The inhibitory neurotransmitter GABA evokes long‐lasting Ca2+ oscillations in cortical astrocytes

Studies over the last decade provided evidence that in a dynamic interaction with neurons glial cell astrocytes contribut to fundamental phenomena in the brain. Most of the knowledge on this derives, however, from studies monitoring the astrocyte Ca²⁺ response to glutamate. Whether astrocytes can similarly respond to other neurotransmitters, including the inhibitory neurotransmitter GABA, is relatively unexplored. By using confocal and two photon laser‐scanning microscopy the astrocyte response to GABA in the mouse somatosensory and temporal cortex was studied. In slices from developing (P15‐20) and adult (P30‐60) mice, it was found that in a subpopulation of astrocytes GABA evoked somatic Ca²⁺ oscillations. This response was mediated by GABA_B receptors and involved both G_i/o protein and inositol 1,4,5‐trisphosphate (IP₃) signalling pathways. In vivo experiments from young adult mice, revealed that also cortical astrocytes in the living brain exibit GABA_B receptor‐mediated Ca²⁺ elevations. At all astrocytic processes tested, local GABA or Baclofen brief applications induced long‐lasting Ca²⁺ oscillations, suggesting that all astrocytes have the potential to respond to GABA. Finally, in patch‐clamp recordings it was found that Ca²⁺ oscillations induced by Baclofen evoked astrocytic glutamate release and slow inward currents (SICs) in pyramidal cells from wild type but not IP₃R2^?/? mice, in which astrocytic GABA_B receptor‐mediated Ca²⁺ elevations are impaired. These data suggest that cortical astrocytes in the mouse brain can sense the activity of GABAergic interneurons and through their specific recruitment contribut to the distinct role played on the cortical network by the different subsets of GABAergic interneurons. GLIA 2016;64:363–373     

Astrocyte–neuron interaction in the substantia gelatinosa of the spinal cord dorsal horn via P2X7 receptor‐mediated release of glutamate and reactive oxygen species

The substantia gelatinosa (SG) of the spinal cord processes incoming painful information to ascending projection neurons. Whole‐cell patch clamp recordings from SG spinal cord slices documented that in a low Ca²⁺/no Mg²⁺ (low X²⁺) external medium adenosine triphosphate (ATP)/dibenzoyl‐ATP, Bz‐ATP) caused inward current responses, much larger in amplitude than those recorded in a normal X²⁺‐containing bath medium. The effect of Bz‐ATP was antagonized by the selective P2X7 receptor antagonist A‐438079. Neuronal, but not astrocytic Bz‐ATP currents were strongly inhibited by a combination of the ionotropic glutamate receptor antagonists AP‐5 and CNQX. In fact, all neurons and some astrocytes responded to NMDA, AMPA, and muscimol with inward current, demonstrating the presence of the respective receptors. The reactive oxygen species H₂O₂ potentiated the effect of Bz‐ATP at neurons but not at astrocytes. Hippocampal CA1 neurons exhibited a behavior similar to, but not identical with SG neurons. Although a combination of AP‐5 and CNQX almost abolished the effect of Bz‐ATP, H₂O₂ was inactive. A Bz‐ATP‐dependent and A‐438079‐antagonizable reactive oxygen species production in SG slices was proven by a microelectrode biosensor. Immunohistochemical investigations showed the colocalization of P2X7‐immunoreactivity with microglial (Iba1), but not astrocytic (GFAP, S100β) or neuronal (MAP2) markers in the SG. It is concluded that SG astrocytes possess P2X7 receptors; their activation leads to the release of glutamate, which via NMDA‐ and AMPA receptor stimulation induces cationic current in the neighboring neurons. P2X7 receptors have a very low density under resting conditions but become functionally upregulated under pathological conditions. GLIA 2014;62:1671–1686     

P2Y1 receptor inhibits GABA transport through a calcium signalling‐dependent mechanism in rat cortical astrocytes

