Sodium channel currents in rat hippocampal NG2 glia: characterization and contribution to resting membrane potential.

We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I(Na)) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7-21 rats express low density I(Na) (9.5-15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC(50) value of 39.3 nM. The I(Na) expression correlated with a approximately 25 mV more depolarized resting membrane potential (RMP) as compared with non-I(Na)-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 microM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I(Na)-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I(Na) expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 microM TTX. Analysis of the I(Na) activation/inactivation curves yields an I(Na) "window current" at -40+/-20 mV, implying a persistent I(Na) component being active around the NG2 glia RMP of approximately -45 mV. According to the constant-field equation analysis, this active I(Na) component leads to a pNa/pK ratio of 0.14 at RMP which is approximately threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I(Na) component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.

Water homeostasis in the brain: basic concepts.

The mammalian CNS is separated from the blood by tight junctions, collectively termed the blood-brain barrier (BBB). This imposes unique features of solvent and water movement into and out of the CNS. The basic equations for water fluxes driven by osmotic gradients are presented. The anatomy of the BBB and the physiology of the transport processes for cerebrospinal fluid production, extracellular fluid production and intercellular water and solute transport are then described. A quantitative analysis of the need for aquaporin-based water movements to accompany the known rates of CSF production is also presented. Finally, the mechanisms and roles of cellular and vasogenic edema in the CNS, especially in relation to aquaporins, are described.

Freshly isolated hippocampal CA1 astrocytes comprise two populations differing in glutamate transporter and AMPA receptor expression.

We have shown previously that process-bearing GFAP+ astrocytes freshly isolated from rat hippocampus CA1 and CA3 regions are heterogeneous in ion channel expression and K(+) uptake capabilities, such that two distinct populations of astrocytes can be described (Zhou and Kimelberg, 2000). In the present study, we report that glutamate transporter (GT) currents can only be measured from one type of these freshly isolated hippocampal CA1 astrocytes [variably rectifying astrocytes (VRAs)] but were not detectable in the second type of astrocyte [outwardly rectifying astrocytes (ORAs)]. The GT currents showed a strict Na(+) dependency and high affinity for glutamate (EC(50) of 4 +/- 1.1 microm). The astrocytes lacking GT currents (ORAs) showed an AMPA receptor current density (55 pA/pF) that was 42-fold higher than VRAs (1.3 pA/pF). In contrast, the GABA(A) currents were of comparable current density in both types. The specificity of these differences makes it unlikely that they are attributable to preparative damage. Therefore, these findings strongly indicate that, within a single region of the hippocampus, GFAP+ astrocytes comprise a functionally diverse population that are qualitatively different in their functional glutamate transporter and quantitatively different in their functional AMPA receptor expression. This heterogeneity implies that GFAP+ astrocytes may participate in or modulate glutamate synaptic transmission differently.

Developmental expression of metabotropic P2Y(1) and P2Y(2) receptors in freshly isolated astrocytes from rat hippocampus.

There are at least three subtypes of cloned metabotropic P2 receptors linked to intracellular Ca(2+) rises in rat brain cells, namely, P2Y(1), P2Y(2) and P2Y(4). In this study we explore the subtypes of the metabotropic P2 receptors seen in freshly isolated astrocytes (FIAs) from P8-P25 rats. We found by single cell RT-PCR that in process-bearing FIAs from hippocampi of P8-P12 rats, 31% of the glial fibrillary acidic protein (GFAP) mRNA (+) cells expressed P2Y(1) mRNA while only 5% of the cells tested expressed P2Y(2) mRNA. The expression of P2Y(1) receptor mRNA was not changed in FIAs from the hippocampi of P18-P25 rats, but 38% of the GFAP mRNA (+) cells in the P18-P25 age group then showed P2Y(2) mRNA. We also studied whether the mRNA was expressing functional receptor protein by measuring Ca(2+) responses to specific agonists for P2Y(1) and P2Y(2). We found that similar proportions of GFAP mRNA (+) FIAs responded to ATP or UTP as showed mRNAs for P2Y (1) and P2Y(2,) respectively. Total tissue RNA from P9 and P24 rat hippocampus showed a 2.8-fold increase in P2Y(2) mRNA levels from P9 to P24 with a decrease in P2Y(1) mRNA. Thus, this study shows a marked up-regulation of mRNA for P2Y(2) from 9 to 24 days in rat hippocampus, and some of this increase is likely due to the protoplasmic astrocytes which is being translated into functional receptor protein in these cells.

