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The Journal of Neuroscience, August 6, 2003, 23(18):7176-7182
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Functional Changes of Glial Glutamate Transporter GLT-1 during Ischemia: An In Vivo Study in the Hippocampal CA1 of Normal Mice and Mutant Mice Lacking GLT-1
Akira Mitani1 andKohichi Tanaka2,3 1College of Medical Technology, Kyoto University, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan, 2Laboratory of Molecular Neuroscience, School of Biomedical Science and Medical Research Institute, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8579, Japan, and 3Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan
Abstract
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Abstract
Introduction
Glutamate transporters remove glutamate from the extracellular space and maintain it below neurotoxic levels under normal conditions. However, the dynamics under ischemic conditions remain to be determined. In the present study, we evaluated the function of the glial glutamate transporter (GLT-1) during brain ischemia by using an in vivo brain microdialysis technique in GLT-1 mutant mice. A microdialysis probe was placed in the hippocampal CA1 of GLT-1 mutant and wild-type mice, and glutamate levels were measured during 5 and 20 min ischemia. The glutamate levels in mice lacking GLT-1 were significantly higher than the corresponding glutamate levels in wild-type mice during 5 min ischemia. Delayed neuronal death was induced in the CA1 of the mice lacking GLT-1 but not in the CA1 of the wild-type mice. When ischemia was elongated to the duration of 20 min, the glutamate levels in wild-type mice were significantly higher than the corresponding glutamate levels in mice lacking GLT-1 during the last 12.5 min of 20 min ischemia. Acute neuronal death was also observed in the CA1 of wild-type mice. These results suggest that GLT-1 takes up extracellular glutamate to protect neurons in the early stage of ischemia and then releases glutamate, triggering acute neuronal death, when ischemic conditions are elongated. The function of GLT-1 may change from neuroprotective to neurodegenerative during ischemia.
Key words: glutamate transporter; glia; GLT-1; knock-out mouse; hippocampal CA1; ischemia; neuronal death; microdialysis
Introduction
Increased extracellular glutamate during ischemia triggers the death of neurons (Rothman and Olney, 1986; Choi and Rothman, 1990
The genes for glutamate transporters have been cloned, and at least five subtypes have been identified: GLAST-EAAT1 (Storck et al., 1992
Several studies have examined the role of GLT-1 in ischemia using specific pharmacological blockers or antisense oligodeoxynucleotides; however, the results are controversial. A pharmacological study has shown that the GLT-1 blocker reduces the ischemia-induced glutamate release in rat cortical superfusates (Phillis et al., 2000
Recently, we generated mice lacking GLT-1 by using homologous recombination (Tanaka et al., 1997
Materials and Methods
Animals and surgery. The experiments were conducted in accordance with the guidelines for animal experimentation at Ehime University School of Medicine and College of Medical Technology (Kyoto University). In principle, the method was the same as described in previous studies (Mitani et al., 1992Ischemia and brain microdialysis. In the present study, bilateral occlusion of the common carotid arteries (BCCA) was used to induce ischemia in the hippocampus. Generally, satisfactory ischemia with blood flow below 10% of the baseline (Panahian et al., 1996
A thermocouple needle probe of 0.4 mm diameter (TN-800S; Unique Medical Company) and a thermocouple meter (TME-300; Unique Medical Company) were used to monitor brain temperature. The thermocouple probe was inserted in the brain (1.2 mm rostral to the bregma, 1.5 mm lateral to the midline, and depth of 1.8 mm from the cortical surface) at a 15° angle rostral to the vertical plane. An identical thermocouple needle probe and thermocouple meter assembly was used to monitor the rectal temperature. The body and brain temperatures were regulated at 37 ± 0.3°C with a heating blanket (Harvard Apparatus, Kent, England) and overhead heating fan (Panasonic, Tokyo, Japan) during the experiments.
