Tonic release of glutamate by a DIDS-sensitive mechanism in rat hippocampal slices
1 Department of Physiology, University College London, Gower Street, London, WC1E 6BT, UK
Abstract
Tonic release of glutamate into the extracellular space of the hippocampus and striatum is non-vesicular, and has been attributed largely to a cystine–glutamate exchanger which is blockable by the glutamate analogue (S)-4-carboxyphenylglycine (CPG). Tonic glutamate release may be functionally important: modulation of this release in the striatum has been suggested to underlie relapse in the use of cocaine. We monitored tonic glutamate release in area CA1 of hippocampal slices by measuring the glutamate receptor-mediated current evoked in pyramidal cells on block of Na+-dependent glutamate uptake with DL-threo-benzyloxyaspartate (TBOA). Superfused cystine increased tonic glutamate release, and this increase was blocked by CPG, but CPG did not affect tonic glutamate release in the absence of superfused cystine. Tonic glutamate release was not affected by blocking gap junctional hemichannels with 18-glycyrrhetinic acid, blocking ATP receptors with pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), blocking Ca2+-dependent exocytosis from neurones with Cd2+ or bafilomycin, blocking Ca2+-dependent release from glia with indomethacin, or blocking anion channels with 5-nitro-2-(3-phenylpropyl amino) benzoic acid (NPPB) or tamoxifen. However tonic glutamate release was reduced by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), and was potentiated by inhibiting astrocytic conversion of glutamate to glutamine with methionine sulfoximine. These data suggest that although cystine–glutamate exchange is present in the hippocampus it does not generate significant tonic release of glutamate when the extracellular [cystine] is at a physiological level, and that tonic glutamate release is at least partly from astrocytes and is mediated by a DIDS-sensitive mechanism. Theoretical calculations suggest that a significant fraction of tonic glutamate release in hippocampal slices could occur via diffusion of glutamate across lipid membranes.
, 百拇医药
Introduction
In the brain, a low extracellular concentration of neurotransmitter is normally maintained between times of exocytotic transmission, in order to prevent continual activation or desensitization of receptors and, for the excitatory transmitter glutamate, to avoid excitotoxicity (reviewed by Cavelier et al. 2005). However, tonic activation of receptors by the ambient transmitter concentration has been reported for high affinity GABAA receptors in the cerebellum and hippocampus, where it decreases excitability and alters information flow through the tissue (Kaneda et al. 1995; Tia et al. 1996; Brickley et al. 1996; Wall & Usowicz, 1997; Stell & Mody, 2002; Hamann et al. 2002; Rossi et al. 2003; Semyanov et al. 2003), and for NMDA and metabotropic glutamate receptors in hippocampus, where it increases excitability and modulates presynaptic release of transmitter (Sah et al. 1989; Forsythe & Clements, 1990; McBain et al. 1994; Dalby & Mody, 2003).
, 百拇医药
What generates this tonic activation of receptors In the absence of glutamate release, the ionic stoichiometry of glutamate transporters provides sufficient accumulative power to lower the extracellular glutamate concentration, [glu]o, to 2 nM (Zerangue & Kavanaugh, 1996; Levy et al. 1998), but microdialysis experiments in vivo report a [glu]o of 2 μM (Benveniste et al. 1984; Hagberg et al. 1985; Phillis et al. 1994; Wahl et al. 1994) suggesting a constant release of glutamate. Surprisingly, this tonic glutamate release does not reflect either action potential evoked or spontaneous exocytosis of glutamate since, in cultured hippocampal slices, it is not blocked by TTX nor by blocking exocytosis with botulinum A or tetanus neurotoxin (Jabaudon et al. 1999).
, 百拇医药
Baker et al. (2002) have suggested, from microdialysis experiments, that most (60%) of the tonic glutamate release in the striatum is generated by cystine–glutamate exchange (in which glutamate is released in exchange for cystine taken up to make glutathione: Bannai, 1984; Cho & Bannai, 1990; Warr et al. 1999). Further, Baker et al. (2003) proposed that a reduction of cystine–glutamate exchange after withdrawal from cocaine use causes relapse into drug-seeking behaviour, highlighting the possible functional significance of this tonic release. However, cystine–glutamate exchange requires a high [cystine]o (its EC50 for cystine is 100 μM: Warr et al. 1999), while in the extracellular space of the striatum and in the CSF [cystine]o is only around 0.13 μM (Perry et al. 1975; Baker et al. 2003) and will activate cystine–glutamate exchange to less than 0.2% of its maximum rate. Apart from cystine–glutamate exchange, there are several other unusual modes of transmitter release that are also candidates for mediating the tonic release of glutamate. In astrocytes, a rise of [Ca2+]i can release glutamate by a prostaglandin-dependent mechanism (Bezzi et al. 1998), and three distinct ion channels have been shown to release glutamate: swelling-activated anion channels (Rutledge et al. 1998), gap junctional hemichannels (Contreras et al. 2002; Ye et al. 2003), and P2X7 receptors gated by ATP (Sperlagh et al. 2002; Wang et al. 2002; Duan et al. 2003). In this paper we have re-investigated the possible contribution of cystine–glutamate exchange to setting the ambient [glu]o, and tested the role of these novel astrocyte release mechanisms.
, 百拇医药
Methods
Preparation of brain slices
Coronal brain slices (225 μm) containing the hippocampus were obtained from Sprague-Dawley rats (11–13 days old), killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act 1986. The slices were made in an oxygenated (95% O2–5% CO2) solution at 4°C containing (mM): 126 NaCl, 2.5 KCl, 2 MgCl2, 26 NaHCO3, 1 NaH2PO4, 2.5 CaCl2, 10 glucose and 1 sodium kynurenate (to block glutamate receptors), pH 7.4; they were stored for 1 h at 34°C before they were used for the experiments.
, 百拇医药
Electrophysiology
CA1 pyramidal cells were whole-cell voltage-clamped with a pipette solution containing (mM): 135 CsCl, 4 NaCl, 0.5 CaCl2,10 Hepes, 5 Na2EGTA, 2 MgATP and 0.5 Na2GTP, 10 QX-314 (to suppress voltage-gated sodium currents), pH set to 7.2 with CsOH. Electrode junction potentials (–3 mV) were corrected for. Electrodes were pulled from thin-walled borosilicate tube and had a resistance in external solution of 2–4 M. Cells were considered acceptable if the holding current at –33 mV was < 400 pA, with an access resistance < 10 M. External solution, as for brain slicing but without sodium kynurenate, was superfused at 25°C, or in some experiments at 35°C as stated in the text. Excitatory postsynaptic currents were evoked by placing a concentric stimulating electrode at a standard location among the Schaffer collaterals and stimulating at 100 V.
, 百拇医药
Drugs and chemicals
Tetrodotoxin (TTX, 0.1–1 μM, see text), picrotoxin (300 μM), strychnine (5 μM) and glycine (100 μM) were normally present in the bathing fluid to block action potentials, to block GABAA and glycine receptors, and to fully activate the glycine-binding site on NMDA receptors, but TTX was omitted when studying synaptic currents. Glutamate (Glu), DL-threo-benzyloxyaspartate (TBOA), cadmium (CdCl2), indomethacin, D-2-amino-5-phosphonopentanoic acid (AP5), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB), 18-glycyrrhetinic acid, cystine, (S)-4-carboxy-phenylglycine (CPG), tamoxifen and L-methionine sulfoximine (MSO) were applied as indicated. TBOA, NPPB, CPG and bafilomycin were purchased from Tocris (Bristol, UK); picrotoxin, glutamate, DIDS, PPADS, CdCl2, 18-glycyrrhetinic acid, cystine, tamoxifen, veratridine and MSO were from Sigma; TTX was from Alomone labs. In order to maintain a constant extracellular chloride concentration in experiments involving propionate, we used a solution which was obtained from the normal solution by substituting 20 mM sodium methanesulphonate for 20 mM NaCl. Sodium propionate was then substituted for an equivalent amount of sodium methanesulphonate. To pretreat slices with bafilomycin to deplete glutamate from synaptic vesicles, slices were incubated in a solution (without sodium kynurenate) to which 4 μM bafilomycin was added for over 2.5 h, and for 5 min at the start of this period 10 μM veratridine was also present to depolarize neurones and increase vesicle turnover. Control slices were incubated for the same period in solution containing sodium kynurenate (and were also exposed to veratridine for 5 min).
, 百拇医药
Analysis
Data are presented as means ± S.E.M. Glutamate release was assessed as the current produced by activation of glutamate receptors which occurred when glutamate uptake was blocked with TBOA. TBOA-evoked currents in the presence of a putative glutamate release-blocking drug were compared with the average of the values of the current in control solution before applying and after washout of the drug. Levels of significance were assessed using Student's two-tailed t test. Differences in means were considered significant when the P-value was less than 0.05.
, 百拇医药
When putative blockers of glutamate release mechanisms were applied, we carried out control experiments to check that they did not affect the glutamate receptors being used to sense the glutamate concentration, by assessing the effect of the drug on the response to exogenous glutamate. Only if a drug blocked the response to TBOA more than it blocked the response to exogenous glutamate did we conclude that glutamate release had been reduced.
We considered the possibility that exogenously applied glutamate might act on cells in the slice to release further glutamate. This does not invalidate our approach of comparing the effect of a putative release-blocking drug on the responses to TBOA and to exogenous glutamate, as is shown in the following analysis, because any secondary glutamate release evoked by exogenous glutamate will also be produced by the glutamate that accumulates (as a result of tonic release) when transporters are blocked with TBOA. To see this, suppose that a glutamate concentration ([glu]) produced in the extracellular space either exogenously ([glu]applied) or by inhibiting uptake with TBOA ([glu]TBOA), produces extra glutamate release through two mechanisms, one mechanism which is blocked by the drug being investigated and one mechanism which is not affected by the drug. If, in response to the [glu]applied, the [glu] in the extracellular space is amplified by a factor Fb via the mechanism which is blocked (b) by the drug, and amplified by a factor Fnb via the mechanism which is not blocked (nb) by the drug, then in the absence of the drug the total glutamate concentration produced by applied glutamate is:
, 百拇医药
and in the presence of the drug (when Fb = 0):
Thus, the ratio of the glutamate concentrations produced in the presence and absence of drug is:
When TBOA is applied the same equations apply except that [glu]applied is replaced by a glutamate concentration proportional to the glutamate release rate. We express the tonic glutamate release as:
where Rb is the rate of release by the mechanism that is blocked by the drug under consideration, and Rnb is the rate of release by the non-blocked release mechanism. These release mechanisms will generate a glutamate concentration that we assume to be proportional to the release rate (because diffusion and transporters operate with a linear dependence on [glu] at the low concentrations being considered), which will then be amplified as described above for exogenous glutamate application. Thus, in the absence of the blocking drug:
, http://www.100md.com
where k is a constant, and in the presence of the blocking drug (when Rb and Fb are zero):
Thus, the ratio of the glutamate concentrations produced in the presence and absence of drug is:
Comparing eqns (1) and (2), we see that the blocking drug reduces the extracellular glutamate concentration produced by TBOA application more than it reduces the [glu] produced by exogenous glutamate application (and so will reduce the glutamate receptor generated response more as well). The extra factor by which the TBOA response is multiplied is:
, 百拇医药
i.e. the decrease (relative to the decrease of the response to exogenous glutamate) reflects only the fraction of the glutamate release which is suppressed (and is not affected by the parameters Fb and Fnb describing secondary release of glutamate). Thus, if a drug blocks glutamate release, then it will reduce the response to TBOA by a larger factor than it reduces the response to externally applied glutamate. Consequently, we can determine whether a drug blocks glutamate release, even if there is secondary glutamate release, by comparing the effect of the drug on the response to TBOA with the effect on the response to applied glutamate.
, 百拇医药
Transient currents caused putatively by transient glutamate release from astrocytes (Angulo et al. 2004; Fellin et al. 2004) were defined as inward currents with an amplitude > 20 pA, a rise time > 10 ms and a decay time > 100 ms, and their rate of occurrence in 1-min periods in control solution or around the peak of the response to 1.5 min TBOA exposure was quantified. TBOA did not significantly increase the rate of these events (0.33 ± 0.13 events min–1 in 8 cells in TBOA versus 0.22 ± 0.05 events min–1 in 10 control cells, P = 0.4).
, 百拇医药
Results
Blocking Na+-dependent glutamate transporters activates an NMDA receptor current in pyramidal cells
We monitored tonic glutamate release into the extracellular space of hippocampal slices by applying a non-transported blocker of Na+-dependent glutamate transporters, DL-TBOA (Shimamoto et al. 1998, 2000), and detecting the resulting rise of extracellular glutamate concentration, [glu]o, from the membrane current it produced in whole-cell clamped CA1 pyramidal cells (Jabaudon et al. 1999). All experiments were done with action potentials blocked by TTX, GABAA and glycine receptors blocked with picrotoxin and strychnine, and the glycine site of NMDA receptors activated with glycine. Cells were clamped to –33 mV to remove Mg2+ block of NMDA receptor channels and thus maximize the current generated in response to a rise of [glu]o. DL-TBOA blocks the hippocampal glutamate transporters GLAST/EAAT1, GLT-1/EAAT2 and EAAC1/EAAT3 with Ki values of 42, 5.7 and 8.0 μM, respectively (Shimamoto et al. 1998, 2000), so the concentration of TBOA used here (200 μM) should reduce uptake of a low concentration of extracellular glutamate by these transporters by 83%, 97% and 96%, respectively.
, http://www.100md.com
Figure 1A shows that TBOA produced a slowly developing inward current, which reached 84.4 ± 3.6 pA after 1.5 min in 100 cells. The magnitude of the current was not significantly reduced by increasing the concentration of TTX present from 0.1 to 1 μM TTX (75.9 ± 5.3 pA in 34 cells and 88.8 ± 4.6 pA in 66 cells, respectively, P = 0.07), ruling out incompletely blocked action potentials in the lower TTX concentration as a source of tonic glutamate. The TBOA-evoked current was almost completely absent (blocked by 98.4 ± 1.6% in 6 cells, P = 1.3 x 10–6) when TBOA was applied in the presence of the NMDA receptor blocker D-AP5 (50 μM), and recovered when the D-AP5 was washed out (Fig. 1B), implying that the current is generated by tonic release of glutamate activating NMDA receptors (Jabaudon et al. 1999).
, 百拇医药
A, blocking glutamate uptake with TBOA evokes an inward current at –33 mV that is blocked by D-AP5, in an area CA1 pyramidal cell. B, mean current amplitude data for experiments as in A, in 6 cells, normalized to initial amplitude before D-AP5. C, NMDA (200 μM) evokes a desensitizing inward current at –33 mV. D, the effect of the voltage-gated calcium channel blocker Cd2+ on the response of pyramidal cells to glutamate (10 μM, chosen to generate a current similar in amplitude to that evoked by TBOA). E, effect of Cd2+ on the response to TBOA. F, mean amplitude of currents evoked by glutamate (4 cells) and TBOA (4 cells) in Cd2+ (normalized to values without Cd2+ present). G, effect of bafilomycin pretreatment (see Methods) on the amplitude of the EPSC evoked at –33 mV by stimulating the Schaffer collaterals (at 100 V). H, effect of bafilomycin pretreatment on the response of pyramidal cells to 10 μM glutamate. I, effect of bafilomycin pretreatment on the response to 200 μM TBOA.
