Changes of pyridoxal kinase expression and activity in the gerbil hippocampus following transient forebrain ischemia

Neuroscience 128 (2004) 511–518

CHANGES OF PYRIDOXAL KINASE EXPRESSION AND ACTIVITY IN THE GERBIL HIPPOCAMPUS FOLLOWING TRANSIENT FOREBRAIN ISCHEMIA I. K. HWANG,a K.-Y. YOO,a D. S. KIM,a W. S. EUM,b J.-K. PARK,c J. PARK,b O.-S. KWON,d T.-C. KANG,a S. Y. CHOIb* AND M. H. WONa*

Key words: pyridoxal kinase, GABA, astrocytes, hippocampus, transient ischemia, gerbil.

a Department of Anatomy, College of Medicine, Hallym University, Chunchon 200-702, South Korea

GABA is synthesized from two glutamic acid decarboxylase (GAD) isoforms, GAD65 and GAD67. GAD65 is important for the local control of GABA synthesis at the synaptic sites, whereas GAD67 is responsible for maintaining GABA baseline levels for both neurotransmitter and metabolite (Esclapez et al., 1994; Esclapez and Houser, 1999; Martin et al., 2000; Qu et al., 1998). Recently we observed the increased GAD 65 and 67 expressions in GABAergic neurons of the hippocampal CA1 region of the gerbil after transient forebrain ischemia (Kang et al., 2002b). This report suggested that temporal alterations of GAD immunoreactivities reflect the sequential changes of the GABAergic neuronal discharge after ischemic damage (Choi et al., 1999). However, the alterations of GAD isoforms immunoreactivities induced by ischemic insult showed different temporal patterns. Briefly, at 30 min post-ischemia, the immunoreactivities of the both GAD isoforms were markedly elevated. At 3 h after ischemic insult, GAD65 immunoreactivity was recovered at the sham level in the CA1 region. However, increased GAD67 immunoreactivity was detected at 24 h after ischemic insult. These temporal alterations of GAD immunoreactivities reflect the sequential changes of the GABAergic neuronal discharge after ischemia, because GAD67 plays a major role during tonic neuronal activity, whereas GAD65 is particularly recruited and phosphorylated during increased phasic activity (Choi et al., 1999; Kang et al., 2002a; Kouyoumdjian and Ebadi, 1981). However, these changes in GAD expression did not supply direct evidence concerning the increase of GABA release or synthesis, because GAD isoforms differ substantially in their interactions with cofactor pyridoxal 5=-phosphate (PLP), which is catalyzed by pyridoxal kinase (PLK; Choi et al., 1999; Costa et al., 1998; Erlander et al., 1991; Erlander and Tobin, 1991; Fukuda et al., 1998; Houser and Esclapez, 1994; Laming et al., 1989; Weiner and Molinoff, 1994). On the basis of PLP concentration ratios, GAD65 makes up the majority of the inactive apoGAD reservoir in GABAergic neurons, in contrast GAD67 is nearly saturated with PLP as a holoenzyme (Costa et al., 1998; Erlander and Tobin, 1991; Esclapez and Houser, 1999; Robin and Kalloniatis, 1992). Therefore, it is plausible that alterations of both GAD isoforms induced by ischemia would be regulated in response to alterations in PLK expression, although altered PLK expression and its activity have not

b

Department of Genetic Engineering, College of Life Science, Hallym University, Chunchon 200-702, South Korea c

Bio-Research group, KT & G Central Research Institute, Daejon 305-345, South Korea

d Department of Biochemistry, College of Natural Sciences, Kyungpook National University, Taegu 702-701, South Korea

