Role of P2Y1 receptor in astroglia‐to‐neuron signaling at dorsal spinal cord

Several studies have shown that astrocytes release neurotransmitters into the extracellular space that may then activate receptors on nearby neurons. In the present study, the actions of adenosine 5′‐O‐(2‐thiodiphosphate) (ADPbetaS)‐activated astrocyte conditioned medium (ADPbetaS‐ACM) on cultured dorsal spinal cord neurons were evaluated by using confocal laser scanning microscopy and whole‐cell patch‐clamp recording. ADPbetaS caused astrocytic glutamate efflux (43 μM), which in turn induced inward currents in dorsal horn neurons with short time in culture. The inward currents were abolished by 2‐amino‐5‐phosphonlanoicacid (AP‐5; NMDAR antagonist) plus 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX; non‐NMDAR antagonist) but were unaffected by MRS2179 (selective P2Y₁ receptor antagonist). Furthermore, N6‐methyl‐2′‐deoxyadenosine‐3′,5′‐bisphosphate (MRS2179) was used to block glutamate release from astrocytes. As a result, ADPbetaS‐ ACM‐induced inward currents in neurons were significantly blocked. On the other hand, both NMDAR and non‐NMDAR were involved in ADPbetaS‐ACM (concentration was diluted to one‐tenth)‐evoked small [Ca²⁺]_i transients in neurons. Under this condition, the values of glutamate concentrations in the medium are close to values for extracellular glutamate concentrations under physiological conditions. For this reason, it is possible that astrocyte‐derived glutamate is important for distant neuron under physiological conditions at dorsal spinal cord. These observations indicate that astrocytic P2Y₁ receptor activation triggered glutamate efflux, which acts on distant neurons to elevate calcium levels or acts on nearby neurons to evoke inward current. Finally, our results support the conclusion that the astrocytic P2Y₁ receptor plays an important role in bidirectional communication between astrocytes and neurons. © 2009 Wiley‐Liss, Inc.     

Astrocytes in 17beta-estradiol treated mixed hippocampal cultures show attenuated calcium response to neuronal activity

Glial cells in the brain are capable of responding to hormonal signals. The ovarian steroid hormone 17beta-estradiol, in addition to its actions on neurons, can directly affect glial cells. Estrogen receptors have been described on both neurons and astrocytes, suggesting a complex interplay between these two in mediating the effects of the hormone. Astrocytes sense and respond to neuronal activity with a rise in intracellular calcium concentration ([Ca(2+)](i)). Using simultaneous electrophysiology and calcium imaging techniques, we monitored neuronal activity evoked astrocyte ([Ca(2+)](i)) changes in mixed hippocampal cultures loaded with fluo-3 AM. Action potential firing in neurons, elicited by injecting depolarizing current pulses, was associated with ([Ca(2+)](i)) elevations in astrocytes, which could be blocked by 200 microM MCPG and also 1 microM TTX. We compared astrocytic ([Ca(2+)](i)) transients in control and 24-hour estradiol treated cultures. The amplitude of the ([Ca(2+)](i)) transient, the number of responsive astrocytes, and the ([Ca(2+)](i)) wave velocity were all significantly reduced in estradiol treated cultures. ([Ca(2+)](i)) rise in astrocytes in response to local application of the metabotropic glutamate receptor (mGluR) agonist t-ACPD was attenuated in estradiol treated cultures, suggesting functional changes in the astrocyte mGluR following 24-h treatment with estradiol. Since astrocytes can modulate synaptic transmission by release of glutamate, the attenuated ([Ca(2+)](i)) response seen following estradiol treatment could have functional consequences on astrocyte-neuron signaling.     

Acute treatment with 17beta-estradiol attenuates astrocyte-astrocyte and astrocyte-neuron communication