, 百拇医药
D-AP5 itself has been reported to block a large inward current in hippocampal pyramidal cells (Sah et al. 1989: 200 pA at –35 mV in 400 μm thick slices (at 30°C, age not specified); < 60 pA in 100 μm slices), which was attributed to activation of NMDA receptors by the ambient glutamate level present. However, we found (in 225 μm slices, at –33 mV) that D-AP5 only blocked an inward current of 5.4 ± 1.4 pA in seven cells at 25°C and a current of 20.8 ± 4.0 pA in six slices at 35°C (data not shown).
, http://www.100md.com
Applying a saturating concentration of NMDA (200 μM; Fig. 1C) activated a peak current of 3.6 ± 0.4 nA in six cells (at 25°C), which decreased to a slowly declining plateau current of 2.7 ± 1.2 nA after 1.5 min when receptor desensitization had occurred (as will presumably also occur in response to the ambient [glu]o and when raising [glu]o with TBOA). Thus, the AP5-suppressible current (5.4 pA) produced by the baseline [glu]o is 0.2% of the current produced (after 1.5 min) by a saturating NMDA concentration (2.7 nA), or 0.13–0.17% of the current that would be produced by a saturating glutamate concentration, since glutamate produces a maximum current that is 1.2- to 1.5-fold larger than that produced by NMDA (via NR1/NR2A or NR1/NR2B receptors: Priestley et al. 1995). Similarly, the rise of [glu]o produced by TBOA activates NMDA receptors to produce approximately 3.3% (84.1 + 5.4 pA = 89.5 pA) of the maximum NMDA-evoked current (after 1.5 min), or about 2.2–2.8% of the current that would be produced by a saturating glutamate concentration. Using the dose–response curve for NMDA receptors, I = Imax[glu]n/{[glu]n + EC50n}, measured for cultured mouse hippocampal neurones by Patneau & Mayer (1990), who found a Hill coefficient, n, of 1.5 and an EC50 of 2.3 μM, these fractional activations translate into an estimated baseline extracellular glutamate concentration of 27–33 nM, and a concentration after 1.5 min TBOA of 0.18–0.22 μM. These experiments were at 25°C. Carrying out similar experiments at 35°C (at which glutamate release, but also glutamate uptake, may be faster), and assuming for simplicity no change in the EC50 for glutamate activating NMDA receptors, we estimated a baseline extracellular glutamate concentration of 77–89 nM, and a concentration after 1.5 min TBOA of 0.60–0.71 μM. Thus, increasing the temperature increases glutamate release somewhat more than it raises the rate of glutamate uptake, and (in the absence of TBOA) a new equilibrium is reached at a higher [glu]o.
, 百拇医药
Exocytosis does not contribute to tonic glutamate release
The experiment of Fig. 1A and B was done with action potentials blocked, but spontaneous Ca2+-dependent exocytosis might still release glutamate, so we tested the effect of blocking voltage-gated Ca2+ channels with 200 μM Cd2+, with the aim of confirming the conclusion of Jabaudon et al. (1999), for cultured slices, that tonic glutamate release is not via Ca2+-dependent exocytosis. This concentration of Cd2+ abolishes somatic Ca2+ currents in cultured pyramidal cells (Jabaudon et al. 1999) and reduces transmitter release at the CA3–CA1 synapse (assessed from the magnitude of the field EPSP) by about 70% (Wu & Saggau, 1994).
, 百拇医药
Cd2+ is known to block the NMDA receptors we are using to sense the released glutamate (Riveros & Orrego, 1986; Mayer et al. 1989), so we first tested the effect of Cd2+ on the response to superfusion of a glutamate concentration (10 μM) chosen to generate a current similar to that evoked by TBOA. Cd2+ reduced the response to glutamate (Fig. 1D), on average by 47 ± 7% (Fig. 1F, P = 0.005, n = 4). Cd2+ also reduced the current evoked by TBOA (Fig. 1E) but by approximately the same fraction as it blocked the NMDA sensor current (Fig. 1F, 40 ± 5%, P = 0.008). There was no significant difference between the inhibition of the glutamate- and of the TBOA-evoked currents (P = 0.46). Thus, the effect of Cd2+ on the TBOA-evoked current is entirely attributable to its inhibition of the NMDA receptors used to sense glutamate release, and we conclude that Cd2+ had no effect on the tonic release of glutamate. Subsequent experiments use this format for displaying the results, i.e. showing the effect of a drug first on the glutamate sensitivity of the NMDA receptor sensors, and then the effect on tonic release detected as a TBOA-evoked current mediated by the NMDA receptors. In order to conclude that a drug blocks tonic glutamate release we would need to demonstrate a significantly greater reduction of the TBOA-evoked current than of the glutamate-evoked current (see Methods).
, 百拇医药
To rule out a contribution of Ca2+-independent exocytosis to tonic glutamate release, we used bafilomycin pretreatment to block the vesicular H+-ATPase: this depletes vesicles of transmitter and thus prevents spontaneous and evoked exocytotic transmitter release (Zhou et al. 2000; Rossi et al. 2003; Allen et al. 2004; Allen & Attwell, 2004). To check that bafilomycin pretreatment was indeed depleting glutamate from vesicles, we evoked EPSCs in pyramidal cells by stimulating the Schaffer collateral input from CA3. Bafilomycin pretreatment reduced the amplitude of the EPSC evoked by a 100 V stimulus by 91% (Fig. 1G). In contrast bafilomycin had no effect on the cells' response to glutamate (Fig. 1H) or to TBOA (Fig. 1I). We conclude that exocytosis does not contribute significantly to tonic glutamate release onto hippocampal pyramidal cells.
, http://www.100md.com
Jabaudon et al. (1999) also found that, in cultured slices, inhibition of exocytosis with botulinum A or tetanus neurotoxin (which abolished spontaneous miniature EPSCs) did not reduce tonic glutamate release, again ruling out the possibility that tonic release is by Ca2+-independent spontaneous vesicular release.
Ca2+-evoked glial glutamate release does not contribute significantly to tonic activation of glutamate receptors
Transient release of glutamate from glia, probably by exocytosis, can activate NMDA receptors in hippocampal pyramidal cells (Fellin et al. 2004; Angulo et al. 2004). These transient release events were reported to occur at a very low rate (0.16–0.82 min–1 at room temperature to 34°C) and to have an amplitude of 100 pA and a decay time of 600 ms. We also observe these rare transient events, at a mean frequency of 0.22 ± 0.05 min–1 in 10 slices at 25°C (mean amplitude 61.7 ± 1.1 pA, mean rise time 169 ± 42 ms, mean decay time 891 ± 380 ms), but they contributed only a small fraction to the time averaged tonic activation of NMDA receptors (3 ± 1% in 10 cells).
, http://www.100md.com
Cystine–glutamate exchange does not generate the tonic glutamate release
To test the possible contribution of cystine–glutamate exchange to tonic glutamate release, we first activated the exchange by applying 300 μM exogenous cystine. The EC50 for activation of the exchange by external cystine is 100 μM (Wyatt et al. 1996; Warr et al. 1999) so the concentration used is sufficient to activate the exchange to about 75% of its maximum level. As reported previously for cerebellar Purkinje cells and neocortical pyramidal cells, cystine sometimes evoked a small inward current itself (arrow in middle panel of Fig. 2A), reflecting a rise of glutamate concentration activating glutamate receptors (Warr et al. 1999). Cystine did not significantly alter the response to 10 μM glutamate (reduced by 6 ± 4% in 6 cells, P = 0.22, Fig. 2D), but increased the response to TBOA (Fig. 2A) by a factor of 2.26 ± 0.49 in 11 cells (P = 0.025, Fig. 2A and D). Thus, cystine–glutamate exchange is present in the hippocampus, and can contribute significantly to glutamate release when the extracellular cystine concentration is high enough.
, http://www.100md.com
A, cystine potentiates the TBOA-evoked current (without affecting the response to glutamate: see D), implying that it increases glutamate release. Arrow shows a small inward current produced by cystine. B, the cystine–glutamate exchange blocker (S)-4-CPG abolished the potentiation produced by cystine, consistent with cystine releasing glutamate via cystine–glutamate exchange. C, in the absence of cystine, CPG has no effect on the TBOA-evoked current, implying cystine–glutamate exchange does not normally release a significant amount of glutamate. D, mean effects on the glutamate- and TBOA-evoked currents of cystine (6 cells for glutamate, 11 cells for TBOA), cystine in the presence of CPG (6 cells for TBOA; glutamate not studied), and CPG (5 cells each for glutamate and TBOA). Data are normalized to the value with no drug (for cystine alone or CPG alone), or to the value in CPG (for application of cystine in CPG).
, 百拇医药
To determine the contribution of cystine–glutamate exchange to glutamate release in the absence of superfused cystine (in which situation the [cystine]o is presumably closer to its in vivo value (measured in the striatum) of 0.16 μM: Baker et al. 2003), we employed the non-transported cystine–glutamate exchange blocker CPG ((S)-4-carboxyphenylglycine, 50 μM: Ye et al. 1999; Patel et al. 2004). The Ki of CPG is 5 μM (Patel et al. 2004), so for a low external cystine and glutamate concentration 50 μM CPG should inhibit the exchange by 91%. First we verified that CPG did block cystine–glutamate exchange, by repeating the experiment of Fig. 2A in the presence of CPG. CPG abolished the increase of tonic glutamate release evoked by exogenous cystine (Fig. 2B and D; in the presence of CPG, cystine increased the TBOA-evoked current by 0.8 ± 4.5%, P = 0.76, in 6 cells). Then we tested the effect of CPG on the TBOA-evoked current in the absence of exogenous cystine. CPG had no effect on the NMDA receptors used to sense the rise of [glu]o evoked by TBOA (the response to 10 μM glutamate was increased insignificantly by 6.9 ± 3.4% in 5 cells, P = 0.1, Fig. 2D). CPG also had no significant effect on the TBOA-evoked current (increased by 3.8 ± 13.3%, P = 0.77, Fig. 2C and D). Thus, cystine–glutamate exchange does not contribute significantly to tonic glutamate release in the absence of an artificially raised extracellular cystine concentration.
, 百拇医药
Effect of blocking glutamine synthetase on tonic glutamate release
The astrocyte-specific enzyme (Norenberg & Martinez-Hernandez, 1979) glutamine synthetase maintains a low glutamate concentration in astrocytes by converting glutamate to glutamine (Ottersen et al. 1992). Jabaudon et al. (1999) found that blocking this enzyme, and presumably raising astrocyte [glu]i, increased tonic glutamate release in cultured slices, suggesting that at least some tonic glutamate release is from astrocytes. Since astrocytes can change their properties in culture (Thio et al. 1993; Schultz-Suchting & Wolburg, 1994; Kimelberg et al. 1997; Zelenaia et al. 2000), we wanted to check that this result holds in acute slices, before investigating the possible release mechanism of glutamate from astrocytes.
, 百拇医药
We tested the effect of applying the glutamine synthetase blocker L-methionine sulfoximine (MSO, 5 mM) for 1 min on the response to glutamate and TBOA. MSO had no effect on the response to glutamate (Fig. 3A and C, increased by 10.1 ± 7.7%, P = 0.3 in 3 cells), but produced a small inward current itself in the absence of TBOA (12.8 ± 3.0 pA in 5 cells), as expected if a rise of [glu]i in astrocytes increased glutamate release, and increased the response to TBOA by 53.5 ± 5.6% (P = 0.003, n = 4, Fig. 3B and C). The increase in the response to TBOA was significantly larger than that for the response to glutamate (P = 0.01). Thus, as in culture, methionine sulfoximine increases the tonic release of glutamate.
, http://www.100md.com
A, MSO has no effect on the NMDA receptors used to sense glutamate. B, MSO potentiates tonic glutamate release, assessed as a TBOA-evoked current. C, mean data for experiments shown in A and B, normalized to the current value in the absence of MSO (3 cells for glutamate, 4 cells for TBOA).
We were surprised that even a minute's application of MSO was sufficient to potentiate glutamate release, since this agent is often applied for hours. The rapid effect of exposure to MSO suggests that it can enter the cells and inhibit glutamine synthetase, and allow [glu]i to rise, relatively rapidly. A similar rapid regulation of transmitter release by modulation of intracellular transmitter concentration has been demonstrated for GABAergic synaptic transmission: blocking uptake of precursor glutamate decreases intracellular [GABA] and reduces postsynaptic IPSCs within minutes (Mathews & Diamond, 2003).
, http://www.100md.com
Tonic indomethacin-sensitive glutamate release from astrocytes is not significant
A possible contribution to tonic glutamate release of prostaglandin- and Ca2+-dependent glutamate release from astrocytes was assessed by blocking this release mechanism with 100 μM indomethacin, which inhibits glutamate release by > 80% (Bezzi et al. (1998) showed that 1 μM indomethacin blocks 80% of the release). Indomethacin was applied at least 2 min before glutamate or TBOA (10–100 μM indomethacin produces 50–66% of its prostaglandin-blocking effect in 2 min (Ko et al. 2002; Yerxa et al. 2002)). Indomethacin did not significantly alter the response to glutamate (increased by 8.8 ± 8.2% in 6 cells, P = 0.35) and did not reduce the response to TBOA (increased by 38 ± 13% in 6 cells, P = 0.03, Fig. 4A), and the effects on the response to glutamate and TBOA were not significantly different (P = 0.094), implying that the prostaglandin-dependent mechanism does not contribute significantly to tonic glutamate release.
, http://www.100md.com
Effects on the currents evoked by glutamate or TBOA of A, indomethacin (6 cells for glutamate, 6 cells TBOA), B, NPPB (5 cells glutamate, 4 cells TBOA), C, tamoxifen (3 cells glutamate, 3 cells TBOA), D, 18-glycyrrhetinic acid (18-GA, 7 cells glutamate, 4 cells TBOA), and E, PPADS (5 cells glutamate, 5 cells TBOA). Data are normalized to currents in the absence of the drugs.
Tonic glutamate release via astrocyte swelling-activated anion channels, gap junctional hemichannels and P2X7 receptors is not significant
, 百拇医药
Glutamate release from astrocytes can occur via swelling activated anion channels that are blocked by NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) and tamoxifen. These drugs were applied at least 2 min before glutamate or TBOA. NPPB (100 μM) blocks the channels mediating swelling-evoked release by 90% within 1 min (Rutledge et al. 1998; Crepel et al. 1998) and tamoxifen (15 μM) blocks these channels by 80% within 1.5 min: Rutledge et al. 1998; Ransom et al. 2001). Neither NPPB (100 μM, 4 cells) nor tamoxifen (pooled data from 15 μM on 2 cells and 30 μM on 1 cell) had a significant effect (< 4% change, P > 0.35) on the pyramidal cell response to either glutamate or TBOA (Fig. 4B and C).