Astrocytes express a variety of purinergic (P2) receptors, involved in astrocytic communication through fast increases in [Ca²⁺]_i. Of these, the metabotropic ATP receptors (P2Y) regulate cytoplasmic Ca²⁺ levels through the PLC‐PKC pathway. GABA transporters are a substrate for a number of Ca²⁺‐related kinases, raising the possibility that calcium signalling in astrocytes impact the control of extracellular levels of the major inhibitory transmitter in the brain. To access this possibility we tested the influence of P2Y receptors upon GABA transport into astrocytes. Mature primary cortical astroglial‐enriched cultures expressed functional P2Y receptors, as evaluated through Ca²⁺ imaging, being P2Y₁ the predominant P2Y receptor subtype. ATP (100 μM, for 1 min) caused an inhibition of GABA transport through either GAT‐1 or GAT‐3 transporters, decreasing the V_max kinetic constant. ATP‐induced inhibition of GATs activity was still evident in the presence of adenosine deaminase, precluding an adenosine‐mediated effect. This, was mimicked by a specific agonist for the P2Y_1,12,13 receptor (2‐MeSADP). The effect of 2‐MeSADP on GABA transport was blocked by the P2 (PPADS) and P2Y₁ selective (MRS2179) receptor antagonists, as well as by the PLC inhibitor (U73122). 2‐MeSADP failed to inhibit GABA transport in astrocytes where intracellular calcium had been chelated (BAPTA‐AM) or where calcium stores were depleted (α‐cyclopiazonic acid, CPA). In conclusion, P2Y₁ receptors in astrocytes inhibit GABA transport through a mechanism dependent of P2Y₁‐mediated calcium signalling, suggesting that astrocytic calcium signalling, which occurs as a consequence of neuronal firing, may operate a negative feedback loop to enhance extracellular levels of GABA. GLIA 2014;62:1211–1226     

Astrocytic P2Y1 receptor is involved in the regulation of cytokine/chemokine transcription and cerebral damage in a rat model of cerebral ischemia

After brain ischemia, significant amounts of adenosine 5′-triphosphate are released or leaked from damaged cells, thus activating purinergic receptors in the central nervous system. A number of P2X/P2Y receptors have been implicated in ischemic conditions, but to date the P2Y₁ receptor (P2Y₁R) has not been implicated in cerebral ischemia. In this study, we found that the astrocytic P2Y₁R, via phosphorylated-RelA (p-RelA), has a negative effect during cerebral ischemia/reperfusion. Intracerebroventricular administration of the P2Y₁R agonist, MRS 2365, led to an increase in cerebral infarct volume 72 hours after transient middle cerebral artery occlusion (tMCAO). Administration of the P2Y₁R antagonist, MRS 2179, significantly decreased infarct volume and led to recovered motor coordination. The effects of MRS 2179 occurred within 24 hours of tMCAO, and also markedly reduced the expression of p-RelA and interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein-1/chemokine (C-C motif) ligand 2 (CCL2), and interferon-inducible protein-10/chemokine (C-X-C motif) ligand 10 (CXCL10) mRNA. P2Y₁R and p-RelA were colocalized in glial fibrillary acidic protein-positive astrocytes, and an increase in infarct volume after MRS 2365 treatment was inhibited by the nuclear factor (NF)-κB inhibitor ammonium pyrrolidine dithiocarbamate. These results provide evidence that the P2Y₁R expressed in cortical astrocytes may help regulate the cytokine/chemokine response after tMCAO/reperfusion through a p-RelA-mediated NF-κB pathway.     

G protein‐coupled receptor 37‐like 1 modulates astrocyte glutamate transporters and neuronal NMDA receptors and is neuroprotective in ischemia

We show that the G protein‐coupled receptor GPR37‐like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1^–/– mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1‐mediated signalling inhibited astrocyte glutamate transporters and – surprisingly, given its lack of expression in neurons – reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D‐serine or TNF‐α, two astrocyte‐derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by ～40% in Gpr37l1^–/– brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation.     