Astrocytes are now recognized as dynamic signaling elements in the brain. Bidirectional communication between neurons and astrocytes involves integration of neuronal inputs by astrocytes and release of gliotransmitters that modulate neuronal excitability and synaptic transmission. The ovarian steroid hormone, 17beta-estradiol, in addition to its rapid actions on neuronal electrical activity can rapidly alter astrocyte intracellular calcium concentration ([Ca2+]i) through a membrane-associated estrogen receptor. Using calcium imaging and electrophysiological techniques, we investigated the functional consequences of acute treatment with estradiol on astrocyte-astrocyte and astrocyte-neuron communication in mixed hippocampal cultures. Mechanical stimulation of an astrocyte evoked a [Ca2+]i rise in the stimulated astrocyte, which propagated to the surrounding astrocytes as a [Ca2+]i wave. Following acute treatment with estradiol, the amplitude of the [Ca2+]i elevation in astrocytes around the stimulated astrocyte was attenuated. Further, estradiol inhibited the [Ca2+]i rise in individual astrocytes in response to the metabotropic glutamate receptor agonist, trans-(+/-)-1-amino-1,3-cyclopentanedicarboxylic acid. Mechanical stimulation of astrocytes induced [Ca2+]i elevations and electrophysiological responses in adjacent neurons. Estradiol rapidly attenuated the astrocyte-evoked glutamate-mediated [Ca2+]i rise and slow inward current in neurons. Also, the incidence of astrocyte-induced increase in spontaneous postsynaptic current frequency was reduced in the presence of estradiol. The effects of estradiol were stereo-specific and reversible following washout. These findings may indicate that the regulation of neuronal excitability and synaptic transmission by astrocytes is sensitive to rapid estradiol-mediated hormonal control.     

Astrocyte calcium signals at Schaffer collateral to CA1 pyramidal cell synapses correlate with the number of activated synapses but not with synaptic strength

Glial cells respond to neuronal activity by transient increases in their intracellular calcium concentration. At hippocampal Schaffer collateral to CA1 pyramidal cell synapses, such activity-induced astrocyte calcium transients modulate neuronal excitability, synaptic activity, and LTP induction threshold by calcium-dependent release of gliotransmitters. Despite a significant role of astrocyte calcium signaling in plasticity of these synapses, little is known about activity-dependent changes of astrocyte calcium signaling itself. In this study, we analyzed calcium transients in identified astrocytes and NG2-cells located in the stratum radiatum in response to different intensities and patterns of Schaffer collateral stimulation. To this end, we employed multiphoton calcium imaging with the low-affinity indicator dye Fluo-5F in glial cells, combined with extracellular field potential recordings to monitor postsynaptic responses to the afferent stimulation. Our results confirm that somata and processes of astrocytes, but not of NG2-cells, exhibit intrinsic calcium signaling independent of evoked neuronal activity. Moderate stimulation of Schaffer collaterals (three pulses at 50 Hz) induced calcium transients in astrocytes and NG2-cells. Astrocyte calcium transients upon this three-pulse stimulation could be evoked repetitively, increased in amplitude with increasing stimulation intensity and were dependent on activation of metabotropic glutamate receptors. Activity-induced transients in NG2-cells, in contrast, showed a rapid run-down upon repeated three-pulse stimulation. Theta burst stimulation and stimulation for 5 min at 1 Hz induced synaptic potentiation and depression, respectively, as revealed by a lasting increase or decrease in population spike amplitudes upon three-pulse stimulation. Synaptic plasticity was, however, not accompanied by corresponding alterations in the amplitude of astrocyte calcium signals. Taken together, our results suggest that the amplitude of astrocyte calcium signals reflects the number of activated synapses but does not correlate with the degree of synaptic potentiation or depression at Schaffer collateral to CA1 pyramidal cell synapses.     

Astrocyte membrane responses and potassium accumulation during neuronal activity

Older studies suggest that astrocytes act as potassium electrodes and depolarize with the potassium efflux accompanying neuronal activity. Newer studies suggest that astrocytes depolarize in response to neuronal glutamate release and the activity of electrogenic glial glutamate transporters, thus casting doubt on the fidelity with which astrocytes might sense extracellular potassium rises. Any K(+)-induced astrocyte depolarization might reflect a spatial buffering effect of astrocytes during neuronal activity. For these reasons, we studied stimulus-evoked currents in hippocampal CA1 astrocytes. Hippocampal astrocytes exhibited stimulus-evoked transient glutamate transporter currents and slower Ba(2+)-sensitive inward rectifier potassium (K(ir)) currents. In whole-cell astrocyte recordings, Ba(2+) blocked a very weakly rectifying component of the astrocyte membrane conductance. The slow stimulus-elicited current, like measurements from K(+)-sensitive electrodes under the same conditions, predicted small bulk [K(+)](o) increases (<0.5 mM) following the termination of short-stimulus trains. These currents indicate the potential for astrocyte spatial K(+) buffering. However, Ba(2+) did not significantly affect resting [K(+)](o) or the [K(+)](o) rises detected by the K(+)-sensitive electrode. To test whether local K(+) rises may be significantly higher than those detected by glial recordings or by K(+) electrodes, we assayed EPSCs and fiber volleys, two measures very sensitive to K(+) increases. We found that Ba(2+) had little effect on neuronal axonal or synaptic function during short-stimulus trains, indicating that K(ir)s do not influence local [K(+)](o) rises enough, under these conditions to affect synaptic transmission. In conclusion, our results indicate that hippocampal astrocytes are faithful sensors of [K(+)](o) rises, but we find little evidence for physiologically relevant spatial K(+) buffering during brief bursts of presynaptic activity.     