, 百拇医药
To test for tonic glutamate release via gap junctional hemichannels, which are opened by low [Ca2+]o and metabolic inhibition but could have a non-zero open probability under normal conditions (Contreras et al. 2002; Ye et al. 2003), we applied 18-glycyrrhetinic acid (25 μM, applied at least 2 min before glutamate or TBOA was applied), which blocks hemichannel permeability by > 80% (Ye et al. 2003), and acts in 10 s (Takeda et al. 2005). This did not significantly alter the pyramidal cell response to glutamate (decreased by 8.3 ± 6.0% in 7 cells, P = 0.22) and increased (rather than decreased) the response to TBOA (increased by 22.2 ± 4.0% in 4 cells, P = 0.01), implying that there is no significant contribution to tonic glutamate release from these channels (Fig. 4D).
, 百拇医药
ATP-gated P2X7 receptors can mediate transmitter release, either via transmitter diffusing through the large pore that these receptors form or by activation of a transporter mechanism (Sperlagh et al. 2002; Wang et al. 2002; Duan et al. 2003). In the presence of 30 μM PPADS (pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, applied 2 min before glutamate or TBOA), which reduces P2X7 receptor activation by more than 75–86% in 1 min (Duan et al. 2003; Sun et al. 1999), the response to glutamate was reduced by 27.9 ± 8.6% (P = 0.047, 5 cells), implying a small inhibition of NMDA receptors (Fig. 4E), but there was no significant change in the response to TBOA (reduced by 10.2 ± 7.8% in 5 cells, P = 0.19), and there was no significant difference when comparing the effects of PPADS on the glutamate and TBOA evoked currents (P = 0.16), implying that PPADS did not inhibit tonic glutamate release.
, 百拇医药
Tonic glutamate release is reduced by DIDS
4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) has been found to block numerous anion transporters and channels via which glutamate might exit the cell. Applying 1 mM DIDS evoked an outward current in pyramidal cells (69.1 ± 9.5 pA in 9 cells at –33 mV), and increased the response to glutamate by 32.7 ± 10.9% (P = 0.04) in 5 cells (Fig. 5A and C). Tauskela et al. (2003) previously suggested that 1 mM DIDS decreased NMDA receptor-mediated currents; however, they applied DIDS only in the presence of NMDA and the apparent decrease of the NMDA-evoked current that they reported was apparently an artefactual result caused by the outward DIDS-evoked current shown in Fig. 5A.
, 百拇医药
A, DIDS evokes an outward current and increases the current evoked by glutamate. B, DIDS decreases the TBOA-evoked current. C, mean data for experiments shown in A and B, normalized to current values in the absence of DIDS (5 cells for glutamate, 6 cells for TBOA). D, propionate (which, like DIDS, is expected to evoke an intracellular acidification) also evokes an outward current and increases the glutamate-evoked current. E, unlike DIDS, propionate slightly increases the TBOA-evoked current. F, mean data from experiments shown in D and E (5 cells each for glutamate and TBOA).
, http://www.100md.com
DIDS inhibits pH-regulating transporters and makes the intracellular pH go acid (Schwiening & Boron, 1994; Baxter & Church, 1996; Bonnet et al. 2000). Sodium propionate (20 mM), which enters across the cell membrane in neutral form and then releases a proton intracellularly, is expected to mimic this acidification (Rorig et al. 1996; Lee et al. 1996). We found that sodium propionate also induced an outward current (37.7 ± 2.7 pA in 7 cells at –33 mV) and increased the response to glutamate by 13.4 ± 3.6% in five cells (P = 0.016; Fig. 5D and F), suggesting that the outward current and the potentiation of the glutamate-evoked NMDA response seen in DIDS are produced by intracellular acidification.
, 百拇医药
Propionate also increased the response to TBOA (by 28.5 ± 4.7% in 5 cells, P = 0.0017, Fig. 5E and F). By contrast, DIDS decreased the response to TBOA by 47.2 ± 5.3% in six cells (P = 0.0002, Fig. 5B and C), which was statistically significantly different from the DIDS-evoked change in the glutamate response (P = 0.00067), implying that DIDS decreases the tonic release of glutamate. The fact that DIDS decreases the response to TBOA, while propionate increases it, suggests that the suppressive action of DIDS on tonic glutamate release (unlike its actions on the baseline current and the response to glutamate, which mimic those of propionate) is not produced by an intracellular acidification.
, http://www.100md.com
DIDS has been reported to inhibit a glutamate transporter (probably GLAST) in Bergmann glial cells (Ruiz & Ortega, 1995). We considered the possibility that the inhibition of the response to TBOA in Fig. 5B and C was a result of DIDS having already inhibited glutamate transporters present in the slice, so that superimposed TBOA produced less uptake inhibition. If this were the case we would expect that application of DIDS would generate a rise of [glu]o that would partially activate NMDA receptors. This cannot be assessed from the change of current evoked by DIDS, because that change clearly also involves an outward (probably acidification-evoked) component. We therefore applied 50 μM D-AP5 to determine whether there was more tonic activation of NMDA receptors present in DIDS. The current change measured (2.7 ± 1.2 pA in 6 cells) was not larger (P = 0.18) than that seen in absence of DIDS (5.4 ± 1.4 pA in 7 cells, see above), showing that inhibition of glutamate transporters by DIDS cannot explain the results in Fig. 5B and C.
, 百拇医药
Discussion
The value of the baseline extracellular glutamate concentration, [glu]o, in the CNS could have a major effect on neuronal excitability and synaptic transmission, but what sets this value is poorly understood. The data in this paper from acutely isolated hippocampal slices extend earlier results on tonic non-synaptic glutamate release in cultured slices (Jabaudon et al. 1999) which showed using tetanus toxin that this release is not via exocytosis, a result we have confirmed in acute slices using bafilomycin to deplete vesicles of glutamate. We have demonstrated that this release is not via cystine–glutamate exchange, contrary to current ideas based on microdialysis work in striatum (Baker et al. 2002), and that it is not via four other glutamate release mechanisms previously characterized in astrocytes, but that it is inhibited by DIDS. Below, we assess the implications of these results. Throughout this discussion it should be remembered that, although we discuss release of glutamate, it is possible that the release of other agents acting on NMDA receptors, such as aspartate, might also contribute to the currents recorded.
, 百拇医药
Cystine–glutamate exchange does not significantly contribute to the ambient [glu]o
In our experiments the cystine–glutamate exchange blocker CPG had no effect on tonic glutamate release unless an unphysiologically high concentration of external cystine was superfused. This is consistent with the low level of cystine present in the extracellular space of the brain. Baker et al. (2003) measured an extracellular [cystine] value of 0.16 μM, which is much less than the EC50 for external cystine activating cystine–glutamate exchange (100 μM, Wyatt et al. 1996; Warr et al. 1999) and will activate the exchanger to less than 0.2% of its maximum rate.
, 百拇医药
There are three possible explanations for the difference between our results and those of Baker et al. (2002). First, in our experiments on slices the diffusional access to the bulk solution (and lack of a cystine/cysteine supply from the blood) might reduce the cystine concentration in the extracellular spaces of the preparation to a value even smaller than the 0.16 μM that Baker et al. (2003) measured in vivo, leading to cystine–glutamate exchange being deactivated. If so, then the cystine–glutamate exchange could be important in vivo (despite the very low [cystine]o), as Baker et al. (2002, 2003) suggest, but not in slice experiments. Alternatively, in the experiments of Baker et al. (2002) the microdialysis cannula might have caused leakage of cystine into the extracellular space from the blood where the cystine concentration is 80 μM (Battistin et al. 1971), artefactually raising the local [cystine]o (Westergren et al. 1995). In this case, slice experiments might better assess the true role of cystine–glutamate exchange, which could be negligible as a source of glutamate in vivo. Finally, Baker et al. (2002) relied on CPG being a specific blocker of cystine–glutamate exchange, but this glutamate analogue is also an antagonist at mGluR1 and mGluR5 metabotropic glutamate receptors and an agonist at mGluR2 (Hayashi et al. 1994; Kingston et al. 1995) and is expected to reduce neuronal excitability and glutamate release by both of these mechanisms (Lingenhohl et al. 1993; Ugolini & Bordi, 1995; Moroni et al. 1998). Baker et al. (2002) checked that an action of CPG at group I mGluRs was not how CPG inhibited cystine uptake in their experiments, but they did not check that CPG still reduced tonic glutamate release when mGluRs were blocked. Thus, CPG may have reduced [glu]o in their experiments by acting on mGluRs and reducing spontaneous exocytotic glutamate release rather than by blocking cystine–glutamate exchange.
, 百拇医药
What mechanism mediates tonic glutamate release other than cystine–glutamate exchange
We found, like Jabaudon et al. (1999), that the glutamine synthetase inhibitor methionine sulfoximine (MSO) increased tonic glutamate release. MSO is expected to raise the glutamate concentration in astrocytes, so the increase in tonic release could be explained if at least some of the release occurs from astrocytes and a raised astrocyte [glutamate] potentiates this release. However, our experiments ruled out a contribution to tonic glutamate release from four previously characterized glutamate release mechanisms present in astrocytes: prostaglandin- and Ca2+-dependent release, swelling-activated anion channels, gap junctional hemichannels and P2X7 receptors. The only agent we found to significantly reduce tonic glutamate release was DIDS, which blocks many transporters and channels that allow the movement of negatively charged molecules like glutamate.
, 百拇医药
In the Appendix, we consider the possibility that some of the tonic glutamate release results from passive diffusion of glutamate across cell membranes. Using the measured diffusion coefficients for charged and neutral amino acids crossing lipid bilayers, we estimate the baseline [glu]o that could be produced by diffusive glutamate release when Na+-dependent uptake is functioning normally ([glu]o 31 nM) or when it is inhibited with TBOA ([glu]o 0.5 μM). Although these estimates depend on parameters for diffusion and uptake which are only approximately known, they are quite similar in magnitude to the values we estimated experimentally (30 nM and 0.2 μM, respectively, at 25°C, see Results). Thus, it appears that diffusive glutamate efflux may contribute significantly to determining the baseline [glu]o in hippocampal slices. Potentiation of the TBOA-evoked current by MSO (Fig. 3) could then just reflect more diffusive efflux when the intracellular glutamate concentration is raised in astrocytes, but it is unclear how DIDS might modulate diffusive glutamate efflux (Chakrabarti & Deamer, 1992 reported that pH changes had little effect on the diffusion of amino acids across membranes), suggesting a release mechanism in addition to passive diffusion across the lipid bilayer.
, 百拇医药
Functional significance of tonic glutamate release
The micromolar levels of ambient glutamate found in microdialysis experiments should activate NMDA and mGluR receptors, and induce desensitization of NMDA, AMPA and kainate receptors, implying that alteration of the ambient [glu]o may have profound consequences for the information processing carried out by neurones. Indeed, in cultured hippocampal neurones, Forsythe & Clements (1990) found that less than 1 μM glutamate depressed the EPSC amplitude by 40%, and Zorumski et al. (1996) found similar effects of low micromolar levels of glutamate on both the AMPA and the NMDA components of the EPSC. Sah et al. (1989) and Dalby & Mody (2003) reported that NMDA receptors in cells in hippocampal slices could be tonically activated by the ambient glutamate level, generating an inward current which increases the excitability of the neurones. However, the size of the tonically activated NMDA receptor (D-AP5 suppressible) current found in the present study was much smaller than that found in pyramidal cells by Sah et al. (1989), suggesting a resting [glu]o of around 30–80 nM at 25–35°C (see Results) rather than the 2 μM found in microdialysis experiments. The reason for the difference in D-AP5 suppressible current is unclear, but it could reflect the sharp electrode recordings made by Sah et al. (1989) being from cells very deep in the slice, possibly at a location with compromised oxygen supply, while those we studied with patch-clamp techniques are from more superficial cells. Whether our resting [glu]o is artefactually lowered in the slice preparation, or the microdialysis value is artefactually raised by damage caused by the microdialysis procedure (see Westergren et al. 1995), remains an important question to be answered.
, 百拇医药
The small size of the D-AP5-sensitive current we record without TBOA present (5 pA at –33 mV at 25°C), compared to that seen when uptake is blocked by TBOA (84 pA), highlights the fact that the functional significance of the tonic release of glutamate will depend critically on the prevailing level of Na+-dependent glutamate uptake.
Appendix
Estimate of the contribution of diffusion across lipid membranes to the ambient [glu]o
, http://www.100md.com
We considered the possibility that some of the tonic glutamate efflux detected when TBOA is applied results from passive diffusion of glutamate across cell membranes. Charged and neutral amino acids cross lipid bilayers with a permeability coefficient of P = 5–20 x 10–14 m s–1 (depending on the saturation of the membrane lipid molecules: Chakrabarti & Deamer, 1992). The membrane area per unit volume of hippocampus is 14 μm2/μm3, so in 1 litre there is A = 1.4 x 104 m2 membrane area (Lehre & Danbolt, 1998). Thus, for a mean intracellular glutamate concentration of [glu]i = 3 mM (Attwell et al. 1993), the diffusive glutamate flux into the extracellular space will be Rdiffusion = PA[glu]i = 2.1–8.4 nmol l–1 s –1. The [glu]o this would produce can be estimated by noting that, if this is the only glutamate release mechanism, the release rate must equal the uptake rate by Na+-dependent transporters. Equating an Rdiffusion of (say) 5 nmol l–1 s–1 to an uptake rate, described by Michaelis-Menten kinetics as Umax[glu]o/([glu]o + Km), where the maximum uptake rate, Umax, in brain slices is 0.2 μmol ml–1 min–1 or 3.33 μmol l–1 s–1 and Km is 20 μM (Hertz, 1979; Schousboe, 1981), we predict a baseline [glu]o of 31 nM, which is similar to the 30 nM we estimate from the size of the AP5-suppressible current. When TBOA is added, the [glu]o rise will depend critically on the magnitude of glutamate uptake remaining unblocked in TBOA. Assuming that 21% of hippocampal uptake is due to GLAST and 79% to GLT-1 (Lehre & Danbolt, 1998), i.e. ignoring the contribution of EAAC1 (Haugeto et al. 1996), the expected 83% and 97% inhibition of GLAST and GLT-1 in the presence of 200 μM TBOA (see Results) will lead to only 6% of glutamate uptake remaining functional in TBOA. Equating an Rdiffusion of 5 nmol l–1 s–1 to an uptake rate of 0.06.Umax[glu]o/([glu]o + Km) predicts an equilibrium value for [glu]o in TBOA of 0.5 μM. This is slightly higher than the 0.2 μM [glu]o we estimate to be produced when [glu]o is still rising after 1.5 min of TBOA exposure (see Results). Aspartate could be released by the same mechanism, and would contribute to the activation of NMDA receptors detected.
, http://www.100md.com
References
Allen NJ & Attwell D (2004). The effect of simulated ischaemia on spontaneous GABA release in area CA1 of the juvenile rat hippocampus. J Physiol 561, 485–498.
Allen NJ, Rossi DJ & Attwell D (2004). Sequential release of GABA by exocytosis and reversed uptake leads to neuronal swelling in simulated ischemia of hippocampal slices. J Neurosci 24, 3837–3849.
Angulo MC, Kozlov AS, Charpak S & Audinat E (2004). Glutamate released from glial cells synchronizes neuronal activity in the hippocampus. J Neurosci 24, 6920–6927.