Astrocytic poly(ADP‐ribose) polymerase‐1 activation leads to bioenergetic depletion and inhibition of glutamate uptake capacity

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is a ubiquitous nuclear enzyme involved in genomic stability. Excessive oxidative DNA strand breaks lead to PARP‐1‐induced depletion of cellular NAD⁺, glycolytic rate, ATP levels, and eventual cell death. Glutamate neurotransmission is tightly controlled by ATP‐dependent astrocytic glutamate transporters, and thus we hypothesized that astrocytic PARP‐1 activation by DNA damage leads to bioenergetic depletion and compromised glutamate uptake. PARP‐1 activation by the DNA alkylating agent, N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), caused a significant reduction of cultured cortical astrocyte survival (EC₅₀ = 78.2 ± 2.7 μM). HPLC revealed MNNG‐induced time‐dependent reductions in NAD⁺ (98%, 4 h), ATP (71%, 4 h), ADP (63%, 4 h), and AMP (66%, 4 h). The maximal [³H]glutamate uptake rate (V_max) also declined in a manner that corresponded temporally with ATP depletion, falling from 19.3 ± 2.8 in control cells to 2.1 ± 0.8 nmol/min/mg protein 4 h post‐MNNG. Both bioenergetic depletion and loss of glutamate uptake capacity were attenuated by genetic deletion of PARP‐1, directly indicating PARP‐1 involvement, and by adding exogenous NAD⁺ (10 mM). In mixed neurons/astrocyte cultures, MNNG neurotoxicity was partially mediated by extracellular glutamate and was reduced by co‐culture with PARP‐1^−/− astrocytes, suggesting that impairment of astrocytic glutamate uptake by PARP‐1 can raise glutamate levels sufficiently to have receptor‐mediated effects at neighboring neurons. Taken together, these experiments showed that PARP‐1 activation leads to depletion of the total adenine nucleotide pool in astrocytes and severe reduction in neuroprotective glutamate uptake capacity. © 2009 Wiley‐Liss, Inc.     

Astrocytes expressing ALS‐linked mutant FUS induce motor neuron death through release of tumor necrosis factor‐alpha

Mutations in fused in sarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease affecting both upper and lower motor neurons. While it is established that astrocytes contribute to the death of motor neurons in ALS, the specific contribution of mutant FUS (mutFUS) through astrocytes has not yet been studied. Here, we used primary astrocytes expressing a N‐terminally GFP tagged R521G mutant or wild‐type FUS (WTFUS) and show that mutFUS‐expressing astrocytes undergo astrogliosis, damage co‐cultured motor neurons via activation of an inflammatory response and produce conditioned medium (ACM) that is toxic to motor neurons in isolation. Time lapse imaging shows that motor neuron cultures exposed to mutFUS ACM, but not WTFUS ACM, undergo significant cell loss, which is preceded by progressive degeneration of neurites. We found that Tumor Necrosis Factor‐Alpha (TNFα) is secreted into ACM of mutFUS‐expressing astrocytes. Accordingly, mutFUS astrocyte‐mediated motor neuron toxicity is blocked by targeting soluble TNFα with neutralizing antibodies. We also found that mutant astrocytes trigger changes to motor neuron AMPA receptors (AMPAR) that render them susceptible to excitotoxicity and AMPAR‐mediated cell death. Our data provide the first evidence of astrocytic involvement in FUS‐ALS, identify TNFα as a mediator of this toxicity, and provide several potential therapeutic targets to protect motor neurons in FUS‐linked ALS.     

Pharmacological characterization of purinergic inhibitory neuromuscular transmission in the human colon

Background In the present study, we further characterize the purinergic receptors mediating the inhibitory junction potential (IJP) and smooth muscle relaxation in the human colon using a new, potent and selective agonist (MRS2365), and antagonists (MR2279 and MRS2500) of the P2Y₁ receptor. The P2Y₁₂ antagonist AR‐C66096 was tested as well. Using this pharmacological approach, we tested whether β‐nicotinamide adenine dinucleotide (β‐NAD) fulfilled the criteria to be considered an inhibitory neurotransmitter in the human colon. Methods We carried out muscle bath and microelectrode experiments on circular strips from the human colon and calcium imaging recordings on HEK293 cells, which constitutively express the human P2Y₁ receptor. Key Results Both the fast component of IJP and non‐nitrergic relaxation was concentration‐dependently inhibited by MRS2279 and MRS2500. This antagonism was confirmed in HEK293 cells. However, AR‐C66096 did not modify either inhibitory response. Adenosine 5′‐Ο‐2‐thiodiphosphate and MRS2365 caused a smooth muscle hyperpolarization and transient inhibition of spontaneous motility that was antagonized by MRS2279 and MRS2500. β‐Nicotinamide adenine dinucleotide inhibited the spontaneous motility (IC₅₀ = 3.3 mmol L^?1). Nevertheless, this effect was not antagonized by high concentrations of P2Y₁ antagonists. Conclusions & Inferences Inhibitory purinergic neuromuscular transmission in the human colon was pharmacologically assessed by the use of new P2Y₁ receptor antagonists MRS2179, MRS2279, and MRS2500. The rank order of potency of the P2Y₁ antagonists is MRS2500 > MRS2279 > MRS2179. We found that β‐NAD partially fulfills the criteria to be considered an inhibitory neurotransmitter in the human colon, but the relative contribution of each purine (ATP/ADP vsβ‐NAD) requires further studies.     