Astrocytes selectively enhance N-type calcium current in hippocampal neurons

Astrocytes influence neuronal development, synapse formation, and synaptic transmission, partly through affecting neuronal calcium signals. In order to elucidate the extent to which astrocytes modulate neuronal voltage-gated calcium currents, we performed a whole-cell patch clamp analysis of neurons in astrocyte-deplete and astrocyte-enriched conditions. We demonstrate that hippocampal neurons in an astrocyte-enriched environment show augmentation of voltage-gated calcium current at 1-3 days in vitro. Further study in pairs of adjacent neurons showed that the augmentation in calcium current was dependent on direct contact with the astrocyte. Pharmacological analysis demonstrated the augmentation is selective for the N-type calcium current, although immunochemical labeling of the alpha1(B) subunit of the N-type calcium channel was unchanged. These findings show that astrocytes regulate neuronal voltage-gated calcium currents in a contact-dependent manner. The specificity of the effect for the N-type calcium current at early days in culture has special significance regarding the role of astrocytes in hippocampal synaptogenesis.     

Activated microglia impairs neuroglial interaction by opening Cx43 hemichannels in hippocampal astrocytes

Glia plays an active role in neuronal functions and dysfunctions, some of which depend on the expression of astrocyte connexins, the gap junction channel and hemichannel proteins. Under neuroinflammation triggered by the endotoxin lipopolysacharide (LPS), microglia is primary stimulated and releases proinflammatory agents affecting astrocytes and neurons. Here, we investigate the effects of such microglial activation on astrocyte connexin‐based channel functions and their consequences on synaptic activity in an ex vivo model. We found that LPS induces astroglial hemichannel opening in acute hippocampal slices while no change is observed in gap junctional communication. Based on pharmacological and genetic approaches we found that the LPS‐induced hemichannel opening is mainly due to Cx43 hemichannel activity. This process primarily requires a microglial stimulation resulting in the release of at least two proinflammatory cytokines, IL‐1β and TNF‐α. Consequences of the hemichannel‐mediated increase in membrane permeability are a calcium rise in astrocytes and an enhanced glutamate release associated to a reduction in excitatory synaptic activity of pyramidal neurons in response to Schaffer's collateral stimulation. As a whole our findings point out astroglial hemichannels as key determinants of the impairment of synaptic transmission during neuroinflammation. GLIA 2015;63:795–811     

Impaired glutamate recycling and GluN2B‐mediated neuronal calcium overload in mice lacking TGF‐β1 in the CNS

Transforming growth factor β1 (TGF‐β1) is a pleiotropic cytokine expressed throughout the CNS. Previous studies demonstrated that TGF‐β1 contributes to maintain neuronal survival, but mechanistically this effect is not well understood. We generated a CNS‐specific TGF‐β1‐deficient mouse model to investigate the functional consequences of TGF‐β1‐deficiency in the adult mouse brain. We found that depletion of TGF‐β1 in the CNS resulted in a loss of the astrocyte glutamate transporter (GluT) proteins GLT‐1 (EAAT2) and GLAST (EAAT1) and decreased glutamate uptake in the mouse hippocampus. Treatment with TGF‐β1 induced the expression of GLAST and GLT‐1 in cultured astrocytes and enhanced astroglial glutamate uptake. Similar to GLT‐1‐deficient mice, CNS‐TGF‐β1‐deficient mice had reduced brain weight and neuronal loss in the CA1 hippocampal region. CNS‐TGF‐β1‐deficient mice showed GluN2B‐dependent aberrant synaptic plasticity in the CA1 area of the hippocampus similar to the glutamate transport inhibitor DL‐TBOA and these mice were highly sensitive to excitotoxic injury. In addition, hippocampal neurons from TGF‐β1‐deficient mice had elevated GluN2B‐mediated calcium signals in response to extrasynaptic glutamate receptor stimulation, whereas cells treated with TGF‐β1 exhibited reduced GluN2B‐mediated calcium signals. In summary, our study demonstrates a previously unrecognized function of TGF‐β1 in the CNS to control extracellular glutamate homeostasis and GluN2B‐mediated calcium responses in the mouse hippocampus.     