Tamoxifen inhibits nitrotyrosine formation after reversible middle cerebral artery occlusion in the rat.

Tamoxifen (TAM), a widely used non-steroidal anti-estrogen, has recently been shown to be neuroprotective in a rat model of reversible middle cerebral artery occlusion (rMCAo). Tamoxifen has several potential mechanisms of action including inhibition of the release of excitatory amino acids (EAA) and nitric oxide synthase (NOS) activity. The question addressed in this study was whether TAM reduces ischemia-induced production of nitrotyrosine, considered as a footprint of the product of nitric oxide and superoxide, peroxynitrite. In rat brain, 2 h rMCAo produced a time-dependent increase in nitrotyrosine content in the cerebral cortex, as measured by Western blot analysis. Compared with vehicle, TAM significantly reduced nitrotyrosine levels in the ischemic cortex at 24 h. The neuronal (n)NOS inhibitor, 7-nitroindazole also tended to reduce nitrotyrosine, but this reduction was not statistically significant. Immunostaining for nitrotyrosine was seen in cortical neurons in the MCA territory and this immunostaining was reduced by TAM. In vitro, TAM and the calmodulin inhibitor trifluoperazine inhibited, with similar EC(50) values, the activity of recombinant nNOS as well as NOS activity in brain homogenates, measured by conversion of [(3)H]arginine to [(3)H]citrulline. There was marginal inhibition of recombinant inducible (i)NOS activity up to 100 microM TAM. These data suggest that TAM is an effective inhibitor of Ca(2+)/calmodulin-dependent NOS and the derived peroxynitrite production in transient focal cerebral ischemia and this may be one mechanism for its neuroprotective effect following rMCAo.

Freshly isolated astrocytes from rat hippocampus show two distinct current patterns and different [K(+)](o) uptake capabilities.

Whether astrocytes predominantly express ohmic K(+) channels in vivo, and how expression of different K(+) channels affects [K(+)](o) homeostasis in the CNS have been long-standing questions for how astrocytes function. In the present study, we have addressed some of these questions in glial fibrillary acidic protein [GFAP(+)], freshly isolated astrocytes (FIAs) from CA1 and CA3 regions of P7-15 rat hippocampus. As isolated, these astrocytes were uncoupled allowing a higher resolution of electrophysiological study. FIAs showed two distinct ion current profiles, with neither showing a purely linear I-V relationship. One population of astrocytes had a combined expression of outward potassium currents (I(Ka), I(Kd)) and inward sodium currents (I(Na)). We term these outwardly rectifying astrocytes (ORA). Another population of astrocytes is characterized by a relatively symmetric potassium current pattern, comprising outward I(Kdr), I(Ka), and abundant inward potassium currents (I(Kin)), and a larger membrane capacitance (C(m)) and more negative resting membrane potential (RMP) than ORAs. We term these variably rectifying astrocytes (VRA). The I(Kin) in 70% of the VRAs was essentially insensitive to Cs(+), while I(Kin) in the remaining 30% of VRAs was sensitive. The I(Ka) of VRAs was most sensitive to 4-aminopyridine (4-AP), while I(Kdr) of ORAs was more sensitive to tetraethylammonium (TEA). ORAs and VRAs occurred approximately equally in FIAs isolated from the CA1 region (52% ORAs versus 48% VRAs), but ORAs were enriched in FIAs isolated from the CA3 region (71% ORAs versus 29% VRAs), suggesting an anatomical segregation of these two types of astrocytes within the hippocampus. VRAs, but not ORAs, showed robust inward currents in response to an increase in extracellular K(+) from 5 to 10 mM. As VRAs showed a similar current pattern and other passive membrane properties (e.g., RMP, R(in)) to "passive astrocytes"in situ (i.e., these showing linear I-V curves), such passive astrocytes possibly represent VRAs influenced by extensive gap-junction coupling in situ. Thus, our data suggest that, at least in CA1 and CA3 regions from P7-15 rats, there are two classes of GFAP(+) astrocytes which possess different K(+) currents. Only VRAs seem suited to uptake of extracellular K(+) via I(Kin) channels at physiological membrane potentials and increases of [K(+)](o). ORAs show abundant outward potassium currents with more depolarized RMP. Thus VRAs and ORAs may cooperate in vivo for uptake and release of K(+), respectively.