Glutamate‐dependent astrocyte modulation of synaptic transmission between cultured hippocampal neurons

The idea that astrocytes merely provide structural and trophic support for neurons has been challenged by the demonstration that astrocytes can regulate neuronal calcium levels. However, the physiological consequences of astrocyte–neuron signalling are unknown. Using mixed cultures of rat hippocampal astrocytes and neurons we have determined functional consequences of elevating astrocyte calcium levels on co-cultured neurons. Electrical or mechanical stimulation of astrocytes to increase their calcium level caused a glutamate-dependent slow inward current (SIC) in associated neurons. Microinjection of 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) into astrocytes to prevent the stimulus-dependent increase in astrocyte calcium level, blocks the appearance of the neuronal SIC. Pharmacological manipulations indicate that this astrocyte-dependent SIC is mediated by extracellular glutamate acting on N-methyl-d -aspartate (NMDA) and non-NMDA glutamate receptors. Additionally, stimulation of astrocytes reduced the magnitude of action potential-evoked excitatory and inhibitory postsynaptic currents through the activation of metabotropic glutamate receptors. The demonstration that astrocytes modulate neuronal currents and synaptic transmission raises the possibility that astrocytes play a neuromodulatory role by controlling the extracellular level of glutamate.     

Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast‐derived iPS cells using zinc‐finger nuclease technology

Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast‐derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human‐derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc‐finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte‐specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno‐associated virus integration site 1 (AAVS1) locus in disease and control‐derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP‐iPS astrocytes exhibited a well‐described functional astrocytic activity in vitro characterized by neuron‐dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP‐iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP‐iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte‐driven disease pathogenesis and therapy. GLIA 2016;64:63–75     

P2Y2 receptor antagonists as anti‐allodynic agents in acute and sub‐chronic trigeminal sensitization: Role of satellite glial cells

Trigeminal (TG) pain often lacks a satisfactory pharmacological control. A better understanding of the molecular cross‐talk between TG neurons and surrounding satellite glial cells (SGCs) could help identifying innovative targets for the development of more effective analgesics. We have previously demonstrated that neuronal pro‐algogenic mediators upregulate G protein‐coupled nucleotide P2Y receptors (P2YRs) expressed by TG SGCs in vitro. Here, we have identified the specific P2YR subtypes involved (i.e., the ADP‐sensitive P2Y₁R and the UTP‐responsive P2Y₂R subtypes), and demonstrated the contribution of neuron‐derived prostaglandins to their upregulation. Next, we have translated these data to an in vivo model of TG pain (namely, rats injected with Complete Freund's adjuvant in the temporomandibular joint), by demonstrating activation of SGCs and upregulation of P2Y₁R and P2Y₂R in the ipsi‐lateral TG. To unequivocally link P2YRs to the development of facial allodynia, we treated animals with various purinergic antagonists. The selective P2Y₂R antagonist AR‐C118925 completely inhibited SGCs activation, exerted a potent anti‐allodynic effect that lasted over time, and was still effective when administration was started 6‐days post induction of allodynia, i.e. under subchronic pain conditions. Conversely, the selective P2Y₁R antagonist MRS2179 was completely ineffective. Moreover, similarly to the anti‐inflammatory drug acetylsalicylic acid and the known anti‐migraine agent sumatriptan, the P2X/P2Y nonselective antagonist PPADS was only partially effective, and completely lost its activity under sub‐chronic conditions. Taken together, our results highlight glial P2Y₂Rs as potential “druggable” targets for the successful management of TG‐related pain. GLIA 2015;63:1256–1269     

