes detected. To optimize the detection of astrocytic genes, we employed a novel strategy consisting of the localization of pre-determined astrocyte and neuronal gene clusters in publicly available whole-brain transcriptomes. Specifically, we used cortical transcriptomes from 766 individuals, including cognitively normal subjects (Controls), and people diagnosed with mild cognitive impairment (MCI) or dementia due to AD. Samples came from three independent cohorts organized by the Mount Sinai Hospital, the Mayo Clinic, and the Religious Order Study/Memory and Aging Project (ROSMAP). Astrocyte- and neuron-specific gene clusters were generated from human brain cell-type specific RNAseq data using hierarchical clustering and cell-type enrichment scoring. Genes from each cluster were manually annotated according to cell-type specific functional Categories. Gene Set Variation Analysis (GSVA) and Principal Component Analysis (PCA) were used to establish changes in these functional categories among clinical cohorts. We highlight three novel findings of the study. First, individuals with the same clinical diagnosis were molecularly heterogeneous. Particularly in the Mayo Clinic and ROSMAP cohorts, over 50% of Controls presented down-regulation of genes encoding synaptic proteins typical of AD, whereas 30% of patients diagnosed with dementia due to AD presented Control-like transcriptomic profiles. Second, down-regulation of neuronal genes related to synaptic proteins coincided, in astrocytes, with up-regulation of genes related to perisynaptic astrocytic processes (PAP) and down-regulation of genes encoding endolysosomal and mitochondrial proteins. Third, down-regulation of astrocytic mitochondrial genes inversely correlated with the disease stages defined by Braak and CERAD scoring. Finally, we interpreted these changes as maladaptive or adaptive from the point of view of astrocyte biology in a model of the phenotypical transformation of astrocytes in AD. The main prediction is that early malfunction of the astrocytic endolysosomal system, associated with progressive mitochondrial dysfunction, contribute to Alzheimer's disease. If this prediction is correct, therapies preventing organelle dysfunction in astrocytes may be beneficial in preclinical and clinical AD.

Uncoupling the Excitatory Amino Acid Transporter 2 From Its C-Terminal Interactome Restores Synaptic Glutamate Clearance at Corticostriatal Synapses and Alleviates Mutant Huntingtin-Induced Hypokinesia

Rapid removal of glutamate from the sites of glutamate release is an essential step in excitatory synaptic transmission. However, despite many years of research, the molecular mechanisms underlying the intracellular regulation of glutamate transport at tripartite synapses have not been fully uncovered. This limits the options for pharmacological treatment of glutamate-related motor disorders, including Huntington’s disease (HD). We therefore investigated the possible binding partners of transgenic EAAT2 and their alterations under the influence of mutant huntingtin (mHTT). Mass spectrometry analysis after pull-down of striatal YFP-EAAT2 from wild-type (WT) mice and heterozygote (HET) Q175 mHTT-knock-in mice identified a total of 148 significant (FDR < 0.05) binders to full-length EAAT2. Of them 58 proteins exhibited mHTT-related differences. Most important, in 26 of the 58 mHTT-sensitive cases, protein abundance changed back toward WT levels when the mice expressed a C-terminal-truncated instead of full-length variant of EAAT2. These findings motivated new attempts to clarify the role of astrocytic EAAT2 regulation in cortico-basal movement control. Striatal astrocytes of Q175 HET mice were targeted by a PHP.B vector encoding EAAT2 with different degree of C-terminal modification, i.e., EAAT2-S506X (truncation at S506), EAAT2-4KR (4 lysine to arginine substitutions) or EAAT2 (full-length). The results were compared to HET and WT injected with a tag-only vector (CTRL). It was found that the presence of a C-terminal-modified EAAT2 transgene (i) increased the level of native EAAT2 protein in striatal lysates and perisynaptic astrocyte processes, (ii) enhanced the glutamate uptake of transduced astrocytes, (iii) stimulated glutamate clearance at individual corticostriatal synapses, (iv) increased the glutamate uptake of striatal astrocytes and (iv) alleviated the mHTT-related hypokinesia (open field indicators of movement initiation). In contrast, over-expression of full-length EAAT2 neither facilitated glutamate uptake nor locomotion. Together, our results support the new hypothesis that preventing abnormal protein-protein interactions at the C-terminal of EAAT2 could eliminate the mHTT-related deficits in corticostriatal synaptic glutamate clearance and movement initiation.

Neuronal contact upregulates astrocytic sphingosine-1-phosphate receptor 1 to coordinate astrocyte-neuron cross communication.

Astrocytes, the most abundant glial cells in the mammalian brain, directly associate with and regulate neuronal processes and synapses and are important regulators of brain development. Yet little is known of the molecular mechanisms that control the establishment of astrocyte morphology and the bi-directional communication between astrocytes and neurons. Here we show that neuronal contact stimulates expression of S1PR1, the receptor for the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P), on perisynaptic astrocyte processes and that S1PR1 drives astrocyte morphological complexity and morphogenesis. Moreover, the S1P/S1PR1 axis increases neuronal contact-induced expression of astrocyte secreted synaptogenic factors SPARCL1 and thrombospondin 4 that are involved in neural circuit assembly. Our findings have uncovered new functions for astrocytic S1PR1 signaling in regulation of bi-directional astrocyte-neuron crosstalk at the nexus of astrocyte morphogenesis and synaptogenesis.