Existence and distinction of acid‐evoked currents in rat astrocytes

Astrocytes are vital structures that support and/or protect neighboring neurons from pathology. Although it is generally accepted that glutamate receptors mediate most astrocyte effects, acid‐evoked currents have recently attracted attention for their role in this regard. Here, we identified the existence and characteristics of acid‐sensing ion channels (ASICs) and the transient receptor potential vanilloid type 1 (TRPV1) in astrocytes. There were two types of currents recorded under the application of acidic solution (pH 6.0) in cultured rat astrocytes. Transient currents were exhibited by 10% of the astrocytes, and sustained currents were exhibited by the other 90%, consistent with the features of ASIC and TRPV1 currents, respectively. Western blotting and immunofluorescence confirmed the expression of ASIC1, ASIC2a, ASIC3, and TRPV1 in cultured and in situ astrocytes. Unlike the ASICs expressed in neurons, which were mainly distributed in the cell membrane/cytoplasm, most of the ASICs in astrocytes were expressed in the nucleus. TRPV1 was more permeable to Na⁺ in cultured astrocytes, which differed from the typical neuronal TRPV1 that was mainly permeable to Ca²⁺. This study demonstrates that there are two kinds of acid‐evoked currents in rat astrocytes, which may provide a new understanding about the functions of ligand‐gated ion channels in astrocytes. © 2010 Wiley‐Liss, Inc.     

Glutamate-induced Release of the Nitric Oxide Precursor, Arginine, From Glial Cells

Arginine, the nitric oxide precursor, is predominantly localized in glial cells, whereas the constitutive nitric oxide synthase is mainly found in neurons. Therefore, a transfer of arginine from glial cells to neurons is needed to replenish the neuronal precursor pool. This is further supported by the finding that arginine is released upon selective pathway stimulation both in vitro and in vivo . We investigated the mechanism underlying this glial-neuronal interaction by analysing the effect of glutamate receptor agonists on the extracellular [³H]arginine level in cerebellar and cortical slices and in cultures of either cortical astroglial cells or neurons. We present data indicating that arginine is released from cerebellar and cortical slices and astroglial cell cultures upon activation of ionotropic non-NMDA glutamate receptors. Glutamate had no effect on the extracellular [³H]arginine level in neuronal cultures. Moreover, the effect of glutamate in cerebellar slices was tetrodotoxin-insensitive, and the calcium ionophore A23187 evoked the release of [³H]arginine from astroglial cell cultures. Thus, nitric oxide synthesis and nitric oxide transmission may be based on the glial-neuronal transfer of arginine which is induced by activation of excitatory amino acid receptors on glial cells.     

Regulation of brain-derived neurotrophic factor and nerve growth factor mRNA in primary cultures of hippocampal neurons and astrocytes.

Brain-derived neurotrophic factor (BDNF) and NGF are both expressed by neurons in the hippocampus. In previous studies, it has been demonstrated that both BDNF and NGF mRNA levels are regulated by neuronal activity. Upregulation is predominantly regulated by the glutamate (NMDA and non-NMDA receptors); downregulation, predominantly by the GABA system (Zafra et al., 1990, 1991). In neuronal cultures of the rat hippocampus, potassium depolarization and kainic acid-mediated increases in BDNF and NGF mRNA were eliminated in a dose-dependent manner by the calcium channel blocker nifedipine. Conversely, calcium ionophores (Bay-K8644 and ionomycin) augmented BDNF and NGF mRNA levels by a calmodulin-mediated mechanism. In view of the fact that many potential modulators (conventional transmitters and neuropeptides) of neuronal and astrocytic BDNF and NGF mRNA synthesis may act via the adenylate cyclase system, we studied the effect of forskolin, an activator of adenylate cyclase. Indeed, forskolin enhanced the effects of calcium ionophores and kainic acid on BDNF and NGF mRNA levels. Cytokines, such as interleukin-1 and transforming growth factor-beta 1, which have previously been shown to increase NGF mRNA markedly in astrocytes, were without effect on neuronal BDNF and NGF mRNA levels. In contrast to neuronal cultures, where the regulation of BDNF and NGF mRNA was generally very similar, the regulation in astrocytes was distinctly different. All the cytokines that produce a marked increase in NGF mRNA were without effect on astrocyte BDNF mRNA levels, which under basic conditions were below the detection limit. However, norepinephrine produced a marked elevation of BDNF mRNA in astrocytes, an effect that was further enhanced by glutamate receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glutamate controls tPA recycling by astrocytes, which in turn influences glutamatergic signals