, http://www.100md.com
Attwell D, Barbour B & Szatkowski M (1993). Nonvesicular release of neurotransmitter. Neuron 11, 401–407.
Baker DA, McFarland K, Lake RW, Shen H, Tang XC, Toda S & Kalivas PW (2003). Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse. Nature Neurosci 6, 743–749.
Baker DA, Xi Z, Shen H, Swanson CJ & Kalivas PW (2002). The origin and neuronal function of in vivo nonsynaptic glutamate. J Neurosci 22, 9134–9141.
, http://www.100md.com
Bannai S (1984). Transport of cystine and cysteine in mammalian cells. Biochim Biophys Acta 779, 289–306.
Battistin L, Grynbaum A & Lajtha A (1971). The uptake of various amino acids by the mouse brain in vivo. Brain Res 29, 85–99.
Baxter KA & Church J (1996). Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol 493, 457–470.
Benveniste H, Drejer J, Schousboe A & Diemer NH (1984). Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 43, 1369–1374.
, http://www.100md.com
Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL, Pozzan T & Volterra A (1998). Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391, 281–285.
Bonnet U, Leniger T & Wiemann M (2000). Alteration of intracellular pH and activity of CA3-pyramidal cells in guinea pig hippocampal slices by inhibition of transmembrane acid extrusion. Brain Res 872, 116–124.
Brickley SG, Cull-Candy SG & Farrant M (1996). Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol 497, 753–759.
, 百拇医药
Cavelier P, Hamann M, Rossi DJ, Mobbs P & Attwell D (2005). Tonic excitation and inhibition of neurons. Prog Biophys Molec Biol 87, 3–16.
Chakrabarti AC & Deamer DW (1992). Permeability of lipid bilayers to amino acids and phosphate. Biochim Biophys Acta 1111, 171–177.
Cho Y & Bannai S (1990). Uptake of glutamate and cystine in C-6 glioma cells and in cultured astrocytes. J Neurochem 55, 2091–2097.
, http://www.100md.com
Contreras JE, Sanchez HA, Eugenin EA, Speidel D, Theis M, Willecke K, Bukauskas FF, Bennett MV & Saez JC (2002). Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proc Natl Acad Sci U S A 99, 495–500.
Crepel V, Panenka W, Kelly MEM & MacVicar BA (1998). Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current. J Neurosci 18, 1196–1206.
, 百拇医药
Dalby NO & Mody I (2003). Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release. J Neurophysiol 90, 786–797.
Duan S, Anderson CM, Keung EC, Chen Y, Chen Y & Swanson RA (2003). P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci 23, 1320–1328.
Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG & Carmignoto G (2004). Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729–743.
, 百拇医药
Forsythe ID & Clements JD (1990). Presynaptic glutamate receptors depress excitatory monosynaptic transmission between mouse hippocampal neurones. J Physiol 429, 1–16.
Hagberg H, Lehmann A, Sandberg M, Nystrom B, Jacobson I & Hamberger A (1985). Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 5, 413–419.
Hamann M, Rossi D & Attwell D (2002). Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33, 625–633.
, 百拇医药
Haugeto , Ullensvang K, Levy LM, Chaudhry FH, Honore T, Nielsen M, Lehre KP & Danbolt NC (1996). Brain glutamate transporter proteins form homomultimers. J Biol Chem 271, 27715–27722.
Hayashi Y, Sekiyama N, Nakanishi S, Jane DE, Sunter DC, Birse EF, Udvarhelyi PM & Watkins JC (1994). Analysis of agonist and antagonist activities of phenylglycine derivatives for different cloned metabotropic glutamate receptor subtypes. J Neurosci 14, 3370–3377.
, http://www.100md.com
Hertz L (1979). Functional interactions between neurons and astrocytes I. Turnover and metabolism of putative amino acid transmitters. Prog Neurobiol 13, 277–323.
Jabaudon D, Shimamoto K, Yasuda-Kamatani Y, Scanziani M, Ghwiler BH & Gerber U (1999). Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin. Proc Natl Acad Sci U S A 96, 8733–8738.
Kaneda M, Farrant M & Cull-Candy SG (1995). Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum. J Physiol 485, 419–435.
, 百拇医药
Kimelberg HK, Cai Z, Rastogi P, Charniga CJ, Goderie S, Dave V & Jalonen TA (1997). Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture. J Neurochem 68, 1088–1098.
Kingston AE, Burnett JP, Mayne NG & Lodge D (1995). Pharmacological analysis of 4-carboxyphenylglycine derivatives: comparison of effects on mGluR1 and mGluR5 subtypes. Neuropharmacol 34, 887–894.
Ko WH, Law VW, Yip WC, Yue GG, Lau CW, Chen ZY & Huang Y (2002). Stimulation of chloride secretion by baicalein in isolated rat distal colon. Am J Physiol Gastrointest Liver Physiol 282, G508–G518.
, 百拇医药
Lee J, Taira T, Pihlaja P, Ransom BR & Kaila K (1996). Effects of CO2 on excitatory transmission apparently caused by changes in intracellular pH in the rat hippocampal slice. Brain Res 706, 210–216.
Lehre KP & Danbolt NC (1998). The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci 18, 8751–8757.
Levy LM, Warr O & Attwell D (1998). Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. J Neurosci 18, 9620–9628.
, 百拇医药
Lingenhohl K, Olpe HR, Bendali N & Knopfel T (1993). Phenylglycine derivatives antagonize the excitatory response to Purkinje cells to 1S,3R-ACPD: an in vivo and in vitro study. Neurosci Res 18, 229–234.
Mathews GC & Diamond JS (2003). Neuronal glutamate uptake contributes to GABA synthesis and inhibitory synaptic strength. J Neurosci 23, 2040–2048.
Mayer ML, Vyklicky L Jr & Westbrook GL (1989). Modulation of excitatory amino acid receptors by group IIB metal cations in cultured mouse hippocampal neurones. J Physiol 415, 329–350.
, 百拇医药
McBain CJ, DiChiara TJ & Kauer JA (1994). Activation of metabotropic glutamate receptors differentially affects two classes of hippocampal interneurons and potentiates excitatory synaptic transmission. J Neurosci 14, 4433–4445.
Moroni F, Cozzi A, Lombardi G, Sourtcheva S, Leonardi P, Carfi M & Pellicciari R (1998). Presynaptic mGlu1 type receptors potentiate transmitter output in the rat cortex. Eur J Pharmacol 347, 189–195.
, 百拇医药
Norenberg MD & Martinez-Hernandez A (1979). Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161, 303–310.
Ottersen OP, Zhang N & Walberg F (1992). Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 46, 519–534.
Patel SA, Warren BA, Rhoderick JF & Bridges RJ (2004). Differentiation of substrate and non-substrate inhibitors of transport system xc–: an obligate exchanger of L-glutamate and L-cystine. Neuropharmacol 46, 273–284.
, 百拇医药
Patneau D & Mayer ML (1990). Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors. J Neurosci 10, 2385–2399.
Perry TL, Hansen S & Kennedy J (1975). CSF amino acids and plasma-CSF amino acid ratios in adults. J Neurochem 24, 587–589.
Phillis JW, Smith-Barbour M, Perkins LM & O'Regan MH (1994). Characterization of glutamate, aspartate, and GABA release from ischemic rat cerebral cortex. Brain Res Bull 34, 457–466.
, 百拇医药
Priestley T, Laughton P, Myers J, Le Bourdelles B, Kerby J & Whiting PJ (1995). Pharmacological properties of recombinant human N-methyl-D-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fibroblast cells. Mol Pharmacol 48, 841–848.
Ransom CB, O'Neal JTO & Sontheimer H (2001). Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells. J Neurosci 21, 7674–7683.
, 百拇医药
Riveros N & Orrego F (1986). N-methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances. Neuroscience 17, 541–546.
Rorig B, Klausa G & Sutor B (1996). Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones. J Physiol 490, 31–49.
Rossi DJ, Hamann M & Attwell D (2003). Multiple modes of GABAergic inhibition of rat cerebellar granule cells. J Physiol 548, 97–110.
, 百拇医药
Ruiz M & Ortega A (1995). Characterization of an Na+-dependent glutamate/aspartate transporter from cultured Bergmann glia. Neuroreport 6, 2041–2044.
Rutledge EM, Aschner M & Kimelberg HK (1998). Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures. Am J Physiol 274, C1511–C1520.
Sah P, Hestrin S & Nicoll RA (1989). Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons. Science 246, 815–818.
, 百拇医药
Schousboe A (1981). Transport and metabolism of glutamate and GABA in neurons and glial cells. Int Rev Neurobiol 22, 1–45.
Schultz-Suchting F & Wolburg H (1994). Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex. Cell Tissue Res 277, 557–564.
Schwiening CJ & Boron WF (1994). Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na+-dependent Cl––HCO3– exchange. J Physiol 475, 59–67.
, 百拇医药
Semyanov A, Walker MC & Kullmann DM (2003). GABA uptake regulates cortical excitability via cell type-specific tonic inhibition. Nature Neurosci 6, 484–490.
Shimamoto K, Lebrun B, Yasuda-Kamatani Y, Sakaitani M, Shigeri Y, Yumoto N & Nakajima T (1998). DL-threo-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. Mol Pharmacol 53, 195–201.
Shimamoto K, Shigeri Y, Yasuda-Kamatani Y, Lebrun B, Yumoto N & Nakajima T (2000). Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers. Bioorg Med Chem Lett 10, 2407–2410.
, 百拇医药
Sperlagh B, Kofalvi A, Deuchars J, Atkinson L, Milligan CJ, Buckley NJ & Vizi ES (2002). Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus. J Neurochem 81, 1196–1211.
Stell BM & Mody I (2002). Receptors with different affinities mediate phasic and tonic GABAA conductances in hippocampal neurons. J Neurosci 22, RC223.
Sun SH, Lin LB, Hung AC & Kuo JS (1999). ATP-stimulated Ca2+ influx and phospholipase D activities of a rat brain-derived type-2 astrocyte cell line, RBA-2, are mediated through P2X7 receptors. J Neurochem 73, 334–343.
, http://www.100md.com
Takeda Y, Ward SM, Sanders KM & Koh SD (2005). Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells. Am J Physiol Gastrointest Liver Physiol (in press).
Tauskela JS, Mealing G, Comas T, Brunette E, Monette R, Small DL & Morley P (2003). Protection of cortical neurons against oxygen-glucose deprivation and N-methyl-D-aspartate by DIDS and SITS. Eur J Pharmacol 464, 17–25.
Thio CL, Waxman SG & Sontheimer H (1993). Ion channels in spinal cord astrocytes in vitro. III. Modulation of channel expression by coculture with neurons and neuron-conditioned medium. Neurosci Res 18, 229–234.
, http://www.100md.com
Tia S, Wang JF, Kotchabhakdi N & Vicini S (1996). Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABAA receptor 6 subunit. J Neurosci 16, 3630–3640.
Ugolini A & Bordi F (1995). Metabotropic glutamate group II receptors are responsible for the depression of synaptic transmission induced by ACPD in the dentate gyrus. Eur J Pharmacol 294, 403–410.
Wahl F, Obrenovitch TP, Hardy AM, Plotkine M, Boulu R & Symon L (1994). Extracellular glutamate during focal cerebral ischaemia in rats: time course and calcium-dependency. J Neurochem 63, 1003–1011.
, 百拇医药
Wall MJ & Usowicz MM (1997). Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum. Eur J Neurosci 9, 533–548.
Wang CM, Chang YY, Kuo JS & Sun SH (2002). Activation of P2X7 receptors induced [3H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl–/HCO3–-dependent mechanism. Glia 37, 8–18.
Warr O, Takahashi M & Attwell D (1999). Modulation of extracellular glutamate concentration in rat brain slices by cystine-glutamate exchange. J Physiol 514, 783–793.
, http://www.100md.com
Westergren L, Nystrom B, Hamberger A & Johansson BB (1995). Intracerebral dialysis and the blood brain barrier. J Neurochem 64, 229–234.
Wu LG & Saggau P (1994). Presynaptic calcium is increased during normal synaptic transmission and paired-pulse facilitation, but not in long-term potentiation in area CA1 of hippocampus. J Neurosci 14, 645–654.
Wyatt I, Gyte A, Simpson MG, Widdowson PS & Lock EA (1996). The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat. Arch Toxicol 70, 724–735.
, 百拇医药
Ye ZC, Rothstein JD & Sontheimer H (1999). Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange. J Neurosci 19, 10767–10777.
Ye ZC, Wyeth MS, Baltan-Tekkok S & Ransom BR (2003). Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 23, 3588–3596.
Yerxa BR, Sabater JR, Davis CW, Stutts MJ, Lang-Furr M, Picher M, Jones AC, Cowlen M, Dougherty R, Boyer J, Abraham WM & Boucher RC (2002). Pharmacology of INS37217 [P1-(uridine 5') -P4-(2'-deoxycytidine 5') tetraphosphate, tetrasodium salt], a next-generation P2Y2 receptor agonist for the treatment of cystic fibrosis. J Pharmacol Exp Ther 302, 871–880.
, 百拇医药
Zelenaia O, Schlag BD, Gochenauer GE, Ganel R, Song W, Beesley JS, Grinspan JB, Rothstein JD & Robinson MB (2000). Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-B. Mol Pharmacol 57, 667–678.
Zerangue N & Kavanaugh MP (1996). Flux coupling in a neuronal glutamate transporter. Nature 383, 634–637.
, 百拇医药
Zhou Q, Petersen CC & Nicoll RA (2000). Effects of reduced vesicular filling on synaptic transmission in rat hippocampal neurones. J Physiol 525, 195–206.
Zorumski CF, Mennerick S & Que J (1996). Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons. J Physiol 494, 465–477., http://www.100md.com(Pauline Cavelier and Davi)
Abstract
Tonic release of glutamate into the extracellular space of the hippocampus and striatum is non-vesicular, and has been attributed largely to a cystine–glutamate exchanger which is blockable by the glutamate analogue (S)-4-carboxyphenylglycine (CPG). Tonic glutamate release may be functionally important: modulation of this release in the striatum has been suggested to underlie relapse in the use of cocaine. We monitored tonic glutamate release in area CA1 of hippocampal slices by measuring the glutamate receptor-mediated current evoked in pyramidal cells on block of Na+-dependent glutamate uptake with DL-threo-benzyloxyaspartate (TBOA). Superfused cystine increased tonic glutamate release, and this increase was blocked by CPG, but CPG did not affect tonic glutamate release in the absence of superfused cystine. Tonic glutamate release was not affected by blocking gap junctional hemichannels with 18-glycyrrhetinic acid, blocking ATP receptors with pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), blocking Ca2+-dependent exocytosis from neurones with Cd2+ or bafilomycin, blocking Ca2+-dependent release from glia with indomethacin, or blocking anion channels with 5-nitro-2-(3-phenylpropyl amino) benzoic acid (NPPB) or tamoxifen. However tonic glutamate release was reduced by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), and was potentiated by inhibiting astrocytic conversion of glutamate to glutamine with methionine sulfoximine. These data suggest that although cystine–glutamate exchange is present in the hippocampus it does not generate significant tonic release of glutamate when the extracellular [cystine] is at a physiological level, and that tonic glutamate release is at least partly from astrocytes and is mediated by a DIDS-sensitive mechanism. Theoretical calculations suggest that a significant fraction of tonic glutamate release in hippocampal slices could occur via diffusion of glutamate across lipid membranes.