Pharmacological Investigations in Glia Culture Model of Inflammation

Astrocytes and microglia are the main cell population besides neurons in the central nervous system (CNS). Astrocytes support the neuronal network via maintenance of transmitter and ion homeostasis. They are part of the tripartite synapse, composed of pre- and postsynaptic neurons and perisynaptic astrocytic processes as a functional unit. There is an increasing evidence that astroglia are involved in the pathophysiology of CNS disorders such as epilepsy, autoimmune CNS diseases or neuropsychiatric disorders, especially with regard to glia-mediated inflammation. In addition to astrocytes, investigations on microglial cells, the main immune cells of the CNS, offer a whole network approach leading to better understanding of non-neuronal cells and their pathological role in CNS diseases and treatment. An in vitro astrocyte-microglia co-culture model of inflammation was developed by Faustmann et al. (2003), which allows to study the endogenous inflammatory reaction and the cytokine expression under drugs in a differentiated manner. Commonly used antiepileptic drugs (e.g., levetiracetam, valproic acid, carbamazepine, phenytoin, and gabapentin), immunomodulatory drugs (e.g., dexamethasone and interferon-beta), hormones and psychotropic drugs (e.g., venlafaxine) were already investigated, contributing to better understanding mechanisms of actions of CNS drugs and their pro- or anti-inflammatory properties concerning glial cells. Furthermore, the effects of drugs on glial cell viability, proliferation and astrocytic network were demonstrated. The in vitro astrocyte-microglia co-culture model of inflammation proved to be suitable as unique in vitro model for pharmacological investigations on astrocytes and microglia with future potential (e.g., cancer drugs, antidementia drugs, and toxicologic studies).

Calcium Signals in Astrocyte Microdomains, a Decade of Great Advances.

The glial cells astrocytes have long been recognized as important neuron-supporting elements in brain development, homeostasis, and metabolism. After the discovery that the reciprocal communication between astrocytes and neurons is a fundamental mechanism in the modulation of neuronal synaptic communication, over the last two decades astrocytes became a hot topic in neuroscience research. Crucial to their functional interactions with neurons are the cytosolic Ca2+ elevations that mediate gliotransmission. Large attention has been posed to the so-called Ca2+microdomains, dynamic Ca2+ changes spatially restricted to fine astrocytic processes including perisynaptic astrocytic processes (PAPs). With presynaptic terminals and postsynaptic neuronal membranes, PAPs compose the tripartite synapse. The distinct spatial-temporal features and functional roles of astrocyte microdomain Ca2+ activity remain poorly defined. However, thanks to the development of genetically encoded Ca2+ indicators (GECIs), advanced microscopy techniques, and innovative analytical approaches, Ca2+ transients in astrocyte microdomains were recently studied in unprecedented detail. These events have been observed to occur much more frequently (∼50–100-fold) and dynamically than somatic Ca2+ elevations with mechanisms that likely involve both IP3-dependent and -independent pathways. Further progress aimed to clarify the complex, dynamic machinery responsible for astrocytic Ca2+ activity at microdomains is a crucial step in our understanding of the astrocyte role in brain function and may also reveal astrocytes as novel therapeutic targets for different brain diseases. Here, we review the most recent studies that improve our mechanistic understanding of the essential features of astrocyte Ca2+ microdomains.

Heterogeneity and Development of Fine Astrocyte Morphology Captured by Diffraction-Limited Microscopy.

The fine processes of single astrocytes can contact many thousands of synapses whose function they can modulate through bi-directional signaling. The spatial arrangement of astrocytic processes and neuronal structures is relevant for such interactions and for the support of neuronal signaling by astrocytes. At the same time, the geometry of perisynaptic astrocyte processes is variable and dynamically regulated. Studying these fine astrocyte processes represents a technical challenge, because many of them cannot be fully resolved by diffraction-limited microscopy. Therefore, we have established two indirect parameters of astrocyte morphology, which, while not fully resolving local geometry by design, provide statistical measures of astrocyte morphology: the fraction of tissue volume that astrocytes occupy and the density of resolvable astrocytic processes. Both are straightforward to obtain using widely available microscopy techniques. We here present the approach and demonstrate its robustness across various experimental conditions using mainly two-photon excitation fluorescence microscopy in acute slices and in vivo as well as modeling. Using these indirect measures allowed us to analyze the morphology of relatively large populations of astrocytes. Doing so we captured the heterogeneity of astrocytes within and between the layers of the hippocampal CA1 region and the developmental profile of astrocyte morphology. This demonstrates that volume fraction (VF) and segment density are useful parameters for describing the structure of astrocytes. They are also suitable for online monitoring of astrocyte morphology with widely available microscopy techniques.