Tissue-type plasminogen activator (tPA) regulates physiological processes in the brain, such as learning and memory, and plays a critical role in neuronal survival and neuroinflammation in pathological conditions. Here we demonstrate, by combining mouse in vitro and in vivo data, that tPA is an important element of the cross talk between neurons and astrocytes. The data show that tPA released by neurons is constitutively endocytosed by astrocytes via the low-density lipoprotein-related protein receptor, and is then exocytosed in a regulated manner. The exocytotic recycling of tPA by astrocytes is inhibited in the presence of extracellular glutamate. Kainate receptors of astrocytes act as sensors of extracellular glutamate and, via a signaling pathway involving protein kinase C, modulate the exocytosis of tPA. Further, by thus capturing extracellular tPA, astrocytes serve to reduce NMDA-mediated responses potentiated by tPA. Overall, this work provides the first demonstration that the neuromodulator, tPA, may also be considered as a gliotransmitter.     

Heterosynaptic long‐term depression mediated by ATP released from astrocytes

Heterosynaptic long‐term depression (hLTD) at untetanized synapses accompanying the induction of long‐term potentiation (LTP) spatially sharpens the activity‐induced synaptic potentiation; however, the underlying mechanism remains unclear. We found that hLTD in the hippocampal CA1 region is caused by stimulation‐induced ATP release from astrocytes that suppresses transmitter release from untetanized synaptic terminals via activation of P2Y receptors. Selective stimulation of astrocytes expressing channelrhodopsin‐2, a light‐gated cation channel permeable to Ca²⁺, resulted in LTD of synapses on neighboring neurons. This synaptic modification required Ca²⁺ elevation in astrocytes and activation of P2Y receptors, but not N‐methyl‐D ‐aspartate receptors. Furthermore, blocking P2Y receptors or buffering astrocyte intracellular Ca²⁺ at a low level prevented hLTD without affecting LTP induced by SC stimulation. Thus, astrocyte activation is both necessary and sufficient for mediating hLTD accompanying LTP induction, strongly supporting the notion that astrocytes actively participate in activity‐dependent synaptic plasticity of neural circuits. © 2012 Wiley Periodicals, Inc.     

Dihydrokainate-sensitive neuronal glutamate transport is required for protection of rat cortical neurons in culture against synaptically released glutamate

Glutamate transport in nearly pure rat cortical neurons in culture (less than 0.2% astrocytes) is potently inhibited by dihydrokainate, l -serine-O-sulphate, but not by l -α-amino-adipate. This system allows for a test of the hypothesis that glutamate transport is important for protecting neurons against the toxicity of endogenous synaptically released glutamate. In support of this hypothesis, a 20–24 h exposure to 1 mm dihydrokainate reduced cell survival to only 14.8 ± 9.8% in neuronal cultures (P < 0.001;n = 3), although it had no effect on neuronal survival in astrocyte-rich cultures (P > 0.05;n = 3). Dihydrokainate also significantly caused accumulation of glutamate in the extracellular medium of cortical neuronal cultures (6.6 ± 4.9 μm , compared to 1.2 ± 0.3 μm in control, n = 14, P < 0.01). The neurotoxicity of dihydrokainate was blocked by 10 μm MK-801, 10 μm tetrodotoxin, and an enzyme system that degrades extracellular glutamate. The latter two also abolished the accumulation of glutamate in the extracellular medium. Dihydrokainate (1 mm ) inhibited the ⁴⁵calcium uptake stimulated by 30 μm N-methyl-d -aspartate (NMDA), but not by higher concentrations consistent with a weak antagonist action of dihydrokainate at the NMDA receptor. Whole cell recordings showed that 1 mm dihydrokainate produced ≈ 25% inhibition of 30 μm NMDA-induced current in cortical neurons. Dihydrokainate (1 mm ) alone generated a small current (17% of the current produced by 30 μm NMDA) that was blocked by 30 μm 5,7-dichlorokynurenate and only weakly by 10 μm 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These results suggest that the toxicity of dihydrokainate in neuronal cultures is due to its ability to block glutamate transport in these cultures, and that dihydrokainate-sensitive neuronal glutamate transport may be important in protecting neurons against the toxicity of synaptically released glutamate.     