Acute treatment with tamoxifen reduces ischemic damage following middle cerebral artery occlusion.

Inhibitors of cell-swelling-activated anion channels, including the antiestrogenic compound tamoxifen (TAM), have been shown to attenuate the increase in excitatory amino acids (EAA) during ischemia. Since TAM enters the CNS we tested whether it provides protection from damage due to reversible middle cerebral artery occlusion (rMCAo) in rats. TAM (5 mg/kg, i.v.) infused 25 min before ischemia, potently reduced the total volume of the infarct from 328 +/- 34 mm3 to 41 +/- 21 mm3, a reduction of 87%, as measured by TTC staining. It was equally effective when infused starting at 1 h after reperfusion, i.e. 3 h after initiation of rMCAo. Protection of neurons was also found histologically. TAM had no effect on CBF as measured by hydrogen clearance. This appears to be the first report of a marked neuroprotective effect of TAM. Further studies are needed to determine whether its effects are due to inhibition of EAA release and/or other potential neuroprotective sites of action.

Freshly isolated astrocyte (FIA) preparations: a useful single cell system for studying astrocyte properties.

Astrocytes are cell constituents of the mammalian CNS whose intricate relationships with neurons, blood vessels and meninges in situ are well documented. These relationships and their complex morphologies imply numerous functions. Over the past quarter century or so, however, the main experimental basis for determining which roles are likely have been derived from studies on primary astrocyte cultures, usually prepared from neonatal rodent brains. We list a number of examples where these cultures have shown quantitative and qualitative differences from the properties exhibited by astrocytes in situ. The absence of an adequate reliable database makes proposals of likely hypotheses of astrocyte function difficult to formulate. In this article we describe representative studies from our laboratory showing that freshly isolated astrocytes (FIAs), can be used to determine the properties of astrocytes that seem more in concordance with the properties exhibited in situ. Although the cells are most easily isolated from < or =15 day old rat hippocampi they can be isolated from up to 30 day old rats. The examples we describe are that several different types of K(+) currents can be determined by patch clamp electrophysiology, of all the mGluRs only mGluR3 and 5 were detected by single cell RT-PCR, and that single cell Ca(2+) imaging shows that the mGluR5 receptor is functional. It was found that the frequency of cells expressing mGluR5 declines with the age of the animal with the mGluR5b type splice variant replacing the mGluR5a type, as occurs in the intact brain. It is concluded that FIAs can be used to determine the individual characteristics of astrocytes and their properties without the problems of indirect effects inherent in a heterogeneous system such as the slice, and without the problem of cultures unpredictably reflecting the in situ state. The FIAs obviously cannot be used to study interactions of astrocytes with the other CNS components but we propose that they will provide a good database on which hypotheses regarding such interactions can be tested in slices. FIAs can also be isolated from brain slices or intact brain after various pharmacological or electrophysiological perturbations to determine the changes in astrocyte properties that correlate with the perturbations.

Acutely isolated astrocytes as models to probe astrocyte functions.

Neuroscientists have become increasingly aware and accepting of the concept that astrocytes likely have many important functions in the CNS. One limitation in establishing these functions is the usual problem of what constitutes suitable experimental approaches. A major experimental step for functional studies of astrocytes has been the widespread use of primary astrocyte cultures, an approach that Leif Hertz pioneered. However, it is now becoming clear that, building on this work, an experimental paradigm shift is now needed. Namely, to increasingly study preparations corresponding to in situ conditions, such as slices. An alternative experimental system where the cells have some of the technical advantages of primary astrocyte cultures is freshly isolated astrocytes. Recent experiments from our laboratory have shown metabotropic glutamate receptor expression by such cells. Examples are given of how functional receptor studies and channel activity measured by patch clamp electrophysiology can be combined with single cell RT-PCR to define further the receptor or channel type are described to illustrate the uses of such preparations.