, 百拇医药
Introduction
In the brain, a low extracellular concentration of neurotransmitter is normally maintained between times of exocytotic transmission, in order to prevent continual activation or desensitization of receptors and, for the excitatory transmitter glutamate, to avoid excitotoxicity (reviewed by Cavelier et al. 2005). However, tonic activation of receptors by the ambient transmitter concentration has been reported for high affinity GABAA receptors in the cerebellum and hippocampus, where it decreases excitability and alters information flow through the tissue (Kaneda et al. 1995; Tia et al. 1996; Brickley et al. 1996; Wall & Usowicz, 1997; Stell & Mody, 2002; Hamann et al. 2002; Rossi et al. 2003; Semyanov et al. 2003), and for NMDA and metabotropic glutamate receptors in hippocampus, where it increases excitability and modulates presynaptic release of transmitter (Sah et al. 1989; Forsythe & Clements, 1990; McBain et al. 1994; Dalby & Mody, 2003).
, 百拇医药
What generates this tonic activation of receptors In the absence of glutamate release, the ionic stoichiometry of glutamate transporters provides sufficient accumulative power to lower the extracellular glutamate concentration, [glu]o, to 2 nM (Zerangue & Kavanaugh, 1996; Levy et al. 1998), but microdialysis experiments in vivo report a [glu]o of 2 μM (Benveniste et al. 1984; Hagberg et al. 1985; Phillis et al. 1994; Wahl et al. 1994) suggesting a constant release of glutamate. Surprisingly, this tonic glutamate release does not reflect either action potential evoked or spontaneous exocytosis of glutamate since, in cultured hippocampal slices, it is not blocked by TTX nor by blocking exocytosis with botulinum A or tetanus neurotoxin (Jabaudon et al. 1999).
, 百拇医药
Baker et al. (2002) have suggested, from microdialysis experiments, that most (60%) of the tonic glutamate release in the striatum is generated by cystine–glutamate exchange (in which glutamate is released in exchange for cystine taken up to make glutathione: Bannai, 1984; Cho & Bannai, 1990; Warr et al. 1999). Further, Baker et al. (2003) proposed that a reduction of cystine–glutamate exchange after withdrawal from cocaine use causes relapse into drug-seeking behaviour, highlighting the possible functional significance of this tonic release. However, cystine–glutamate exchange requires a high [cystine]o (its EC50 for cystine is 100 μM: Warr et al. 1999), while in the extracellular space of the striatum and in the CSF [cystine]o is only around 0.13 μM (Perry et al. 1975; Baker et al. 2003) and will activate cystine–glutamate exchange to less than 0.2% of its maximum rate. Apart from cystine–glutamate exchange, there are several other unusual modes of transmitter release that are also candidates for mediating the tonic release of glutamate. In astrocytes, a rise of [Ca2+]i can release glutamate by a prostaglandin-dependent mechanism (Bezzi et al. 1998), and three distinct ion channels have been shown to release glutamate: swelling-activated anion channels (Rutledge et al. 1998), gap junctional hemichannels (Contreras et al. 2002; Ye et al. 2003), and P2X7 receptors gated by ATP (Sperlagh et al. 2002; Wang et al. 2002; Duan et al. 2003). In this paper we have re-investigated the possible contribution of cystine–glutamate exchange to setting the ambient [glu]o, and tested the role of these novel astrocyte release mechanisms.
, 百拇医药
Methods
Preparation of brain slices
Coronal brain slices (225 μm) containing the hippocampus were obtained from Sprague-Dawley rats (11–13 days old), killed by cervical dislocation in accordance with the UK Animals (Scientific Procedures) Act 1986. The slices were made in an oxygenated (95% O2–5% CO2) solution at 4°C containing (mM): 126 NaCl, 2.5 KCl, 2 MgCl2, 26 NaHCO3, 1 NaH2PO4, 2.5 CaCl2, 10 glucose and 1 sodium kynurenate (to block glutamate receptors), pH 7.4; they were stored for 1 h at 34°C before they were used for the experiments.
, 百拇医药
Electrophysiology
CA1 pyramidal cells were whole-cell voltage-clamped with a pipette solution containing (mM): 135 CsCl, 4 NaCl, 0.5 CaCl2,10 Hepes, 5 Na2EGTA, 2 MgATP and 0.5 Na2GTP, 10 QX-314 (to suppress voltage-gated sodium currents), pH set to 7.2 with CsOH. Electrode junction potentials (–3 mV) were corrected for. Electrodes were pulled from thin-walled borosilicate tube and had a resistance in external solution of 2–4 M. Cells were considered acceptable if the holding current at –33 mV was < 400 pA, with an access resistance < 10 M. External solution, as for brain slicing but without sodium kynurenate, was superfused at 25°C, or in some experiments at 35°C as stated in the text. Excitatory postsynaptic currents were evoked by placing a concentric stimulating electrode at a standard location among the Schaffer collaterals and stimulating at 100 V.
, 百拇医药
Drugs and chemicals
Tetrodotoxin (TTX, 0.1–1 μM, see text), picrotoxin (300 μM), strychnine (5 μM) and glycine (100 μM) were normally present in the bathing fluid to block action potentials, to block GABAA and glycine receptors, and to fully activate the glycine-binding site on NMDA receptors, but TTX was omitted when studying synaptic currents. Glutamate (Glu), DL-threo-benzyloxyaspartate (TBOA), cadmium (CdCl2), indomethacin, D-2-amino-5-phosphonopentanoic acid (AP5), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB), 18-glycyrrhetinic acid, cystine, (S)-4-carboxy-phenylglycine (CPG), tamoxifen and L-methionine sulfoximine (MSO) were applied as indicated. TBOA, NPPB, CPG and bafilomycin were purchased from Tocris (Bristol, UK); picrotoxin, glutamate, DIDS, PPADS, CdCl2, 18-glycyrrhetinic acid, cystine, tamoxifen, veratridine and MSO were from Sigma; TTX was from Alomone labs. In order to maintain a constant extracellular chloride concentration in experiments involving propionate, we used a solution which was obtained from the normal solution by substituting 20 mM sodium methanesulphonate for 20 mM NaCl. Sodium propionate was then substituted for an equivalent amount of sodium methanesulphonate. To pretreat slices with bafilomycin to deplete glutamate from synaptic vesicles, slices were incubated in a solution (without sodium kynurenate) to which 4 μM bafilomycin was added for over 2.5 h, and for 5 min at the start of this period 10 μM veratridine was also present to depolarize neurones and increase vesicle turnover. Control slices were incubated for the same period in solution containing sodium kynurenate (and were also exposed to veratridine for 5 min).
, 百拇医药
Analysis
Data are presented as means ± S.E.M. Glutamate release was assessed as the current produced by activation of glutamate receptors which occurred when glutamate uptake was blocked with TBOA. TBOA-evoked currents in the presence of a putative glutamate release-blocking drug were compared with the average of the values of the current in control solution before applying and after washout of the drug. Levels of significance were assessed using Student's two-tailed t test. Differences in means were considered significant when the P-value was less than 0.05.
, 百拇医药
When putative blockers of glutamate release mechanisms were applied, we carried out control experiments to check that they did not affect the glutamate receptors being used to sense the glutamate concentration, by assessing the effect of the drug on the response to exogenous glutamate. Only if a drug blocked the response to TBOA more than it blocked the response to exogenous glutamate did we conclude that glutamate release had been reduced.
We considered the possibility that exogenously applied glutamate might act on cells in the slice to release further glutamate. This does not invalidate our approach of comparing the effect of a putative release-blocking drug on the responses to TBOA and to exogenous glutamate, as is shown in the following analysis, because any secondary glutamate release evoked by exogenous glutamate will also be produced by the glutamate that accumulates (as a result of tonic release) when transporters are blocked with TBOA. To see this, suppose that a glutamate concentration ([glu]) produced in the extracellular space either exogenously ([glu]applied) or by inhibiting uptake with TBOA ([glu]TBOA), produces extra glutamate release through two mechanisms, one mechanism which is blocked by the drug being investigated and one mechanism which is not affected by the drug. If, in response to the [glu]applied, the [glu] in the extracellular space is amplified by a factor Fb via the mechanism which is blocked (b) by the drug, and amplified by a factor Fnb via the mechanism which is not blocked (nb) by the drug, then in the absence of the drug the total glutamate concentration produced by applied glutamate is:
, 百拇医药
and in the presence of the drug (when Fb = 0):
Thus, the ratio of the glutamate concentrations produced in the presence and absence of drug is:
When TBOA is applied the same equations apply except that [glu]applied is replaced by a glutamate concentration proportional to the glutamate release rate. We express the tonic glutamate release as:
where Rb is the rate of release by the mechanism that is blocked by the drug under consideration, and Rnb is the rate of release by the non-blocked release mechanism. These release mechanisms will generate a glutamate concentration that we assume to be proportional to the release rate (because diffusion and transporters operate with a linear dependence on [glu] at the low concentrations being considered), which will then be amplified as described above for exogenous glutamate application. Thus, in the absence of the blocking drug:
, http://www.100md.com
where k is a constant, and in the presence of the blocking drug (when Rb and Fb are zero):
Thus, the ratio of the glutamate concentrations produced in the presence and absence of drug is:
Comparing eqns (1) and (2), we see that the blocking drug reduces the extracellular glutamate concentration produced by TBOA application more than it reduces the [glu] produced by exogenous glutamate application (and so will reduce the glutamate receptor generated response more as well). The extra factor by which the TBOA response is multiplied is:
, 百拇医药
i.e. the decrease (relative to the decrease of the response to exogenous glutamate) reflects only the fraction of the glutamate release which is suppressed (and is not affected by the parameters Fb and Fnb describing secondary release of glutamate). Thus, if a drug blocks glutamate release, then it will reduce the response to TBOA by a larger factor than it reduces the response to externally applied glutamate. Consequently, we can determine whether a drug blocks glutamate release, even if there is secondary glutamate release, by comparing the effect of the drug on the response to TBOA with the effect on the response to applied glutamate.
, 百拇医药
Transient currents caused putatively by transient glutamate release from astrocytes (Angulo et al. 2004; Fellin et al. 2004) were defined as inward currents with an amplitude > 20 pA, a rise time > 10 ms and a decay time > 100 ms, and their rate of occurrence in 1-min periods in control solution or around the peak of the response to 1.5 min TBOA exposure was quantified. TBOA did not significantly increase the rate of these events (0.33 ± 0.13 events min–1 in 8 cells in TBOA versus 0.22 ± 0.05 events min–1 in 10 control cells, P = 0.4).
, 百拇医药
Results
Blocking Na+-dependent glutamate transporters activates an NMDA receptor current in pyramidal cells
We monitored tonic glutamate release into the extracellular space of hippocampal slices by applying a non-transported blocker of Na+-dependent glutamate transporters, DL-TBOA (Shimamoto et al. 1998, 2000), and detecting the resulting rise of extracellular glutamate concentration, [glu]o, from the membrane current it produced in whole-cell clamped CA1 pyramidal cells (Jabaudon et al. 1999). All experiments were done with action potentials blocked by TTX, GABAA and glycine receptors blocked with picrotoxin and strychnine, and the glycine site of NMDA receptors activated with glycine. Cells were clamped to –33 mV to remove Mg2+ block of NMDA receptor channels and thus maximize the current generated in response to a rise of [glu]o. DL-TBOA blocks the hippocampal glutamate transporters GLAST/EAAT1, GLT-1/EAAT2 and EAAC1/EAAT3 with Ki values of 42, 5.7 and 8.0 μM, respectively (Shimamoto et al. 1998, 2000), so the concentration of TBOA used here (200 μM) should reduce uptake of a low concentration of extracellular glutamate by these transporters by 83%, 97% and 96%, respectively.
, http://www.100md.com
Figure 1A shows that TBOA produced a slowly developing inward current, which reached 84.4 ± 3.6 pA after 1.5 min in 100 cells. The magnitude of the current was not significantly reduced by increasing the concentration of TTX present from 0.1 to 1 μM TTX (75.9 ± 5.3 pA in 34 cells and 88.8 ± 4.6 pA in 66 cells, respectively, P = 0.07), ruling out incompletely blocked action potentials in the lower TTX concentration as a source of tonic glutamate. The TBOA-evoked current was almost completely absent (blocked by 98.4 ± 1.6% in 6 cells, P = 1.3 x 10–6) when TBOA was applied in the presence of the NMDA receptor blocker D-AP5 (50 μM), and recovered when the D-AP5 was washed out (Fig. 1B), implying that the current is generated by tonic release of glutamate activating NMDA receptors (Jabaudon et al. 1999).
, 百拇医药
A, blocking glutamate uptake with TBOA evokes an inward current at –33 mV that is blocked by D-AP5, in an area CA1 pyramidal cell. B, mean current amplitude data for experiments as in A, in 6 cells, normalized to initial amplitude before D-AP5. C, NMDA (200 μM) evokes a desensitizing inward current at –33 mV. D, the effect of the voltage-gated calcium channel blocker Cd2+ on the response of pyramidal cells to glutamate (10 μM, chosen to generate a current similar in amplitude to that evoked by TBOA). E, effect of Cd2+ on the response to TBOA. F, mean amplitude of currents evoked by glutamate (4 cells) and TBOA (4 cells) in Cd2+ (normalized to values without Cd2+ present). G, effect of bafilomycin pretreatment (see Methods) on the amplitude of the EPSC evoked at –33 mV by stimulating the Schaffer collaterals (at 100 V). H, effect of bafilomycin pretreatment on the response of pyramidal cells to 10 μM glutamate. I, effect of bafilomycin pretreatment on the response to 200 μM TBOA.
, 百拇医药
D-AP5 itself has been reported to block a large inward current in hippocampal pyramidal cells (Sah et al. 1989: 200 pA at –35 mV in 400 μm thick slices (at 30°C, age not specified); < 60 pA in 100 μm slices), which was attributed to activation of NMDA receptors by the ambient glutamate level present. However, we found (in 225 μm slices, at –33 mV) that D-AP5 only blocked an inward current of 5.4 ± 1.4 pA in seven cells at 25°C and a current of 20.8 ± 4.0 pA in six slices at 35°C (data not shown).