Astrocytes restrict discharge duration and neuronal sodium loads during recurrent network activity

Influx of sodium ions into active neurons is a highly energy‐expensive process which must be strictly limited. Astrocytes could play an important role herein because they take up glutamate and potassium from the extracellular space, thereby dampening neuronal excitation. Here, we performed sodium imaging in mouse hippocampal slices combined with field potential and whole‐cell patch‐clamp recordings and measurement of extracellular potassium ([K⁺]_o). Network activity was induced by Mg²⁺‐free, bicuculline‐containing saline, during which neurons showed recurring epileptiform bursting, accompanied by transient increases in [K⁺]_o and astrocyte depolarizations. During bursts, neurons displayed sodium increases by up to 22 mM. Astrocyte sodium concentration increased by up to 8.5 mM, which could be followed by an undershoot below baseline. Network sodium oscillations were dependent on action potentials and activation of ionotropic glutamate receptors. Inhibition of glutamate uptake caused acceleration, followed by cessation of electrical activity, irreversible sodium increases, and swelling of neurons. The gliotoxin NaFAc (sodium‐fluoroacetate) resulted in elevation of astrocyte sodium concentration and reduced glial uptake of glutamate and potassium uptake through Na⁺/K⁺‐ATPase. Moreover, NaFAc extended epileptiform bursts, caused elevation of neuronal sodium, and dramatically prolonged accompanying sodium signals, most likely because of the decreased clearance of glutamate and potassium by astrocytes. Our experiments establish that recurrent neuronal bursting evokes sodium transients in neurons and astrocytes and confirm the essential role of glutamate transporters for network activity. They suggest that astrocytes restrict discharge duration and show that an intact astrocyte metabolism is critical for the neurons' capacity to recover from sodium loads during synchronized activity. GLIA 2015;63:936–957     

Selective and AMPA receptor-dependent astrocyte death following prolonged blockade of glutamate reuptake in rat cerebellar cultures

In this study we examined the effects of prolonged l-trans-pyrrolidine-2,4-dicarboxylate (PDC)-induced glutamate reuptake blockade on the viability of glial cells in cerebellar granule cell cultures. Immunofluorescence staining for the glial-specific intermediate filament protein, GFAP, revealed that the PDC- induced increase of extracellular glutamate concentration was accompanied by increased astrocyte death, while neurons and oligodendrocytes remained intact and viable. Astrocytic cell death was manifested as fragmentation of processes and cell bodies. The selective astrocyte death was completely prevented by the competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptor antagonist, NBQX (10 microM), whereas MK-801 (10 microM), a noncompetitive blocker of N-methyl-D-aspartate receptors, gave only partial protection. Double staining for GFAP and the AMPA receptor subunits GluR2/3 showed that astrocytes had much higher immunoreactivity for GluR2/3 than neurons or oligodendrocytes, suggesting that the number of AMPA receptors is likely to be higher on astrocytes. Furthermore, we employed real-time RT-PCR to measure GluR1-4 subunit mRNA expression in control and PDC-exposed cultures. Following treatment with PDC, GluR1 and GluR4 mRNAs were reduced by 40% and GluR3 was reduced by 70% relative to control levels. In contrast, GluR2 expression was not affected by the PDC treatment, indicating that GluR3 was the dominant type of AMPA receptor subunit expressed on astrocytes. Our results show that astrocytes appear to be more vulnerable than neurons or oligodendrocytes to a gradual increase in the extracellular glutamate concentration, suggesting that astrocytes may be critically involved in the pathophysiology of slowly developing chronic neurodegenerative disorders.     

Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ

Astrocytes closely contact neurons where they respond to neuronally released glutamate in immature brain slices. In previous studies, neither metabotropic nor ionotropic glutamate receptor-mediated responses were detected by imaging Ca2+ in astrocytes from mature (P21-P42) animals, suggesting astrocyte glutamate receptors only contribute to hippocampus physiology during development. In contrast to Ca2+ imaging, published electrophysiological experiments suggest P30-P35 astrocytes have alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. For this study, we imaged astrocytes in P31-P38 hippocampal slices to determine if metabotropic and ionotropic glutamate receptor activation elevates intracellular calcium in mature astrocytes. Drugs were perfused while [Ca2+]i was monitored (confocal imaging) in cells loaded with Calcium Green 1-AM. Imaged cells were subsequently identified as astrocytes by GFAP/S-100 immunostaining. Astrocytic Ca2+ increased after glutamate application in the presence of a glutamate uptake inhibitor. An agonist at group I/II metabotropic glutamate receptors, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), elicited Ca2+ increases as did group I agonist 3,5-dihydroxyphenylglycine (DHPG), suggesting that mature astrocytes respond to glutamate via metabotropic glutamate receptors. AMPA also elicited Ca2+ elevations that were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and occurred after treatment with omega-conotoxin MVIIC to block neurotransmitter release. These results demonstrate that astrocytes in mature hippocampus have functional ionotropic and metabotropic glutamate receptors that regulate astrocytic calcium levels. Glutamatergic regulation of astrocytic [Ca2+]i may be involved in synapse modeling, long-term potentiation, excitotoxicity and other events dependent on glutamatergic transmission in adult hippocampus.     

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.     

Thyroid hormone increases astrocytic glutamate uptake and protects astrocytes and neurons against glutamate toxicity

Thyroid hormone (T(3)) regulates the growth and differentiation of rat cerebellar astrocytes. Previously, we have demonstrated that these effects are due, at least in part, to the increased expression of extracellular matrix molecules and growth factors, such as fibroblast growth factor-2. T(3) also modulates neuronal development in an astrocyte-mediated manner. In the mammalian central nervous system, excitatory neurotransmission is mediated mainly by glutamate. However, excessive stimulation of glutamate receptors can lead to excitotoxicity and cell death. Astrocytic glutamate transporters, GLT-1 and GLAST, play an essential role in the clearance of the neuronal-released glutamate from the extracellular space and are essential for maintaining physiological extracellular glutamate levels in the brain. In the present study, we showed that T(3) significantly increased glutamate uptake by cerebellar astrocytes compared with control cultures. Inhibitors of glutamate uptake, such as L-PDC and DL-TBOA, abolished glutamate uptake on control or T(3)-treated astrocytes. T(3) treatment of astrocytes increased both mRNA levels and protein expression of GLAST and GLT-1, although no significant changes on the distribution of these transporters were observed. The gliotoxic effect of glutamate on cultured cerebellar astrocytes was abolished by T(3) treatment of astrocytes. In addition, the neuronal viability against glutamate challenge was enhanced on T(3)-treated astrocytes, showing a putative neuroprotective effect of T(3). In conclusion, our results showed that T(3) regulates extracellular glutamate levels by modulating the astrocytic glutamate transporters. This represents an important mechanism mediated by T(3) on the improvement of astrocytic microenvironment in order to promote neuronal development and neuroprotection.     

Glutamate transporter function of rat hippocampal astrocytes is impaired following the global ischemia

Astroglial glutamate transporters, GLT-1 and GLAST, play an essential role in removing released glutamate from the extracellular space and are essential for maintaining a low concentration of extracellular glutamate in the brain. It was hypothesized that impaired function of glial glutamate transporters induced by transient global ischemia may lead to an elevated level of extracellular glutamate and subsequent excitotoxic neuronal death. To test this hypothesis, in the present study, we performed whole-cell patch-clamp recording of hippocampal CA1 astrocytes in control or postischemic slices, and measured glutamate transporter activity by recording glutamate-evoked transporter currents. Six to 24 h after global ischemia, maximal amplitude of glutamate transporter currents recorded from postischemic CA1 astrocytes was significantly reduced. Western blotting analysis indicated that transient global ischemia decreased the protein level of GLT-1 in the hippocampal CA1 area without affecting GLAST protein level. Further real-time quantitative RT-PCR assays showed that global ischemia resulted in a decrease in GLT-1 mRNA level of hippocampal CA1 region. Global ischemia-induced reduction in GLT-1 expression and glutamate transporter function of CA1 astrocytes precedes the initiation of delayed neuronal death in CA1 pyramidal layer. The present study provides the evidence that transient global ischemia downregulates glutamate transporter function of hippocampal CA1 astrocytes by decreasing mRNA and protein levels of GLT-1.