GFAP mRNA positive glia acutely isolated from rat hippocampus predominantly show complex current patterns.

Electrophysiologically complex glial cells have been widely identified from different regions of the central nervous system and constitute a dominant glial type in juvenile mice or rats. As these cells express several types of ion channels and neurotransmitter channels that were thought to be only present in neurons, this glial cell type has attracted considerable attention. However, the actual classification of these electrophysiologically complex glial cells remains unclear. They have been speculated to be an immature astrocyte because, although these cells show positive staining for the predominantly astrocytic marker S 100beta, it has not been possible to show staining for the commonly accepted mature astrocytic marker, glial fibrillary acidic protein (GFAP). To address the question of whether these cells might express GFAP at the transcript level, we combined patch-clamp electrophysiological recording with single cell RT-PCR for GFAP mRNA in glial cells acutely isolated from 4 to 12 postnatal day rats. In fresh cell suspensions from the CA1 region, complex glial cells were found to be the dominant cell type (65% total cells). We found that the majority of these electrophysiologically complex cells (74%) were positive for GFAP mRNA. We also showed that the complex cells responded to AMPA and GABA application, and these were also GFAP mRNA positive. We also fixed and stained the preparations for GFAP without electrophysiological recording to better preserve GFAP immunoreactively. In agreement with other studies, only 1.5% of these presumed electrophysiologically complex cells, based on morphology, showed immunoreactivity for GFAP. The expression of GFAP at the transcript level indicates GFAP (-)/GFAP mRNA (+) glial cells have an astrocytic identity. As single cell RT-PCR is able to detect both GFAP (-)/GFAP mRNA (+) and GFAP (+)/GFAP mRNA (+) astrocytic subtypes, the present study also suggests it is a feasible approach for astrocytic lineage studies.

Metabotropic glutamate receptors in acutely isolated hippocampal astrocytes: developmental changes of mGluR5 mRNA and functional expression.

We previously found that 82% of glial fibrillary acidic protein (GFAP)-positive hippocampal astrocytes acutely isolated from P1-10 rats responded to glutamate (Glu) with transient intracellular calcium increases via activation of a Group I metabotropic glutamate receptor (mGluR). Fewer cells responded to ATP and none to serotonin (5-HT). In this study we asked the question whether hippocampal astrocytes in older animals retain this relative pattern of expression. We have found that 77% of GFAP (+) cells from P11-20 rats responded to 50 microM Glu, 43% to ATP, and none to 5-HT. Thirty-three percent of GFAP (+) cells from P25-35 rats responded to Glu, 12% to ATP and 3% to 5-HT. In the case of the responses to Glu, pharmacological characterization and single-cell RT-PCR data confirmed that these responses were mediated by the mGluR5 subtype of group I mGluRs. Also, fewer (36%) GFAP mRNA (+) cells from P25-35 rats expressed detectable mGluR5 mRNA than those from P11-20 rats (77%). This number essentially corresponds to the number of GFAP(+) showing a Ca(2+) response to Glu. Both mGluR5a and b were expressed with equal frequency in cells from P11-20 rats, but the b form predominated in cells from older animals. Overall, our studies show that expression of mGluR5 in hippocampal astrocytes decreases with increasing age and the "a" splice variant declines to a greater extent than the "b" splice variant, corresponding to the developmental changes shown in total tissue for mGluR5.

Current methods and approaches to studying astrocytes: a forum position paper.

Astrocytes are becoming increasingly recognized as targets for neurotoxic agents. For neurotoxicologists, as for other neuroscientists, an important concern is how to study astrocytes. In this paper, it is argued that studies of primary astrocyte cultures, while they are convenient as experimental systems and have been of great value to a resurgence of interest in these cell types over the past quarter century, now need to be supplemented to a large degree by studies on preparations where the properties of astrocytes are less likely to deviate from their properties in situ. Different and alternative systems to primary astrocyte cultures are described and critically evaluated in this article.