A microdialysis probe (0.5-mm-long dialysis membrane, 0.22 mm diameter; molecular weight cutoff, 50,000) (A-I-4-005; Eicom, Kyoto, Japan) was positioned perpendicularly in the CA1 (1.8 -2.3 mm caudal to the bregma, 1.5-2.2 mm lateral to the midline, and 1.5-1.6 mm ventral to the cortical surface). Subsequently, the microdialysis probe was perfused with Ringer's solution at a flow rate of 0.6 µl/min by means of a microinfusion pump (Bioresearch Center, Nagoya, Japan). After a 2 hr stabilization period, halothane administration was decreased to 1% and then transient forebrain ischemia of 5 or 20 min duration was induced. The sutures around the two common carotid arteries were pulled using 6 gm weights to occlude the circulation. After ischemia, the sutures were cut and removed to restore blood flow.
Samples (150 sec; 1.5 µl) of the dialysate were collected consecutively. In mice subjected to 5 min ischemia, six samples were collected before ischemia, two samples were collected during ischemia, and 14 samples were collected after ischemia. In mice subjected to 20 min ischemia, six samples were collected before ischemia, eight samples were collected during ischemia, and 16 samples were collected after ischemia. After the collection of dialysate samples, the probes for microdialysis, flowmetry, and thermomonitor were gently removed. All surgical incisions were carefully sutured. The animals were treated with antibiotics, removed from the stereotaxic apparatus, and put into a comfortable position on a warming blanket. After awakening, the animals were returned to individual cages in a room maintained at a constant temperature (26°C) and allowed access to food and water ad libitum. Other sets of wild-type mice served as sham-operated controls in which the animals were subjected to identical procedures, except that the sutures around the common carotid arteries were not pulled.
Glutamate assay. The present enzymatic cycling procedure for glutamate assay was essentially the same as described in previous studies (Mitani et al., 1990
Histological analysis. The animals subjected to 5 min ischemia were deeply anesthetized with pentobarbital and perfused transcardially with heparinized saline (5 ml) and then with 10% formalin in 0.1 M phosphate buffer, pH 7.4 (50 ml), at 1 hr, 24 hr, or 4 d after recirculation. The animals subjected to 20 min ischemia were anesthetized and perfused with the same procedure at 1 hr after recirculation. The brains were removed and processed for paraffin embedding. Serial coronal 5 µm sections were cut and stained with cresyl violet to examine the intensity of ischemic injury and the position of the microdialysis probe. The intensity of the ischemic injury was quantitatively determined by counting the number of normal-appearing neurons (distinct cell membrane, well defined nucleus, and nucleolus) at 400x magnification in a blind manner. Cell counts were performed in the section close to the track of the microdialysis probe but did not include any mechanical damage produced by insertion of the probe, and the number of normal-appearing neurons along a 1 mm linear length of the hippocampal CA1 pyramidal cell layer was calculated.
Data analysis. ANOVA was used for the statistical analysis of glutamate levels and the number of normal-appearing neurons; this analysis included Scheffé's or Dunnett's multiple-comparison procedure. Data were presented as the mean ± SEM. The differences discussed in the text were significant at p < 0.01 or p < 0.05.
Results
It has been known that GLT-1 mutant mice sometimes show spontaneous epileptic seizures and, in most cases, die within a few minutes of seizure onset (Tanaka et al., 1997Blood flow
Microdialysis experiments were performed on 151 C57BL/6 mice (69 GLT-1 mutant mice and 82 wild-type mice, including 20 sham-operated controls), and the blood flow in the hippocampus decreased consistently below 10% of the baseline during occlusion of BCCA in 40 mice (20 GLT-1 mutant mice and 20 wild-type mice) (Fig. 1). In these animals, the blood flow decreased promptly after the onset of BCCA occlusion and remained constant throughout ischemia. No significant differences in the reduction of the blood flow were detected between GLT-1 mutant mice and wild-type mice [8.3 ± 0.3 and 8.1 ± 0.2% of the baseline in mutant mice (n = 12) and wild-type mice (n = 12) subjected to 5 min occlusion, respectively; 7.8 ± 0.4 and 7.9 ± 0.4% of the baseline in mutant mice (n = 8) and wild-type mice (n = 8) subjected to 20 min occlusion, respectively]. After releasing the occlusion, the return of blood flow to the baseline was delayed for5 min after 5 min ischemia and