Abstract—In the previous study, we observed chronological alterations of glutamic acid decarboxylase (GAD), which is the enzyme converting glutamate into GABA. GAD isoforms (GAD65 and GAD67) differ substantially in their interactions with cofactor pyridoxal 5=-phosphate, which is catalyzed by pyridoxal kinase (PLK). In the present study, we examined the chronological changes of PLK expression and activity in the hippocampus after 5 min transient forebrain ischemia in gerbils. PLK immunoreactivity in the sham-operated group was detected weakly in the hippocampus. Ischemia-related change of PLK immunoreactivity in the hippocampus was significant in the hippocampal cornu ammonis (CA1) region, not in the hippocampal CA2/3 region and dentate gyrus. PLK immunoreactivity was observed in non-pyramidal GABAergic neurons at 30 min to 3 h after ischemic insult. At 12 h after ischemic insult, PLK immunoreactivity was shown in many CA1 pyramidal cells as well as some non-pyramidal cells. At this time point, PLK immunoreactivity and protein content was highest after ischemia. Thereafter, PLK immunoreactivity and protein content is decreased time-dependently by 4 days after ischemic insult. Four days after ischemia, some astrocytes expressed PLK in the CA1 region. The specific PLK activity was not altered following ischemic insult up to 2 days after ischemic insult. Thereafter, the specific PLK activity decreased time-dependently. However, total activity of PLK was significantly increased 12–24 h after ischemic insult, and thereafter total activity of PLK decreased. Therefore, we suggest that the over-expression of PLK in the CA1 pyramidal cells at 12 h after ischemia may induce increase of GAD in the CA1 pyramidal cells, which plays an important role in delayed neuronal death via the increase of GABA or enhancement of GABA shunt pathway. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding authors. Tel: ⫹82-33-248-2522; fax: ⫹82-33-256-1614 (M. H. Won); Tel: ⫹82-33-248-2112; fax: 82-33-241-1463 (S. Y. Choi). E-mail addresses: [email protected] (M. H. Won); [email protected] (S. Y. Choi), Abbreviations: CA1, cornu ammonis; GABA-T, GABA transaminase; GAD, glutamic acid decarboxylase; GAT-1, glutamic acid transporter-1; GFAP, glial fibrillary acidic protein; PBS, phosphate-buffered saline; PLK, pyridoxal kinase; PLP, pyridoxal 5=-phosphate; ROD, relative optical density.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.06.061

511

512

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

Fig. 1. Photographs of PLK immunoreactivity in the gerbil hippocampal CA1 region in the sham-operated group (A), at 30 min (B), 3 h (C), 12 h (D) and 4 days (E) after 5 min transient forebrain ischemia. In the sham-operated group (A), PLK immunoreactivity is observed weakly. Some non-pyramidal cells show PLK immunoreactivity at 30 min after ischemic insult (B). At 3 h after ischemia-reperfusion (C), the immunoreactivity and number of PLK-immunoreactive neurons increase. Note that well-stained processes are distributed in the strata radiatum (SR) and oriens (SO). At 12 after ischemic insult (D), many pyramidal cells in the stratum pyramidale (SP) show PLK immunoreactivity. By 4 days after ischemic insult (E), PLK-immunoreactive glial components are distributed in the CA1 region. The NeuN-immunoreactive neurons are evenly distributed in the shamoperated group (F) and 12 h post-ischemic group (G). Note that the neuronal loss is not detected in the 12 h post-ischemic group. Scale bar⫽100 ␮m.

been definitively determined. In the present study, therefore, we examined the expression and chronological changes of PLK in the hippocampus in order to determine the relationship between the neuronal damage and GAD expressions following transient ischemia in gerbils.

EXPERIMENTAL PROCEDURES Experimental animals This study utilized the progeny of Mongolian gerbils (Meriones unguiculatus) obtained from the Experimental Animal Center, Hallym University, Chunchon, South Korea. The animals were housed at constant temperature (23 °C) and relative humidity (60%) with a fixed 12-h light/dark cycle and free access to food and water. Procedures involving animals and their care were

conformed to the institutional guidelines, which are in compliance with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85–23, 1985, revised 1996). All experiment was conducted to minimize the number of animals used and suffering.

Induction of ischemia Male Mongolian gerbils (M. unguiculatus) weighing 66 –75 g were placed under general anesthesia with a mixture of 2.5% isoflurane in 33% oxygen and 67% nitrous oxide. A midline ventral incision was made in the neck. Both common carotid arteries were isolated, freed of nerve fibers, and occluded using non-traumatic aneurysm clips. Complete interruption of blood flow was confirmed by observing the central artery in eyeballs using an ophthalmoscope. After 5 min of occlusion, the aneurysm clips were removed from both of the common carotid arteries. Restoration of

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

513

Fig. 2. The ROD of PLK immunoreactivity in the hippocampal sub-regions (CA1 and CA2/3) and dentate gyrus (DG) after ischemic insult. The results of the quantitative data obtained using image analysis are consistent with the immunohistochemical study (* P⬍0.05, ** P⬍0.01, significant differences from adjacent group). The bars indicate the means⫾S.E.

blood flow (reperfusion) was observed directly under the ophthalmoscope. Sham-operated animals (n⫽10) were subjected to the same surgical procedures except that common carotid arteries were not occluded. Body temperature was monitored and maintained at 37⫾0.5 °C during the surgery and during the immediate postoperative period until the animals recovered fully from anesthesia. At the designated reperfusion times, the sham-operated and operated groups were killed for this study (Kang et al., 2003; Won et al., 2001).