Alteration of glial‐neuronal metabolic interactions in a mouse model of Alexander disease

Alexander disease is a rare and usually fatal neurological disorder characterized by the abundant presence of protein aggregates in astrocytes. Most cases result from dominant missense de novo mutations in the gene encoding glial fibrillary acidic protein (GFAP), but how these mutations lead to aggregate formation and compromise function is not known. A transgenic mouse line (Tg73.7) over‐expressing human GFAP produces astrocytic aggregates indistinguishable from those seen in the human disease, making them a model of this disorder. To investigate possible metabolic changes associated with Alexander disease Tg73.7 mice and controls were injected simultaneously with [1‐¹³C]glucose to analyze neuronal metabolism and [1,2‐¹³C]acetate to monitor astrocytic metabolism. Brain extracts were analyzed by ¹H magnetic resonance spectroscopy (MRS) to quantify amounts of several key metabolites, and by ¹³C MRS to analyze amino acid neurotransmitter metabolism. In the cerebral cortex, reduced utilization of [1,2‐¹³C]acetate was observed for synthesis of glutamine, glutamate, and GABA, and the concentration of the marker for neuronal mitochondrial metabolism, N‐acetylaspartate (NAA) was decreased. This indicates impaired astrocytic and neuronal metabolism and decreased transfer of glutamine from astrocytes to neurons compared with control mice. In the cerebellum, glutamine and GABA content and labeling from [1‐¹³C]glucose were increased. Evidence for brain edema was found in the increased amount of water and of the osmoregulators myo‐inositol and taurine. It can be concluded that astrocyte—neuronal interactions were altered differently in distinct regions. © 2010 Wiley‐Liss, Inc.     

Neurosteroid dehydroepiandrosterone enhances activity and trafficking of astrocytic GLT‐1 via σ1 receptor‐mediated PKC activation in the hippocampal dentate gyrus of rats

Neurosteroid dehydroepiandrosterone (DHEA) has been reported to exert a potent neuroprotective effect against glutamate‐induced excitotoxicity. However, the underlying mechanism remains to be elucidated. One of the possible mechanisms may be an involvement of astrocytic glutamate transporter subtype‐1 (GLT‐1) that can quickly clear spilled glutamate at the synapse to prevent excitotoxicity. To examine the effect of DHEA on GLT‐1 activity, we measured synaptically induced glial depolarization (SIGD) in the dentate gyrus (DG) of adult rats by applying an optical recording technique to the hippocampal slices stained with voltage‐sensitive dye RH155. Bath‐application of DHEA for 10 min dose‐dependently increased SIGD without changing presynaptic glutamate releases, which was sensitive to the GLT‐1 blocker DHK. Patch‐clamp recordings in astrocytes showed that an application of 50 μM DHEA increased glutamate‐evoked inward currents (Iglu) by approximately 1.5‐fold, which was dependent on the GLT‐1 activity. In addition, the level of biotinylated GLT‐1 protein in the surface of astrocytes was significantly elevated by DHEA. The DHEA‐increased SIGD, Iglu, and GLT‐1 translocation to the cell surface were blocked by the σ₁R antagonist NE100 and mimicked by the σ₁R agonist PRE084. DHEA elevated the phosphorylation level of PKC in a σ₁R‐dependent manner. Furthermore, the PKC inhibitor chelerythrine could prevent the DHEA‐increased SIGD, Iglu, and GLT‐1 translocation. Collectively, present results suggest that DHEA enhances the activity and translocation to cell surface of astrocytic GLT‐1 mainly via σ₁R‐mediated PKC cascade.     