Intracellular ATP depletion inhibits swelling-induced D-[3H]aspartate release from primary astrocyte cultures.

Volume expansion-sensing outward rectifier (VSOR) anion channel, also referred to as volume-sensitive organic osmolyte-anion channel (VSOAC), appears to be responsible for cell swelling-induced amino acid release in a variety of cells. One prominent feature of the VSOR/VSOAC is that non-hydrolyzed intracellular ATP binding to the channel or an accessory protein is required for its activation. In this study, the effect of intracellular ATP depletion on the swelling-induced release of D-[3H]aspartate from rat primary astrocyte cultures due to exposure to either high K(+) or hypotonic media was studied. When the cells were pretreated for 10 min with a combination of the metabolic inhibitors 2-deoxyglucose and rotenone, 100 mM K(+) media- or hypotonic media-induced D-[3H]aspartate release was completely suppressed. Added separately, each inhibitor showed only partial or no inhibition of D-[3H]aspartate release, which correlated with its relative effectiveness in decreasing intracellular ATP levels. These data are consistent with the view that during high [K(+)](o) or hypotonic media-induced swelling of primary astrocyte cultures an ATP-dependent swelling-activated VSOAC channel is responsible for D-[3H]aspartate release and close to normal ATP is required for full channel activation.

Volume-dependent taurine release from cultured astrocytes requires permissive [Ca(2+)](i) and calmodulin.

Cell swelling results in regulatory activation of multiple conductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement of extracellular and intracellular Ca(2+) in volume-dependent [(3)H]taurine efflux from primary cultured astrocytes and compare the Ca(2+) sensitivity of this efflux in slow (high K(+) medium induced) and fast (hyposmotic medium induced) cell swelling. Neither Ca(2+)-free medium nor Ca(2+)-channel blockers prevented the volume-dependent [(3)H]taurine release. In contrast, loading cells with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed [(3)H]taurine efflux by 65-70% and 25-30% under high-K(+) and hyposmotic conditions, respectively. Fura 2 measurements confirmed that BAPTA-AM, but not Ca(2+)-free media, significantly reduced resting intracellular Ca(2+) concentration ([Ca(2+)](i)). The calmodulin antagonists trifluoperazine and fluphenazine reversibly and irreversibly, respectively, inhibited the high-K(+)-induced [(3)H]taurine release, consistent with their known actions on calmodulin. In hyposmotic conditions, the effects were less pronounced. These data suggest that volume-dependent taurine release requires minimal basal [Ca(2+)](i) and involves calmodulin-dependent step(s). Quantitative differences in Ca(2+)/calmodulin sensitivity of high-K(+)-induced and hyposmotic medium-induced taurine efflux are due to both the effects of the inhibitors on high-K(+)-induced cell swelling and their effects on transport systems and/or signaling mechanisms determining taurine efflux.

mGluR3 and mGluR5 are the predominant metabotropic glutamate receptor mRNAs expressed in hippocampal astrocytes acutely isolated from young rats.

The metabotropic glutamate receptors (mGluRs) that are expressed and not expressed on astrocytes in the brain have not been defined. While immunohistochemistry and in situ mRNA hybridization have been used on a limited basis to address this question, they do not readily enable the proportion of astrocytes expressing a particular mRNA or protein to be determined. Also, for many receptors, expression by cultured astrocytes does not reflect in situ expression. In this study, therefore, we examined expression of mRNA for all the mGluRs except mGluR6 by single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) in freshly isolated hippocampal astrocytes from postnatal day P1-10 rats, as an additional approach to address the question of which mGluRs are expressed on astrocytes in situ. The astrocytic nature of the cells was supported by simultaneously measuring mRNA for the astrocytic marker glial fibrillary acidic protein (GFAP) from the same cells. In these studies, the percentage of cells showing GFAP mRNA expression was the same as the percentage of cells showing immunocytochemical staining for GFAP. We found that only mGluR3 and mGluR5 mRNAs were significantly present in GFAP mRNA(+) cells. The mGluR5 PCR products were primarily of the "a" splice variant. mGluR1, 2, 4, 7, and 8 were very rarely or never detected. mGluR6 mRNA level was too low in whole rat brain and hippocampus to warrant examination. These results show that interpretation of effects involving mGluR3 or 5 activation in the hippocampus of young rats needs to also consider effects due to astrocytes.