, http://www.100md.com
Applying a saturating concentration of NMDA (200 μM; Fig. 1C) activated a peak current of 3.6 ± 0.4 nA in six cells (at 25°C), which decreased to a slowly declining plateau current of 2.7 ± 1.2 nA after 1.5 min when receptor desensitization had occurred (as will presumably also occur in response to the ambient [glu]o and when raising [glu]o with TBOA). Thus, the AP5-suppressible current (5.4 pA) produced by the baseline [glu]o is 0.2% of the current produced (after 1.5 min) by a saturating NMDA concentration (2.7 nA), or 0.13–0.17% of the current that would be produced by a saturating glutamate concentration, since glutamate produces a maximum current that is 1.2- to 1.5-fold larger than that produced by NMDA (via NR1/NR2A or NR1/NR2B receptors: Priestley et al. 1995). Similarly, the rise of [glu]o produced by TBOA activates NMDA receptors to produce approximately 3.3% (84.1 + 5.4 pA = 89.5 pA) of the maximum NMDA-evoked current (after 1.5 min), or about 2.2–2.8% of the current that would be produced by a saturating glutamate concentration. Using the dose–response curve for NMDA receptors, I = Imax[glu]n/{[glu]n + EC50n}, measured for cultured mouse hippocampal neurones by Patneau & Mayer (1990), who found a Hill coefficient, n, of 1.5 and an EC50 of 2.3 μM, these fractional activations translate into an estimated baseline extracellular glutamate concentration of 27–33 nM, and a concentration after 1.5 min TBOA of 0.18–0.22 μM. These experiments were at 25°C. Carrying out similar experiments at 35°C (at which glutamate release, but also glutamate uptake, may be faster), and assuming for simplicity no change in the EC50 for glutamate activating NMDA receptors, we estimated a baseline extracellular glutamate concentration of 77–89 nM, and a concentration after 1.5 min TBOA of 0.60–0.71 μM. Thus, increasing the temperature increases glutamate release somewhat more than it raises the rate of glutamate uptake, and (in the absence of TBOA) a new equilibrium is reached at a higher [glu]o.
, 百拇医药
Exocytosis does not contribute to tonic glutamate release
The experiment of Fig. 1A and B was done with action potentials blocked, but spontaneous Ca2+-dependent exocytosis might still release glutamate, so we tested the effect of blocking voltage-gated Ca2+ channels with 200 μM Cd2+, with the aim of confirming the conclusion of Jabaudon et al. (1999), for cultured slices, that tonic glutamate release is not via Ca2+-dependent exocytosis. This concentration of Cd2+ abolishes somatic Ca2+ currents in cultured pyramidal cells (Jabaudon et al. 1999) and reduces transmitter release at the CA3–CA1 synapse (assessed from the magnitude of the field EPSP) by about 70% (Wu & Saggau, 1994).
, 百拇医药
Cd2+ is known to block the NMDA receptors we are using to sense the released glutamate (Riveros & Orrego, 1986; Mayer et al. 1989), so we first tested the effect of Cd2+ on the response to superfusion of a glutamate concentration (10 μM) chosen to generate a current similar to that evoked by TBOA. Cd2+ reduced the response to glutamate (Fig. 1D), on average by 47 ± 7% (Fig. 1F, P = 0.005, n = 4). Cd2+ also reduced the current evoked by TBOA (Fig. 1E) but by approximately the same fraction as it blocked the NMDA sensor current (Fig. 1F, 40 ± 5%, P = 0.008). There was no significant difference between the inhibition of the glutamate- and of the TBOA-evoked currents (P = 0.46). Thus, the effect of Cd2+ on the TBOA-evoked current is entirely attributable to its inhibition of the NMDA receptors used to sense glutamate release, and we conclude that Cd2+ had no effect on the tonic release of glutamate. Subsequent experiments use this format for displaying the results, i.e. showing the effect of a drug first on the glutamate sensitivity of the NMDA receptor sensors, and then the effect on tonic release detected as a TBOA-evoked current mediated by the NMDA receptors. In order to conclude that a drug blocks tonic glutamate release we would need to demonstrate a significantly greater reduction of the TBOA-evoked current than of the glutamate-evoked current (see Methods).
, 百拇医药
To rule out a contribution of Ca2+-independent exocytosis to tonic glutamate release, we used bafilomycin pretreatment to block the vesicular H+-ATPase: this depletes vesicles of transmitter and thus prevents spontaneous and evoked exocytotic transmitter release (Zhou et al. 2000; Rossi et al. 2003; Allen et al. 2004; Allen & Attwell, 2004). To check that bafilomycin pretreatment was indeed depleting glutamate from vesicles, we evoked EPSCs in pyramidal cells by stimulating the Schaffer collateral input from CA3. Bafilomycin pretreatment reduced the amplitude of the EPSC evoked by a 100 V stimulus by 91% (Fig. 1G). In contrast bafilomycin had no effect on the cells' response to glutamate (Fig. 1H) or to TBOA (Fig. 1I). We conclude that exocytosis does not contribute significantly to tonic glutamate release onto hippocampal pyramidal cells.
, http://www.100md.com
Jabaudon et al. (1999) also found that, in cultured slices, inhibition of exocytosis with botulinum A or tetanus neurotoxin (which abolished spontaneous miniature EPSCs) did not reduce tonic glutamate release, again ruling out the possibility that tonic release is by Ca2+-independent spontaneous vesicular release.
Ca2+-evoked glial glutamate release does not contribute significantly to tonic activation of glutamate receptors
Transient release of glutamate from glia, probably by exocytosis, can activate NMDA receptors in hippocampal pyramidal cells (Fellin et al. 2004; Angulo et al. 2004). These transient release events were reported to occur at a very low rate (0.16–0.82 min–1 at room temperature to 34°C) and to have an amplitude of 100 pA and a decay time of 600 ms. We also observe these rare transient events, at a mean frequency of 0.22 ± 0.05 min–1 in 10 slices at 25°C (mean amplitude 61.7 ± 1.1 pA, mean rise time 169 ± 42 ms, mean decay time 891 ± 380 ms), but they contributed only a small fraction to the time averaged tonic activation of NMDA receptors (3 ± 1% in 10 cells).
, http://www.100md.com
Cystine–glutamate exchange does not generate the tonic glutamate release
To test the possible contribution of cystine–glutamate exchange to tonic glutamate release, we first activated the exchange by applying 300 μM exogenous cystine. The EC50 for activation of the exchange by external cystine is 100 μM (Wyatt et al. 1996; Warr et al. 1999) so the concentration used is sufficient to activate the exchange to about 75% of its maximum level. As reported previously for cerebellar Purkinje cells and neocortical pyramidal cells, cystine sometimes evoked a small inward current itself (arrow in middle panel of Fig. 2A), reflecting a rise of glutamate concentration activating glutamate receptors (Warr et al. 1999). Cystine did not significantly alter the response to 10 μM glutamate (reduced by 6 ± 4% in 6 cells, P = 0.22, Fig. 2D), but increased the response to TBOA (Fig. 2A) by a factor of 2.26 ± 0.49 in 11 cells (P = 0.025, Fig. 2A and D). Thus, cystine–glutamate exchange is present in the hippocampus, and can contribute significantly to glutamate release when the extracellular cystine concentration is high enough.
, http://www.100md.com
A, cystine potentiates the TBOA-evoked current (without affecting the response to glutamate: see D), implying that it increases glutamate release. Arrow shows a small inward current produced by cystine. B, the cystine–glutamate exchange blocker (S)-4-CPG abolished the potentiation produced by cystine, consistent with cystine releasing glutamate via cystine–glutamate exchange. C, in the absence of cystine, CPG has no effect on the TBOA-evoked current, implying cystine–glutamate exchange does not normally release a significant amount of glutamate. D, mean effects on the glutamate- and TBOA-evoked currents of cystine (6 cells for glutamate, 11 cells for TBOA), cystine in the presence of CPG (6 cells for TBOA; glutamate not studied), and CPG (5 cells each for glutamate and TBOA). Data are normalized to the value with no drug (for cystine alone or CPG alone), or to the value in CPG (for application of cystine in CPG).
, 百拇医药
To determine the contribution of cystine–glutamate exchange to glutamate release in the absence of superfused cystine (in which situation the [cystine]o is presumably closer to its in vivo value (measured in the striatum) of 0.16 μM: Baker et al. 2003), we employed the non-transported cystine–glutamate exchange blocker CPG ((S)-4-carboxyphenylglycine, 50 μM: Ye et al. 1999; Patel et al. 2004). The Ki of CPG is 5 μM (Patel et al. 2004), so for a low external cystine and glutamate concentration 50 μM CPG should inhibit the exchange by 91%. First we verified that CPG did block cystine–glutamate exchange, by repeating the experiment of Fig. 2A in the presence of CPG. CPG abolished the increase of tonic glutamate release evoked by exogenous cystine (Fig. 2B and D; in the presence of CPG, cystine increased the TBOA-evoked current by 0.8 ± 4.5%, P = 0.76, in 6 cells). Then we tested the effect of CPG on the TBOA-evoked current in the absence of exogenous cystine. CPG had no effect on the NMDA receptors used to sense the rise of [glu]o evoked by TBOA (the response to 10 μM glutamate was increased insignificantly by 6.9 ± 3.4% in 5 cells, P = 0.1, Fig. 2D). CPG also had no significant effect on the TBOA-evoked current (increased by 3.8 ± 13.3%, P = 0.77, Fig. 2C and D). Thus, cystine–glutamate exchange does not contribute significantly to tonic glutamate release in the absence of an artificially raised extracellular cystine concentration.
, 百拇医药
Effect of blocking glutamine synthetase on tonic glutamate release
The astrocyte-specific enzyme (Norenberg & Martinez-Hernandez, 1979) glutamine synthetase maintains a low glutamate concentration in astrocytes by converting glutamate to glutamine (Ottersen et al. 1992). Jabaudon et al. (1999) found that blocking this enzyme, and presumably raising astrocyte [glu]i, increased tonic glutamate release in cultured slices, suggesting that at least some tonic glutamate release is from astrocytes. Since astrocytes can change their properties in culture (Thio et al. 1993; Schultz-Suchting & Wolburg, 1994; Kimelberg et al. 1997; Zelenaia et al. 2000), we wanted to check that this result holds in acute slices, before investigating the possible release mechanism of glutamate from astrocytes.
, 百拇医药
We tested the effect of applying the glutamine synthetase blocker L-methionine sulfoximine (MSO, 5 mM) for 1 min on the response to glutamate and TBOA. MSO had no effect on the response to glutamate (Fig. 3A and C, increased by 10.1 ± 7.7%, P = 0.3 in 3 cells), but produced a small inward current itself in the absence of TBOA (12.8 ± 3.0 pA in 5 cells), as expected if a rise of [glu]i in astrocytes increased glutamate release, and increased the response to TBOA by 53.5 ± 5.6% (P = 0.003, n = 4, Fig. 3B and C). The increase in the response to TBOA was significantly larger than that for the response to glutamate (P = 0.01). Thus, as in culture, methionine sulfoximine increases the tonic release of glutamate.
, http://www.100md.com
A, MSO has no effect on the NMDA receptors used to sense glutamate. B, MSO potentiates tonic glutamate release, assessed as a TBOA-evoked current. C, mean data for experiments shown in A and B, normalized to the current value in the absence of MSO (3 cells for glutamate, 4 cells for TBOA).
We were surprised that even a minute's application of MSO was sufficient to potentiate glutamate release, since this agent is often applied for hours. The rapid effect of exposure to MSO suggests that it can enter the cells and inhibit glutamine synthetase, and allow [glu]i to rise, relatively rapidly. A similar rapid regulation of transmitter release by modulation of intracellular transmitter concentration has been demonstrated for GABAergic synaptic transmission: blocking uptake of precursor glutamate decreases intracellular [GABA] and reduces postsynaptic IPSCs within minutes (Mathews & Diamond, 2003).
, http://www.100md.com
Tonic indomethacin-sensitive glutamate release from astrocytes is not significant
A possible contribution to tonic glutamate release of prostaglandin- and Ca2+-dependent glutamate release from astrocytes was assessed by blocking this release mechanism with 100 μM indomethacin, which inhibits glutamate release by > 80% (Bezzi et al. (1998) showed that 1 μM indomethacin blocks 80% of the release). Indomethacin was applied at least 2 min before glutamate or TBOA (10–100 μM indomethacin produces 50–66% of its prostaglandin-blocking effect in 2 min (Ko et al. 2002; Yerxa et al. 2002)). Indomethacin did not significantly alter the response to glutamate (increased by 8.8 ± 8.2% in 6 cells, P = 0.35) and did not reduce the response to TBOA (increased by 38 ± 13% in 6 cells, P = 0.03, Fig. 4A), and the effects on the response to glutamate and TBOA were not significantly different (P = 0.094), implying that the prostaglandin-dependent mechanism does not contribute significantly to tonic glutamate release.
, http://www.100md.com
Effects on the currents evoked by glutamate or TBOA of A, indomethacin (6 cells for glutamate, 6 cells TBOA), B, NPPB (5 cells glutamate, 4 cells TBOA), C, tamoxifen (3 cells glutamate, 3 cells TBOA), D, 18-glycyrrhetinic acid (18-GA, 7 cells glutamate, 4 cells TBOA), and E, PPADS (5 cells glutamate, 5 cells TBOA). Data are normalized to currents in the absence of the drugs.
Tonic glutamate release via astrocyte swelling-activated anion channels, gap junctional hemichannels and P2X7 receptors is not significant
, 百拇医药
Glutamate release from astrocytes can occur via swelling activated anion channels that are blocked by NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid) and tamoxifen. These drugs were applied at least 2 min before glutamate or TBOA. NPPB (100 μM) blocks the channels mediating swelling-evoked release by 90% within 1 min (Rutledge et al. 1998; Crepel et al. 1998) and tamoxifen (15 μM) blocks these channels by 80% within 1.5 min: Rutledge et al. 1998; Ransom et al. 2001). Neither NPPB (100 μM, 4 cells) nor tamoxifen (pooled data from 15 μM on 2 cells and 30 μM on 1 cell) had a significant effect (< 4% change, P > 0.35) on the pyramidal cell response to either glutamate or TBOA (Fig. 4B and C).
, 百拇医药
To test for tonic glutamate release via gap junctional hemichannels, which are opened by low [Ca2+]o and metabolic inhibition but could have a non-zero open probability under normal conditions (Contreras et al. 2002; Ye et al. 2003), we applied 18-glycyrrhetinic acid (25 μM, applied at least 2 min before glutamate or TBOA was applied), which blocks hemichannel permeability by > 80% (Ye et al. 2003), and acts in 10 s (Takeda et al. 2005). This did not significantly alter the pyramidal cell response to glutamate (decreased by 8.3 ± 6.0% in 7 cells, P = 0.22) and increased (rather than decreased) the response to TBOA (increased by 22.2 ± 4.0% in 4 cells, P = 0.01), implying that there is no significant contribution to tonic glutamate release from these channels (Fig. 4D).
, 百拇医药
ATP-gated P2X7 receptors can mediate transmitter release, either via transmitter diffusing through the large pore that these receptors form or by activation of a transporter mechanism (Sperlagh et al. 2002; Wang et al. 2002; Duan et al. 2003). In the presence of 30 μM PPADS (pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid, applied 2 min before glutamate or TBOA), which reduces P2X7 receptor activation by more than 75–86% in 1 min (Duan et al. 2003; Sun et al. 1999), the response to glutamate was reduced by 27.9 ± 8.6% (P = 0.047, 5 cells), implying a small inhibition of NMDA receptors (Fig. 4E), but there was no significant change in the response to TBOA (reduced by 10.2 ± 7.8% in 5 cells, P = 0.19), and there was no significant difference when comparing the effects of PPADS on the glutamate and TBOA evoked currents (P = 0.16), implying that PPADS did not inhibit tonic glutamate release.