Neuron–astrocyte signaling is preserved in the aging brain

Astrocytes play crucial roles in brain homeostasis and are emerging as regulatory elements of neuronal and synaptic physiology by responding to neurotransmitters with Ca²⁺ elevations and releasing gliotransmitters that activate neuronal receptors. Aging involves neuronal and astrocytic alterations, being considered risk factor for neurodegenerative diseases. Most evidence of the astrocyte–neuron signaling is derived from studies with young animals; however, the features of astrocyte–neuron signaling in adult and aging brain remain largely unknown. We have investigated the existence and properties of astrocyte–neuron signaling in physiologically and pathologically aging mouse hippocampal and cortical slices at different lifetime points (0.5 to 20 month‐old animals). We found that astrocytes preserved their ability to express spontaneous and neurotransmitter‐dependent intracellular Ca²⁺ signals from juvenile to aging brains. Likewise, resting levels of gliotransmission, assessed by neuronal NMDAR activation by glutamate released from astrocytes, were largely preserved with similar properties in all tested age groups, but DHPG‐induced gliotransmission was reduced in aged mice. In contrast, gliotransmission was enhanced in the APP/PS1 mouse model of Alzheimer's disease, indicating a dysregulation of astrocyte–neuron signaling in pathological conditions. Disruption of the astrocytic IP₃R2 mediated‐signaling, which is required for neurotransmitter‐induced astrocyte Ca²⁺ signals and gliotransmission, boosted the progression of amyloid plaque deposits and synaptic plasticity impairments in APP/PS1 mice at early stages of the disease. Therefore, astrocyte–neuron interaction is a fundamental signaling, largely conserved in the adult and aging brain of healthy animals, but it is altered in Alzheimer's disease, suggesting that dysfunctions of astrocyte Ca²⁺ physiology may contribute to this neurodegenerative disease. GLIA 2017 GLIA 2017;65:569–580     

Fast Ca2+ responses in astrocyte end‐feet and neurovascular coupling in mice

Cerebral blood flow (CBF) is regulated by the activity of neurons and astrocytes. Understanding how these cells control activity‐dependent increases in CBF is crucial to interpreting functional neuroimaging signals. The relative importance of neurons and astrocytes is debated, as are the functional implications of fast Ca²⁺ changes in astrocytes versus neurons. Here, we used two‐photon microscopy to assess Ca²⁺ changes in neuropil, astrocyte processes, and astrocyte end‐feet in response to whisker pad stimulation in mice. We also developed a pixel‐based analysis to improve the detection of rapid Ca²⁺ signals in the subcellular compartments of astrocytes. Fast Ca²⁺ responses were observed using both chemical and genetically encoded Ca²⁺ indicators in astrocyte end‐feet prior to dilation of arterioles and capillaries. A low dose of the NMDA receptor antagonist (5R,10s)‐(+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine‐hydrogen‐maleate (MK801) attenuated fast Ca²⁺ responses in the neuropil and astrocyte processes, but not in astrocyte end‐feet, and the evoked CBF response was preserved. In addition, a low dose of 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol (THIP), an agonist for the extrasynaptic GABA_A receptor (GABA_AR), increased CBF responses and the fast Ca²⁺ response in astrocyte end‐feet but did not affect Ca²⁺ responses in astrocyte processes and neuropil. These results suggest that fast Ca²⁺ increases in the neuropil and astrocyte processes are not necessary for an evoked CBF response. In contrast, as local fast Ca²⁺ responses in astrocyte end‐feet are unaffected by MK801 but increase via GABA_AR‐dependent mechanisms that also increased CBF responses, we hypothesize that the fast Ca²⁺ increases in end‐feet adjust CBF during synaptic activity.     

Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell‐derived neurons in culture

Human induced pluripotent stem (iPS) cell‐derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell‐derived neural stem cells (NSCs). We first demonstrate human iPS cell‐derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co‐culture on feeder layers of isogenic human iPS cell‐derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor‐channel function and occurred without changes in I_Na, I_K, V_m, and AP properties of iPS cell‐derived neurons. These data demonstrate co‐culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell‐derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell‐derived neurons in culture. We present RNA‐sequencing data for an immature, fetal‐like status of our human iPS cell‐derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co‐culture system.     