Glial cells in neurotoxicity development.

Neuroglial cells of the central nervous system include the astrocytes, oligodendrocytes, and microglia. Their counterparts in the peripheral nervous system are the Schwann cells. The term neuroglia comes from an erroneous concept originally coined by Virchow (1850), in which he envisioned the neurons to be embedded in a layer of connective tissue. The term, or its shortened form--glia, has persisted as the preferred generic term for these cells. A reciprocal relationship exists between neurons and glia, and this association is vital for mutual differentiation, development, and functioning of these cell types. Therefore, perturbations in glial cell function, as well as glial metabolism of chemicals to active intermediates, can lead to neuronal dysfunction. The purpose of this review is to explore neuroglial sites of neurotoxicant actions, discuss potential mechanisms of glial-induced or glial-mediated central nervous system and peripheral nervous system damage, and review the role of glial cells in neurotoxicity development.

[3H]taurine and D-[3H]aspartate release from astrocyte cultures are differently regulated by tyrosine kinases.

Volume-dependent anion channels permeable for Cl- and amino acids are thought to play an important role in the homeostasis of cell volume. Astrocytes are the main cell type in the mammalian brain showing volume perturbations under physiological and pathophysiological conditions. We investigated the involvement of tyrosine phosphorylation in hyposmotic medium-induced [3H]taurine and D-[3H]aspartate release from primary astrocyte cultures. The tyrosine kinase inhibitors tyrphostin 23 and tyrphostin A51 partially suppressed the volume-dependent release of [3H]taurine in a dose-dependent manner with half-maximal effects at approximately 40 and 1 microM, respectively. In contrast, the release of D-[3H]aspartate was not significantly affected by these agents in the same concentration range. The inactive analog tyrphostin 1 had no significant effect on the release of both amino acids. The data obtained suggest the existence of at least two volume-dependent anion channels permeable to amino acids in astrocyte cultures. One of these channels is permeable to taurine and is under the control of tyrosine kinase(s). The other is permeable to both taurine and aspartate, but its volume-dependent regulation does not require tyrosine phosphorylation.

Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker.

BACKGROUND AND PURPOSE: Increased activation of excitatory amino acid (EAA) receptors is considered a major cause of neuronal damage. Possible sources and mechanisms of ischemia-induced EAA release were investigated pharmacologically with microdialysis probes placed bilaterally in rat striatum. METHODS: Forebrain ischemia was induced by bilateral carotid artery occlusion and controlled hypotension in halothane-anesthetized rats. During 30 minutes of ischemia, microdialysate concentrations of glutamate and aspartate were measured in the presence of a nontransportable blocker of the astrocytic glutamate transporter GLT-1, dihydrokinate (DHK), or an anion channel blocker, 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS), administered separately or together through the dialysis probe. RESULTS: In control striata during ischemia, glutamate and aspartate concentrations increased 44+/-13 (mean+/-SEM) times and 19+/-5 times baseline, respectively, and returned to baseline values on reperfusion. DHK (1 mmol/L in perfusate; n=8) significantly attenuated EAA increases compared with control (glutamate peak, 9. 6+/-1.7 versus control, 15.4+/-2.6 pmol/ microL). EAA levels were similarly decreased by 10 mmol/L DHK. DNDS (1 mmol/L; n=5) also suppressed EAA peak increases (glutamate peak, 5.8+/-1.1 versus control, 10.1+/-0.7 pmol/ microL). At a higher concentration, DNDS (10 mmol/L; n=7) further reduced glutamate and aspartate release and also inhibited ischemia-induced taurine release. Together, 1 mmol/L DHK and 10 mmol/L DNDS (n=5) inhibited 83% of EAA release (glutamate peak, 2.7+/-0.7 versus control, 10.9+/-1.2 pmol/ microL). CONCLUSIONS: These findings support the hypothesis that both cell swelling-induced release of EAAs and reversal of the astrocytic glutamate transporter are contributors to the ischemia-induced increases of extracellular EAAs in the striatum as measured by microdialysis.