, 百拇医药
Tonic glutamate release is reduced by DIDS
4,4'-Diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) has been found to block numerous anion transporters and channels via which glutamate might exit the cell. Applying 1 mM DIDS evoked an outward current in pyramidal cells (69.1 ± 9.5 pA in 9 cells at –33 mV), and increased the response to glutamate by 32.7 ± 10.9% (P = 0.04) in 5 cells (Fig. 5A and C). Tauskela et al. (2003) previously suggested that 1 mM DIDS decreased NMDA receptor-mediated currents; however, they applied DIDS only in the presence of NMDA and the apparent decrease of the NMDA-evoked current that they reported was apparently an artefactual result caused by the outward DIDS-evoked current shown in Fig. 5A.
, 百拇医药
A, DIDS evokes an outward current and increases the current evoked by glutamate. B, DIDS decreases the TBOA-evoked current. C, mean data for experiments shown in A and B, normalized to current values in the absence of DIDS (5 cells for glutamate, 6 cells for TBOA). D, propionate (which, like DIDS, is expected to evoke an intracellular acidification) also evokes an outward current and increases the glutamate-evoked current. E, unlike DIDS, propionate slightly increases the TBOA-evoked current. F, mean data from experiments shown in D and E (5 cells each for glutamate and TBOA).
, http://www.100md.com
DIDS inhibits pH-regulating transporters and makes the intracellular pH go acid (Schwiening & Boron, 1994; Baxter & Church, 1996; Bonnet et al. 2000). Sodium propionate (20 mM), which enters across the cell membrane in neutral form and then releases a proton intracellularly, is expected to mimic this acidification (Rorig et al. 1996; Lee et al. 1996). We found that sodium propionate also induced an outward current (37.7 ± 2.7 pA in 7 cells at –33 mV) and increased the response to glutamate by 13.4 ± 3.6% in five cells (P = 0.016; Fig. 5D and F), suggesting that the outward current and the potentiation of the glutamate-evoked NMDA response seen in DIDS are produced by intracellular acidification.
, 百拇医药
Propionate also increased the response to TBOA (by 28.5 ± 4.7% in 5 cells, P = 0.0017, Fig. 5E and F). By contrast, DIDS decreased the response to TBOA by 47.2 ± 5.3% in six cells (P = 0.0002, Fig. 5B and C), which was statistically significantly different from the DIDS-evoked change in the glutamate response (P = 0.00067), implying that DIDS decreases the tonic release of glutamate. The fact that DIDS decreases the response to TBOA, while propionate increases it, suggests that the suppressive action of DIDS on tonic glutamate release (unlike its actions on the baseline current and the response to glutamate, which mimic those of propionate) is not produced by an intracellular acidification.
, http://www.100md.com
DIDS has been reported to inhibit a glutamate transporter (probably GLAST) in Bergmann glial cells (Ruiz & Ortega, 1995). We considered the possibility that the inhibition of the response to TBOA in Fig. 5B and C was a result of DIDS having already inhibited glutamate transporters present in the slice, so that superimposed TBOA produced less uptake inhibition. If this were the case we would expect that application of DIDS would generate a rise of [glu]o that would partially activate NMDA receptors. This cannot be assessed from the change of current evoked by DIDS, because that change clearly also involves an outward (probably acidification-evoked) component. We therefore applied 50 μM D-AP5 to determine whether there was more tonic activation of NMDA receptors present in DIDS. The current change measured (2.7 ± 1.2 pA in 6 cells) was not larger (P = 0.18) than that seen in absence of DIDS (5.4 ± 1.4 pA in 7 cells, see above), showing that inhibition of glutamate transporters by DIDS cannot explain the results in Fig. 5B and C.
, 百拇医药
Discussion
The value of the baseline extracellular glutamate concentration, [glu]o, in the CNS could have a major effect on neuronal excitability and synaptic transmission, but what sets this value is poorly understood. The data in this paper from acutely isolated hippocampal slices extend earlier results on tonic non-synaptic glutamate release in cultured slices (Jabaudon et al. 1999) which showed using tetanus toxin that this release is not via exocytosis, a result we have confirmed in acute slices using bafilomycin to deplete vesicles of glutamate. We have demonstrated that this release is not via cystine–glutamate exchange, contrary to current ideas based on microdialysis work in striatum (Baker et al. 2002), and that it is not via four other glutamate release mechanisms previously characterized in astrocytes, but that it is inhibited by DIDS. Below, we assess the implications of these results. Throughout this discussion it should be remembered that, although we discuss release of glutamate, it is possible that the release of other agents acting on NMDA receptors, such as aspartate, might also contribute to the currents recorded.
, 百拇医药
Cystine–glutamate exchange does not significantly contribute to the ambient [glu]o
In our experiments the cystine–glutamate exchange blocker CPG had no effect on tonic glutamate release unless an unphysiologically high concentration of external cystine was superfused. This is consistent with the low level of cystine present in the extracellular space of the brain. Baker et al. (2003) measured an extracellular [cystine] value of 0.16 μM, which is much less than the EC50 for external cystine activating cystine–glutamate exchange (100 μM, Wyatt et al. 1996; Warr et al. 1999) and will activate the exchanger to less than 0.2% of its maximum rate.
, 百拇医药
There are three possible explanations for the difference between our results and those of Baker et al. (2002). First, in our experiments on slices the diffusional access to the bulk solution (and lack of a cystine/cysteine supply from the blood) might reduce the cystine concentration in the extracellular spaces of the preparation to a value even smaller than the 0.16 μM that Baker et al. (2003) measured in vivo, leading to cystine–glutamate exchange being deactivated. If so, then the cystine–glutamate exchange could be important in vivo (despite the very low [cystine]o), as Baker et al. (2002, 2003) suggest, but not in slice experiments. Alternatively, in the experiments of Baker et al. (2002) the microdialysis cannula might have caused leakage of cystine into the extracellular space from the blood where the cystine concentration is 80 μM (Battistin et al. 1971), artefactually raising the local [cystine]o (Westergren et al. 1995). In this case, slice experiments might better assess the true role of cystine–glutamate exchange, which could be negligible as a source of glutamate in vivo. Finally, Baker et al. (2002) relied on CPG being a specific blocker of cystine–glutamate exchange, but this glutamate analogue is also an antagonist at mGluR1 and mGluR5 metabotropic glutamate receptors and an agonist at mGluR2 (Hayashi et al. 1994; Kingston et al. 1995) and is expected to reduce neuronal excitability and glutamate release by both of these mechanisms (Lingenhohl et al. 1993; Ugolini & Bordi, 1995; Moroni et al. 1998). Baker et al. (2002) checked that an action of CPG at group I mGluRs was not how CPG inhibited cystine uptake in their experiments, but they did not check that CPG still reduced tonic glutamate release when mGluRs were blocked. Thus, CPG may have reduced [glu]o in their experiments by acting on mGluRs and reducing spontaneous exocytotic glutamate release rather than by blocking cystine–glutamate exchange.
, 百拇医药
What mechanism mediates tonic glutamate release other than cystine–glutamate exchange
We found, like Jabaudon et al. (1999), that the glutamine synthetase inhibitor methionine sulfoximine (MSO) increased tonic glutamate release. MSO is expected to raise the glutamate concentration in astrocytes, so the increase in tonic release could be explained if at least some of the release occurs from astrocytes and a raised astrocyte [glutamate] potentiates this release. However, our experiments ruled out a contribution to tonic glutamate release from four previously characterized glutamate release mechanisms present in astrocytes: prostaglandin- and Ca2+-dependent release, swelling-activated anion channels, gap junctional hemichannels and P2X7 receptors. The only agent we found to significantly reduce tonic glutamate release was DIDS, which blocks many transporters and channels that allow the movement of negatively charged molecules like glutamate.
, 百拇医药
In the Appendix, we consider the possibility that some of the tonic glutamate release results from passive diffusion of glutamate across cell membranes. Using the measured diffusion coefficients for charged and neutral amino acids crossing lipid bilayers, we estimate the baseline [glu]o that could be produced by diffusive glutamate release when Na+-dependent uptake is functioning normally ([glu]o 31 nM) or when it is inhibited with TBOA ([glu]o 0.5 μM). Although these estimates depend on parameters for diffusion and uptake which are only approximately known, they are quite similar in magnitude to the values we estimated experimentally (30 nM and 0.2 μM, respectively, at 25°C, see Results). Thus, it appears that diffusive glutamate efflux may contribute significantly to determining the baseline [glu]o in hippocampal slices. Potentiation of the TBOA-evoked current by MSO (Fig. 3) could then just reflect more diffusive efflux when the intracellular glutamate concentration is raised in astrocytes, but it is unclear how DIDS might modulate diffusive glutamate efflux (Chakrabarti & Deamer, 1992 reported that pH changes had little effect on the diffusion of amino acids across membranes), suggesting a release mechanism in addition to passive diffusion across the lipid bilayer.
, 百拇医药
Functional significance of tonic glutamate release
The micromolar levels of ambient glutamate found in microdialysis experiments should activate NMDA and mGluR receptors, and induce desensitization of NMDA, AMPA and kainate receptors, implying that alteration of the ambient [glu]o may have profound consequences for the information processing carried out by neurones. Indeed, in cultured hippocampal neurones, Forsythe & Clements (1990) found that less than 1 μM glutamate depressed the EPSC amplitude by 40%, and Zorumski et al. (1996) found similar effects of low micromolar levels of glutamate on both the AMPA and the NMDA components of the EPSC. Sah et al. (1989) and Dalby & Mody (2003) reported that NMDA receptors in cells in hippocampal slices could be tonically activated by the ambient glutamate level, generating an inward current which increases the excitability of the neurones. However, the size of the tonically activated NMDA receptor (D-AP5 suppressible) current found in the present study was much smaller than that found in pyramidal cells by Sah et al. (1989), suggesting a resting [glu]o of around 30–80 nM at 25–35°C (see Results) rather than the 2 μM found in microdialysis experiments. The reason for the difference in D-AP5 suppressible current is unclear, but it could reflect the sharp electrode recordings made by Sah et al. (1989) being from cells very deep in the slice, possibly at a location with compromised oxygen supply, while those we studied with patch-clamp techniques are from more superficial cells. Whether our resting [glu]o is artefactually lowered in the slice preparation, or the microdialysis value is artefactually raised by damage caused by the microdialysis procedure (see Westergren et al. 1995), remains an important question to be answered.
, 百拇医药
The small size of the D-AP5-sensitive current we record without TBOA present (5 pA at –33 mV at 25°C), compared to that seen when uptake is blocked by TBOA (84 pA), highlights the fact that the functional significance of the tonic release of glutamate will depend critically on the prevailing level of Na+-dependent glutamate uptake.
Appendix
Estimate of the contribution of diffusion across lipid membranes to the ambient [glu]o
, http://www.100md.com
We considered the possibility that some of the tonic glutamate efflux detected when TBOA is applied results from passive diffusion of glutamate across cell membranes. Charged and neutral amino acids cross lipid bilayers with a permeability coefficient of P = 5–20 x 10–14 m s–1 (depending on the saturation of the membrane lipid molecules: Chakrabarti & Deamer, 1992). The membrane area per unit volume of hippocampus is 14 μm2/μm3, so in 1 litre there is A = 1.4 x 104 m2 membrane area (Lehre & Danbolt, 1998). Thus, for a mean intracellular glutamate concentration of [glu]i = 3 mM (Attwell et al. 1993), the diffusive glutamate flux into the extracellular space will be Rdiffusion = PA[glu]i = 2.1–8.4 nmol l–1 s –1. The [glu]o this would produce can be estimated by noting that, if this is the only glutamate release mechanism, the release rate must equal the uptake rate by Na+-dependent transporters. Equating an Rdiffusion of (say) 5 nmol l–1 s–1 to an uptake rate, described by Michaelis-Menten kinetics as Umax[glu]o/([glu]o + Km), where the maximum uptake rate, Umax, in brain slices is 0.2 μmol ml–1 min–1 or 3.33 μmol l–1 s–1 and Km is 20 μM (Hertz, 1979; Schousboe, 1981), we predict a baseline [glu]o of 31 nM, which is similar to the 30 nM we estimate from the size of the AP5-suppressible current. When TBOA is added, the [glu]o rise will depend critically on the magnitude of glutamate uptake remaining unblocked in TBOA. Assuming that 21% of hippocampal uptake is due to GLAST and 79% to GLT-1 (Lehre & Danbolt, 1998), i.e. ignoring the contribution of EAAC1 (Haugeto et al. 1996), the expected 83% and 97% inhibition of GLAST and GLT-1 in the presence of 200 μM TBOA (see Results) will lead to only 6% of glutamate uptake remaining functional in TBOA. Equating an Rdiffusion of 5 nmol l–1 s–1 to an uptake rate of 0.06.Umax[glu]o/([glu]o + Km) predicts an equilibrium value for [glu]o in TBOA of 0.5 μM. This is slightly higher than the 0.2 μM [glu]o we estimate to be produced when [glu]o is still rising after 1.5 min of TBOA exposure (see Results). Aspartate could be released by the same mechanism, and would contribute to the activation of NMDA receptors detected.
, http://www.100md.com
References
Allen NJ & Attwell D (2004). The effect of simulated ischaemia on spontaneous GABA release in area CA1 of the juvenile rat hippocampus. J Physiol 561, 485–498.
Allen NJ, Rossi DJ & Attwell D (2004). Sequential release of GABA by exocytosis and reversed uptake leads to neuronal swelling in simulated ischemia of hippocampal slices. J Neurosci 24, 3837–3849.
Angulo MC, Kozlov AS, Charpak S & Audinat E (2004). Glutamate released from glial cells synchronizes neuronal activity in the hippocampus. J Neurosci 24, 6920–6927.
, http://www.100md.com
Attwell D, Barbour B & Szatkowski M (1993). Nonvesicular release of neurotransmitter. Neuron 11, 401–407.
Baker DA, McFarland K, Lake RW, Shen H, Tang XC, Toda S & Kalivas PW (2003). Neuroadaptations in cystine-glutamate exchange underlie cocaine relapse. Nature Neurosci 6, 743–749.
Baker DA, Xi Z, Shen H, Swanson CJ & Kalivas PW (2002). The origin and neuronal function of in vivo nonsynaptic glutamate. J Neurosci 22, 9134–9141.
, http://www.100md.com
Bannai S (1984). Transport of cystine and cysteine in mammalian cells. Biochim Biophys Acta 779, 289–306.
Battistin L, Grynbaum A & Lajtha A (1971). The uptake of various amino acids by the mouse brain in vivo. Brain Res 29, 85–99.
Baxter KA & Church J (1996). Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol 493, 457–470.
Benveniste H, Drejer J, Schousboe A & Diemer NH (1984). Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 43, 1369–1374.
, http://www.100md.com
Bezzi P, Carmignoto G, Pasti L, Vesce S, Rossi D, Rizzini BL, Pozzan T & Volterra A (1998). Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 391, 281–285.
Bonnet U, Leniger T & Wiemann M (2000). Alteration of intracellular pH and activity of CA3-pyramidal cells in guinea pig hippocampal slices by inhibition of transmembrane acid extrusion. Brain Res 872, 116–124.