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Figure 1. A representative change in hippocampal blood flow in mice subjected to 20 min of BCCA occlusion. Blood flow was recorded from the hippocampus at a distance of
Glutamate changes induced by 5 min ischemia
In wild-type mice subjected to 5 min ischemia (n = 12), stable basal levels of glutamate were observed in six consecutive samples collected before ischemia (Fig. 2). A significant increase in glutamate was detected in the dialysate during ischemia. The increase was acute, and the maximal levels were attained at the end of 5 min ischemia. An
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Figure 2. Changes in dialysate levels of glutamate in the hippocampal CA1 of mice lacking GLT-1 and wild-type mice. The mice were subjected to 5 min ischemia. The data presented are means ± SEM (bars). Glutamate levels in mice lacking GLT-1 (n = 12; -
-
p < 0.01;
Glutamate levels in GLT-1 mutant mice subjected to 5 min ischemia (n = 12) were significantly higher than the corresponding glutamate levels in wild-type mice subjected to 5 min ischemia at all measured points, including before ischemia (p < 0.01; two-way ANOVA; Scheffé's post hoc test) (Fig. 2). In mice lacking GLT-1, stable basal levels of glutamate were observed in the six consecutive samples collected before ischemia. However, the preischemic basal levels of glutamate were approximately three times as high as those in wild-type mice. A significant increase in glutamate was detected during ischemia and the early period of recirculation. The increase was acute and massive, and the maximal levels were attained at the end of 5 min ischemia. The glutamate levels at the end of ischemia showed a sixfold increase and were approximately twice as high as those in wild-type mice. After the onset of recirculation, the glutamate levels in mice lacking GLT-1 decreased gradually, compared with the decline of glutamate levels in wild-type mice. The glutamate levels in mice lacking GLT-1 were significantly higher than the preischemic basal levels for 12.5 min after recirculation, whereas the glutamate levels in wild-type mice were significantly higher than the preischemic basal levels only for 2.5 min after recirculation (Fig. 2). The glutamate levels in mice lacking GLT-1 showed a tendency to be higher than the preischemic basal levels from 12.5 min after recirculation to the end of the measurement; however, the differences were not statistically significant.
Neuronal damage induced by 5 min ischemia
In wild-type mice subjected to 5 min ischemia, no definite neuronal changes were seen in the CA1 at 1 hr, 24 hr, or 4 d after recirculation (Fig. 3a). Dense normal-appearing neurons were observed in the CA1 pyramidal cell layer (Fig. 3b). The neuronal densities at 1 hr, 24 hr, and 4 d after recirculation were 232.2 ± 3.3/mm (n = 4), 230.7 ± 3.1/mm (n = 4), and 234.0 ± 3.3/mm (n = 4), respectively. No significant differences in the number of normal-appearing neurons were detected between wild-type ischemic mice and sham-operated control mice (Fig. 4).
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Figure 3. Photomicrographs showing the track of the microdialysis probe and neuronal death in the hippocampus of mice subjected to 5 min ischemia. In wild-type mice (a,b), neuronal death was not observed 4 d after recirculation (a). The higher magnification of the CA1 pyramidal cell layer shows that normal-appearing CA1 neurons are distributed densely in the layer (b). In mice lacking GLT-1 (c--f), neuronal death was not detected in the CA1 1 hr after recirculation (c). However, neuronal death was induced in the CA1 and also in part of the CA3 (around a filled circle) 24 hr after recirculation (d), and then widespread neuronal death was observed in the CA1 and CA3 4 d after recirculation (e). The high magnification of the CA1 pyramidal cell layer 4 d after recirculation shows that almost all of the CA1 neurons died (f). a, c-e, A portion of the microdialysis membrane (distance between two arrowheads) was placed in the CA1. The arrows indicate the boundary between the CA1 and CA3. Scale bars: a, c, d, e, 0.5 mm; b, f, 50 µm. DG, Dentate gyrus.