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75 antagonists

Two-pore potassium (K_2P) ion channels and P2Y receptors modulate the activity of neurones and are targets for the treatment of neuronal disorders. Here we have characterised their interaction. In cells coexpressing the Gα_i-coupled hP2Y₁₂ receptor, ADP and ATP significantly inhibited hK_2P2.1 currents. This was abolished by pertussis toxin (PTX), the hP2Y₁₂ antagonist AR-C69931MX, the hP2Y₁ antagonist MRS2179 and by mutating potential PKA/PKC phosphorylation sites in the channel C terminal. In cells coexpressing the Gα_q/11-coupled hP2Y₁ receptor, ADP and ATP also inhibited hK_2P2.1 currents, which were abolished by MRS2179, but unaffected by AR-C69931MX and PTX. When both receptors were coexpressed with K_2P2.1 channels, ADP-induced inhibition was antagonised by AR-C69913MX and MRS2179, but not PTX. Thus, both Gα_q/11- and Gα_i-coupled P2Y receptors inhibit K_2P channels and the action of hP2Y₁₂ receptors appears to involve co-activation of endogenous hP2Y₁ receptors. This represents a novel mechanism by which P2Y receptors may modulate neuronal activity.     

MicroRNA‐365 modulates astrocyte conversion into neuron in adult rat brain after stroke by targeting Pax6

Reactive astrocytes induced by ischemia can transdifferentiate into mature neurons. This neurogenic potential of astrocytes may have therapeutic value for brain injury. Epigenetic modifications are widely known to involve in developmental and adult neurogenesis. PAX6, a neurogenic fate determinant, contributes to the astrocyte‐to‐neuron conversion. However, it is unclear whether microRNAs (miRs) modulate PAX6‐mediated astrocyte‐to‐neuron conversion. In the present study we used bioinformatic approaches to predict miRs potentially targeting Pax6, and transient middle cerebral artery occlusion (MCAO) to model cerebral ischemic injury in adult rats. These rats were given striatal injection of glial fibrillary acidic protein targeted enhanced green fluorescence protein lentiviral vectors (Lv‐GFAP‐EGFP) to permit cell fate mapping for tracing astrocytes‐derived neurons. We verified that miR‐365 directly targets to the 3′‐UTR of Pax6 by luciferase assay. We found that miR‐365 expression was significantly increased in the ischemic brain. Intraventricular injection of miR‐365 antagomir effectively increased astrocytic PAX6 expression and the number of new mature neurons derived from astrocytes in the ischemic striatum, and reduced neurological deficits as well as cerebral infarct volume. Conversely, miR‐365 agomir reduced PAX6 expression and neurogenesis, and worsened brain injury. Moreover, exogenous overexpression of PAX6 enhanced the astrocyte‐to‐neuron conversion and abolished the effects of miR‐365. Our results demonstrate that increase of miR‐365 in the ischemic brain inhibits astrocyte‐to‐neuron conversion by targeting Pax6, whereas knockdown of miR‐365 enhances PAX6‐mediated neurogenesis from astrocytes and attenuates neuronal injury in the brain after ischemic stroke. Our findings provide a foundation for developing novel therapeutic strategies for brain injury.     

Mutant disrupted‐in‐schizophrenia 1 in astrocytes: Focus on glutamate metabolism

Disrupted‐in‐schizophrenia 1 (DISC1) is a genetic risk factor that has been implicated in major mental disorders. DISC1 binds to and stabilizes serine racemase to regulate production of D‐serine by astrocytes, contributing to glutamate (GLU) neurotransmission. However, the possible involvement of astrocytic DISC1 in synthesis, metabolism, reuptake, or secretion of GLU remains unexplored. Therefore, we studied the effects of dominant‐negative mutant DISC1 on various aspects of GLU metabolism by using primary astrocyte cultures and hippocampal tissue from transgenic mice with astrocyte‐restricted expression of mutant DISC1. Although mutant DISC1 had no significant effects on astrocyte proliferation, GLU reuptake, glutaminase, or glutamate carboxypeptidase II activity, expression of mutant DISC1 was associated with increased levels of alanine‐serine‐cysteine transporter 2, vesicular glutamate transporters 1 and 3 in primary astrocytes and in the hippocampus, and elevated expression of the NR1 subunit and diminished expression of the NR2A subunit of N‐methyl‐D‐aspartate (NMDA) receptors in the hippocampus, at postnatal day 21. Our findings indicate that decreased D‐serine production by astrocytic mutant DISC1 might lead to compensatory changes in levels of the amino acid transporters and NMDA receptors in the context of tripartite synapse. © 2014 Wiley Periodicals, Inc.