Brickley SG, Cull-Candy SG & Farrant M (1996). Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors. J Physiol 497, 753–759.
, 百拇医药
Cavelier P, Hamann M, Rossi DJ, Mobbs P & Attwell D (2005). Tonic excitation and inhibition of neurons. Prog Biophys Molec Biol 87, 3–16.
Chakrabarti AC & Deamer DW (1992). Permeability of lipid bilayers to amino acids and phosphate. Biochim Biophys Acta 1111, 171–177.
Cho Y & Bannai S (1990). Uptake of glutamate and cystine in C-6 glioma cells and in cultured astrocytes. J Neurochem 55, 2091–2097.
, http://www.100md.com
Contreras JE, Sanchez HA, Eugenin EA, Speidel D, Theis M, Willecke K, Bukauskas FF, Bennett MV & Saez JC (2002). Metabolic inhibition induces opening of unapposed connexin 43 gap junction hemichannels and reduces gap junctional communication in cortical astrocytes in culture. Proc Natl Acad Sci U S A 99, 495–500.
Crepel V, Panenka W, Kelly MEM & MacVicar BA (1998). Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current. J Neurosci 18, 1196–1206.
, 百拇医药
Dalby NO & Mody I (2003). Activation of NMDA receptors in rat dentate gyrus granule cells by spontaneous and evoked transmitter release. J Neurophysiol 90, 786–797.
Duan S, Anderson CM, Keung EC, Chen Y, Chen Y & Swanson RA (2003). P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci 23, 1320–1328.
Fellin T, Pascual O, Gobbo S, Pozzan T, Haydon PG & Carmignoto G (2004). Neuronal synchrony mediated by astrocytic glutamate through activation of extrasynaptic NMDA receptors. Neuron 43, 729–743.
, 百拇医药
Forsythe ID & Clements JD (1990). Presynaptic glutamate receptors depress excitatory monosynaptic transmission between mouse hippocampal neurones. J Physiol 429, 1–16.
Hagberg H, Lehmann A, Sandberg M, Nystrom B, Jacobson I & Hamberger A (1985). Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb Blood Flow Metab 5, 413–419.
Hamann M, Rossi D & Attwell D (2002). Tonic and spillover inhibition of granule cells control information flow through cerebellar cortex. Neuron 33, 625–633.
, 百拇医药
Haugeto , Ullensvang K, Levy LM, Chaudhry FH, Honore T, Nielsen M, Lehre KP & Danbolt NC (1996). Brain glutamate transporter proteins form homomultimers. J Biol Chem 271, 27715–27722.
Hayashi Y, Sekiyama N, Nakanishi S, Jane DE, Sunter DC, Birse EF, Udvarhelyi PM & Watkins JC (1994). Analysis of agonist and antagonist activities of phenylglycine derivatives for different cloned metabotropic glutamate receptor subtypes. J Neurosci 14, 3370–3377.
, http://www.100md.com
Hertz L (1979). Functional interactions between neurons and astrocytes I. Turnover and metabolism of putative amino acid transmitters. Prog Neurobiol 13, 277–323.
Jabaudon D, Shimamoto K, Yasuda-Kamatani Y, Scanziani M, Ghwiler BH & Gerber U (1999). Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin. Proc Natl Acad Sci U S A 96, 8733–8738.
Kaneda M, Farrant M & Cull-Candy SG (1995). Whole-cell and single-channel currents activated by GABA and glycine in granule cells of the rat cerebellum. J Physiol 485, 419–435.
, 百拇医药
Kimelberg HK, Cai Z, Rastogi P, Charniga CJ, Goderie S, Dave V & Jalonen TA (1997). Transmitter-induced calcium responses differ in astrocytes acutely isolated from rat brain and in culture. J Neurochem 68, 1088–1098.
Kingston AE, Burnett JP, Mayne NG & Lodge D (1995). Pharmacological analysis of 4-carboxyphenylglycine derivatives: comparison of effects on mGluR1 and mGluR5 subtypes. Neuropharmacol 34, 887–894.
Ko WH, Law VW, Yip WC, Yue GG, Lau CW, Chen ZY & Huang Y (2002). Stimulation of chloride secretion by baicalein in isolated rat distal colon. Am J Physiol Gastrointest Liver Physiol 282, G508–G518.
, 百拇医药
Lee J, Taira T, Pihlaja P, Ransom BR & Kaila K (1996). Effects of CO2 on excitatory transmission apparently caused by changes in intracellular pH in the rat hippocampal slice. Brain Res 706, 210–216.
Lehre KP & Danbolt NC (1998). The number of glutamate transporter subtype molecules at glutamatergic synapses: chemical and stereological quantification in young adult rat brain. J Neurosci 18, 8751–8757.
Levy LM, Warr O & Attwell D (1998). Stoichiometry of the glial glutamate transporter GLT-1 expressed inducibly in a Chinese hamster ovary cell line selected for low endogenous Na+-dependent glutamate uptake. J Neurosci 18, 9620–9628.
, 百拇医药
Lingenhohl K, Olpe HR, Bendali N & Knopfel T (1993). Phenylglycine derivatives antagonize the excitatory response to Purkinje cells to 1S,3R-ACPD: an in vivo and in vitro study. Neurosci Res 18, 229–234.
Mathews GC & Diamond JS (2003). Neuronal glutamate uptake contributes to GABA synthesis and inhibitory synaptic strength. J Neurosci 23, 2040–2048.
Mayer ML, Vyklicky L Jr & Westbrook GL (1989). Modulation of excitatory amino acid receptors by group IIB metal cations in cultured mouse hippocampal neurones. J Physiol 415, 329–350.
, 百拇医药
McBain CJ, DiChiara TJ & Kauer JA (1994). Activation of metabotropic glutamate receptors differentially affects two classes of hippocampal interneurons and potentiates excitatory synaptic transmission. J Neurosci 14, 4433–4445.
Moroni F, Cozzi A, Lombardi G, Sourtcheva S, Leonardi P, Carfi M & Pellicciari R (1998). Presynaptic mGlu1 type receptors potentiate transmitter output in the rat cortex. Eur J Pharmacol 347, 189–195.
, 百拇医药
Norenberg MD & Martinez-Hernandez A (1979). Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res 161, 303–310.
Ottersen OP, Zhang N & Walberg F (1992). Metabolic compartmentation of glutamate and glutamine: morphological evidence obtained by quantitative immunocytochemistry in rat cerebellum. Neuroscience 46, 519–534.
Patel SA, Warren BA, Rhoderick JF & Bridges RJ (2004). Differentiation of substrate and non-substrate inhibitors of transport system xc–: an obligate exchanger of L-glutamate and L-cystine. Neuropharmacol 46, 273–284.
, 百拇医药
Patneau D & Mayer ML (1990). Structure-activity relationships for amino acid transmitter candidates acting at N-methyl-D-aspartate and quisqualate receptors. J Neurosci 10, 2385–2399.
Perry TL, Hansen S & Kennedy J (1975). CSF amino acids and plasma-CSF amino acid ratios in adults. J Neurochem 24, 587–589.
Phillis JW, Smith-Barbour M, Perkins LM & O'Regan MH (1994). Characterization of glutamate, aspartate, and GABA release from ischemic rat cerebral cortex. Brain Res Bull 34, 457–466.
, 百拇医药
Priestley T, Laughton P, Myers J, Le Bourdelles B, Kerby J & Whiting PJ (1995). Pharmacological properties of recombinant human N-methyl-D-aspartate receptors comprising NR1a/NR2A and NR1a/NR2B subunit assemblies expressed in permanently transfected mouse fibroblast cells. Mol Pharmacol 48, 841–848.
Ransom CB, O'Neal JTO & Sontheimer H (2001). Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells. J Neurosci 21, 7674–7683.
, 百拇医药
Riveros N & Orrego F (1986). N-methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances. Neuroscience 17, 541–546.
Rorig B, Klausa G & Sutor B (1996). Intracellular acidification reduced gap junction coupling between immature rat neocortical pyramidal neurones. J Physiol 490, 31–49.
Rossi DJ, Hamann M & Attwell D (2003). Multiple modes of GABAergic inhibition of rat cerebellar granule cells. J Physiol 548, 97–110.
, 百拇医药
Ruiz M & Ortega A (1995). Characterization of an Na+-dependent glutamate/aspartate transporter from cultured Bergmann glia. Neuroreport 6, 2041–2044.
Rutledge EM, Aschner M & Kimelberg HK (1998). Pharmacological characterization of swelling-induced D-[3H]aspartate release from primary astrocyte cultures. Am J Physiol 274, C1511–C1520.
Sah P, Hestrin S & Nicoll RA (1989). Tonic activation of NMDA receptors by ambient glutamate enhances excitability of neurons. Science 246, 815–818.
, 百拇医药
Schousboe A (1981). Transport and metabolism of glutamate and GABA in neurons and glial cells. Int Rev Neurobiol 22, 1–45.
Schultz-Suchting F & Wolburg H (1994). Astrocytes alter their polarity in organotypic slice cultures of rat visual cortex. Cell Tissue Res 277, 557–564.
Schwiening CJ & Boron WF (1994). Regulation of intracellular pH in pyramidal neurones from the rat hippocampus by Na+-dependent Cl––HCO3– exchange. J Physiol 475, 59–67.
, 百拇医药
Semyanov A, Walker MC & Kullmann DM (2003). GABA uptake regulates cortical excitability via cell type-specific tonic inhibition. Nature Neurosci 6, 484–490.
Shimamoto K, Lebrun B, Yasuda-Kamatani Y, Sakaitani M, Shigeri Y, Yumoto N & Nakajima T (1998). DL-threo-benzyloxyaspartate, a potent blocker of excitatory amino acid transporters. Mol Pharmacol 53, 195–201.
Shimamoto K, Shigeri Y, Yasuda-Kamatani Y, Lebrun B, Yumoto N & Nakajima T (2000). Syntheses of optically pure beta-hydroxyaspartate derivatives as glutamate transporter blockers. Bioorg Med Chem Lett 10, 2407–2410.
, 百拇医药
Sperlagh B, Kofalvi A, Deuchars J, Atkinson L, Milligan CJ, Buckley NJ & Vizi ES (2002). Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus. J Neurochem 81, 1196–1211.
Stell BM & Mody I (2002). Receptors with different affinities mediate phasic and tonic GABAA conductances in hippocampal neurons. J Neurosci 22, RC223.
Sun SH, Lin LB, Hung AC & Kuo JS (1999). ATP-stimulated Ca2+ influx and phospholipase D activities of a rat brain-derived type-2 astrocyte cell line, RBA-2, are mediated through P2X7 receptors. J Neurochem 73, 334–343.
, http://www.100md.com
Takeda Y, Ward SM, Sanders KM & Koh SD (2005). Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells. Am J Physiol Gastrointest Liver Physiol (in press).
Tauskela JS, Mealing G, Comas T, Brunette E, Monette R, Small DL & Morley P (2003). Protection of cortical neurons against oxygen-glucose deprivation and N-methyl-D-aspartate by DIDS and SITS. Eur J Pharmacol 464, 17–25.
Thio CL, Waxman SG & Sontheimer H (1993). Ion channels in spinal cord astrocytes in vitro. III. Modulation of channel expression by coculture with neurons and neuron-conditioned medium. Neurosci Res 18, 229–234.
, http://www.100md.com
Tia S, Wang JF, Kotchabhakdi N & Vicini S (1996). Developmental changes of inhibitory synaptic currents in cerebellar granule neurons: role of GABAA receptor 6 subunit. J Neurosci 16, 3630–3640.
Ugolini A & Bordi F (1995). Metabotropic glutamate group II receptors are responsible for the depression of synaptic transmission induced by ACPD in the dentate gyrus. Eur J Pharmacol 294, 403–410.
Wahl F, Obrenovitch TP, Hardy AM, Plotkine M, Boulu R & Symon L (1994). Extracellular glutamate during focal cerebral ischaemia in rats: time course and calcium-dependency. J Neurochem 63, 1003–1011.
, 百拇医药
Wall MJ & Usowicz MM (1997). Development of action potential-dependent and independent spontaneous GABAA receptor-mediated currents in granule cells of postnatal rat cerebellum. Eur J Neurosci 9, 533–548.
Wang CM, Chang YY, Kuo JS & Sun SH (2002). Activation of P2X7 receptors induced [3H]GABA release from the RBA-2 type-2 astrocyte cell line through a Cl–/HCO3–-dependent mechanism. Glia 37, 8–18.
Warr O, Takahashi M & Attwell D (1999). Modulation of extracellular glutamate concentration in rat brain slices by cystine-glutamate exchange. J Physiol 514, 783–793.
, http://www.100md.com
Westergren L, Nystrom B, Hamberger A & Johansson BB (1995). Intracerebral dialysis and the blood brain barrier. J Neurochem 64, 229–234.
Wu LG & Saggau P (1994). Presynaptic calcium is increased during normal synaptic transmission and paired-pulse facilitation, but not in long-term potentiation in area CA1 of hippocampus. J Neurosci 14, 645–654.
Wyatt I, Gyte A, Simpson MG, Widdowson PS & Lock EA (1996). The role of glutathione in L-2-chloropropionic acid induced cerebellar granule cell necrosis in the rat. Arch Toxicol 70, 724–735.
, 百拇医药
Ye ZC, Rothstein JD & Sontheimer H (1999). Compromised glutamate transport in human glioma cells: reduction-mislocalization of sodium-dependent glutamate transporters and enhanced activity of cystine-glutamate exchange. J Neurosci 19, 10767–10777.
Ye ZC, Wyeth MS, Baltan-Tekkok S & Ransom BR (2003). Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 23, 3588–3596.
Yerxa BR, Sabater JR, Davis CW, Stutts MJ, Lang-Furr M, Picher M, Jones AC, Cowlen M, Dougherty R, Boyer J, Abraham WM & Boucher RC (2002). Pharmacology of INS37217 [P1-(uridine 5') -P4-(2'-deoxycytidine 5') tetraphosphate, tetrasodium salt], a next-generation P2Y2 receptor agonist for the treatment of cystic fibrosis. J Pharmacol Exp Ther 302, 871–880.
, 百拇医药
Zelenaia O, Schlag BD, Gochenauer GE, Ganel R, Song W, Beesley JS, Grinspan JB, Rothstein JD & Robinson MB (2000). Epidermal growth factor receptor agonists increase expression of glutamate transporter GLT-1 in astrocytes through pathways dependent on phosphatidylinositol 3-kinase and transcription factor NF-B. Mol Pharmacol 57, 667–678.
Zerangue N & Kavanaugh MP (1996). Flux coupling in a neuronal glutamate transporter. Nature 383, 634–637.
, 百拇医药
Zhou Q, Petersen CC & Nicoll RA (2000). Effects of reduced vesicular filling on synaptic transmission in rat hippocampal neurones. J Physiol 525, 195–206.
Zorumski CF, Mennerick S & Que J (1996). Modulation of excitatory synaptic transmission by low concentrations of glutamate in cultured rat hippocampal neurons. J Physiol 494, 465–477., http://www.100md.com(Pauline Cavelier and Davi)