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Figure 4. Neuronal density in the CA1 of mice subjected to 5 min ischemia. Normal-appearing neurons were counted in the CA1 of mice lacking GLT-1 (-/-, filled columns), wild-type mice (+/+, open columns), and sham-operated control mice (sham, stippled columns) 1 hr, 24 hr, and 4 d after recirculation, and the density of neurons per millimeter in the pyramidal cell layer was calculated. The sham-operated control mice were +/+. Asterisks indicate a value significantly lower than the corresponding values in wild-type and sham-operated control mice (
In GLT-1 mutant mice subjected to 5 min ischemia, no detectable changes were seen in the CA1 1 hr after recirculation (Fig. 3c). The neuronal density (232.4 ± 3.2/mm; n = 4) was not significantly different from that in wild-type and sham-operated control mice (Fig. 4). Twenty-four hours after recirculation, neuronal loss was detected in the CA1 and also in part of the CA3 (Fig. 3d). Normal-appearing neurons were rarely observed in the CA1 pyramidal cell layer (Fig. 4). Four days after recirculation, neuronal loss was seen throughout the CA1 and the CA3 (Fig. 3e), and normal-appearing neurons were rarely observed in the pyramidal cell layers (Fig. 3f). The neuronal densities in the CA1 pyramidal cell layer 24 hr and 4 d after recirculation were 7.2 ± 1.7/mm (n = 4) and 6.3 ± 1.6/mm (n = 4), respectively. These densities were significantly less than the corresponding densities in wild-type and sham-operated control mice (p < 0.01; one-way ANOVA; Scheffé's post hoc test) (Fig. 4).
Glutamate changes induced by 20 min ischemia
In sham-operated control mice (n = 8), stable low levels of glutamate were observed at all measured points (data not shown). Glutamate levels in GLT-1 mutant mice subjected to 20 min ischemia (n = 8) that were stable before ischemia increased abruptly after the onset of ischemia (Fig. 5). The glutamate levels increased continuously and reached a peak at the end of 20 min ischemia. An
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Figure 5. Changes in dialysate levels of glutamate in the hippocampal CA1 of mice lacking GLT-1 and wild-type mice. The mice lacking GLT-1 (n = 8; p < 0.01;
Glutamate levels in wild-type mice subjected to 20 min ischemia (n = 8) were stable before ischemia. A significant increase in glutamate was detected during ischemia. The increase was acute, but the glutamate levels were significantly lower than the corresponding levels in mice lacking GLT-1 during the first 5 min of 20 min ischemia (Fig. 5), as seen in the experiment of 5 min ischemia. The increase then became steeper, and the glutamate levels crossed the levels in GLT-1 mutant mice. The glutamate levels increased continuously and reached a peak at the end of 20 min ischemia. An
Neuronal damage induced by 20 min ischemia
In GLT-1 mutant mice subjected to 20 min ischemia, neuronal damage was seen in the CA1 1 hr after recirculation. Normal-appearing neurons, which were densely observed in the CA1 pyramidal cell layer in sham-operated control mice (Fig. 6a), were rarely seen in the CA1 (Fig. 6b). Many pyknotic nuclei and damaged neurons were seen. Pyknotic nuclei were surrounded by empty spaces, and numerous dark granules in the cytoplasm (selective chromatolysis) (Kirino and Sano, 1984
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Figure 6. Neuronal death and density in the hippocampal CA1 in mice subjected to 20 min ischemia. Acute neuronal death was observed 1 hr after recirculation in mice subjected to 20 min ischemia. Normal-appearing neurons, which were densely observed in the CA1 pyramidal cell layer in sham-operated control mice (a), were rarely seen in the CA1 of mice lacking GLT-1 (b) and also wild-type mice (c). Scale bar, 50 µm. d, The density of normal-appearing neurons per millimeter of the pyramidal cell layer was calculated in the CA1 in mice lacking GLT-1 (-/-, filled columns), wild-type mice (+/+, open columns), and sham-operated control mice (sham, stippled columns) 1 hr after recirculation. The sham-operated control mice were +/+. Asterisks indicate a value significantly lower than the corresponding value of sham-operated control mice (
In wild-type mice subjected to 20 min ischemia, neuronal damage was seen in the CA1 1 hr after recirculation. Normal-appearing neurons were rarely seen in the CA1 (Fig. 6c). Most CA1 neurons showed darkly stained, shrunken cell bodies with surrounding empty spaces.
No significant differences in the number of normal-appearing neurons were detected between GLT-1 mutant mice (5.1 ± 0.7/mm) and wild-type mice (9.0 ± 2.6/mm), and the neuronal densities in GLT-1 mutant and wild-type mice subjected to 20 min ischemia were significantly less than those observed in sham-operated control mice (p < 0.01; one-way ANOVA; Scheffé's post hoc test) (Fig. 6d).
Discussion
In the present study, we performed an in vivo brain microdialysis study and evaluated the functional role of the glial glutamate transporter during ischemia by the comparison of extracellular glutamate levels in the hippocampal CA1 of GLT-1 mutant mice with those of wild-type mice.Glutamate levels under normal conditions
The present study showed that the preischemic basal levels of extracellular glutamate in the hippocampal CA1 of GLT-1 mutant mice were significantly higher than those of wild-type mice. This result provides direct evidence that GLT-1 removes glutamate from the extracellular fluid in the hippocampal CA1 to maintain extracellular glutamate at low levels under normal conditions, which has been widely presumed but not proved directly.Glutamate levels during 5 min ischemia
The present study demonstrated that glutamate levels in the hippocampal CA1 of wild-type mice were significantly lower than those of mice lacking GLT-1 during 5 min ischemia and quickly decreased immediately after recirculation. The result suggests that GLT-1 normally operates to take up extracellular glutamate during and immediately after 5 min ischemia. In addition, it was shown that delayed neuronal death was induced in the CA1 of GLT-1 mutant mice subjected to 5 min ischemia. In the present study, delayed neuronal death means that morphological damage in ischemic neurons is delayed (Kirino, 1982If GLT-1, which is localized to astrocytes, is not releasing glutamate during 5 min ischemia, what is responsible for the glutamate increase? It may be caused by neurons. Consistent with this idea, no significant increase in extracellular glutamate was observed during 5 min ischemia in the gerbil hippocampal CA1, in which most of the neurons had been eliminated before the experiment (Mitani et al., 1994
The electrophysiological studies have also reported that a GLT-1 blocker and GLT-1 mutation have no effect on the glutamate-evoked current in ischemic neurons (Rossi et al., 2000
Glutamate levels during 20 min ischemia
The experiment of 20 min ischemia revealed that glutamate levels in the hippocampal CA1 of wild-type mice were significantly higher than those of mice lacking GLT-1 during the last 12.5 min of ischemia. In addition, increased glutamate levels in wild-type mice did not remarkably decrease for the first 2.5 min after recirculation, compared with the second 2.5 min after recirculation. If GLT-1 works normally at the end of ischemia, increased glutamate levels would quickly decrease immediately after the onset of recirculation. Therefore, GLT-1 may induce dysfunction. In vitro studies have shown that glutamate release is produced by reversed operation of glutamate transporters under energydepleted conditions (Kauppinen et al., 1988-benzyloxyaspartate reduces the ischemia-induced glutamate release in rat cortical superfusates during 20 min global brain ischemia (Phillis et al., 2000
In GLT-1 mutant mice, the ischemia-induced glutamate level was approximately one-half of that in wild-type mice at the end of 20 min ischemia. However, the histological study did not show any reduction in ischemic neuronal damage at 1 hr after recirculation. The acute death of CA1 neurons was induced in the GLT-1 mutant mice as well as in the wild-type mice. This may be explained by a quantitative dose-toxicity study. It has been shown that neurotoxicity in the cortical cell culture is produced by a 5 min exposure to ED50 of 50 -100 µM glutamate (Choi et al., 1987
In summary, our present results suggest that the function of GLT-1 changes from neuroprotective to neurodegenerative during ischemia: GLT-1 takes up extracellular glutamate to protect neurons against delayed neuronal death during the first 5 min of ischemia and then releases glutamate, triggering acute neuronal death, during the last 12.5 min of 20 min ischemia in the in vivo hippocampal CA1. These findings favor therapeutic strategies aimed at preventing or reducing the excessive release of glutamate in the ischemic brain by modulating glutamate transport. Allosteric GLT-1 activators, such as bromocryptine (Yamashita et al., 1995
Footnotes
Received Mar. 17, 2003; revised Jun. 5, 2003; accepted Jun. 12, 2003.This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science and the Ministry of Health, Labour, and Welfare of Japan. We thank K. Okugawa for help in animal experiments, T. Yagi for photographic help, and D. Shimizu for help in histological experiments.
Correspondence should be addressed to Dr. Akira Mitani, College of Medical Technology, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: amitani{at}itan.kyoto-u.ac.jp.
Copyright © 2003 Society for Neuroscience 0270-6474/03/237176-07$15.00/0
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