Tissue processing and immunohistochemistry All animals were anesthetized with pentobarbital sodium, and perfused transcardially with 0.1 M phosphate-buffered saline (PBS; pH 7.4) followed by 4% paraformaldehyde in 0.1 M PB (pH 7.4) at 30 min (n⫽10), 3 h (n⫽10), 12 h (n⫽10), 1 day (n⫽10), 2 days (n⫽10), and 4 days (n⫽10) after the surgery. Brains were removed, and postfixed in the same fixative for 6 h. The brain tissues were cryoprotected by infiltration with 30% sucrose overnight. Thereafter the brain tissues were frozen and sectioned with a cryostat at 30 ␮m and consecutive sections were collected in six-well plates containing PBS. The sections were sequentially treated with 0.3% hydrogen peroxide (H2O2) in PBS for 30 min and 10% normal horse serum in 0.05 M PBS for 30 min. The sections were next incubated with diluted mouse anti-PLK antibody (diluted 1:200; Choi et al., 1999; Kang et al., 2002a) overnight at room temperature. Thereafter the tissues were exposed to biotinylated horse anti-mouse IgG and streptavidin peroxidase complex (Vector, Burlingame, CA, USA). The sections were visualized with 3,3=-diaminobenzidine in 0.1 M Tris buffer and mounted on the gelatin-coated slides. To confirm the neuronal loss in the hippocampal CA1 region 12 h after ischemic insult, the sections were incubated with diluted mouse anti-NeuN (diluted 1:1000; Chemicon, Temecula, CA, USA). This experiment was conducted parallel to abovementioned immunohistochemistry.

Double immunofluorescence study To confirm the neuronal type containing PLK immunoreactivity, double immunofluorescent staining for both the mouse anti-PLK antiserum (1:25) and the rabbit anti-GAD 67 (GAD67, 1:50;

Chemicon) was performed. We also performed the double immunostaining for both the mouse anti-PLK antiserum (1:25) and the rabbit anti-glial fibrillary acidic protein (GFAP; 1:100; Chemicon, USA) to confirm the glial type. Other brain tissues were incubated in the mixture of antisera overnight at room temperature. After washing three times for 10 min with PBS, the sections were also incubated in a mixture of both FITC-conjugated goat anti-mouse IgG (1:200; Jackson ImmunoResearch, USA) and Cy3 conjugated goat anti-rabbit IgG (1:600; Jackson ImmunoResearch) for 2 h at room temperature. The immunoreactions were observed under the Axioscope microscope attached HBO100 (Carl Zeiss, Germany).

Western blot study Five animals in each group, which were mentioned above, were used in this immunoblot study. After kill and removal of the hippocampal CA1 region, the tissues were homogenized in 50 mM Tris containing 50 mM HEPES (pH 7.4), EGTA (pH 8.0), 0.2% NP-40, 10 mM EDTA (pH 8.0), 15 mM sodium pyrophosphate, 100 mM ␤-glycerophosphate, 50 mM NaF, 150 mM NaCl, 2 mM sodium orthvanadate, 1 mM PMSF and 1 mM DTT. After centrifugation, the protein concentration was determined in the supernatants by using the Micro BCA protein assay kit with bovine serum albumin as the standard (Pierce Chemical, USA). Aliquots containing 20 ␮g total protein were boiled in loading buffer containing 150 mM Tris (pH 6.8), 3 mM DTT, 6% SDS, 0.3% Bromophenol Blue and 30% glycerol. Then, each aliquot was loaded onto a 10% polyacrylamide gel. After electrophoresis, the gels were transferred to nitrocellulose transfer membranes (Schleicher and Schuell, USA). To reduced background staining, the filters were incubated with 5% non-fat dry milk in PBS containing 0.1% Tween 20 for 45 min, followed by incubation with mouse anti-PLK antiserum (1:500), with peroxidase-conjugated horse anti-mouse IgG (Sigma, USA), and then with ECL kit (Amersham, USA).

Enzymatic assay Five animals in each group (the same animals in Western blot study) were used in this enzymatic assay. PLK from gerbil hippocampus was purified according to a procedure of Kerry et al. (1986) using a combination of DEAE-cellulose, and affinity chro-

514

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

Fig. 3. Double immunofluorescent staining for PLK (green, A) and GAD67 (red, B) in the hippocampal CA1 region at 3 h after ischemic insult. PLK immunoreactivity is found to be co-localized with GAD67 immunoreactivity (arrows) in the CA1 region. Note that some GABAergic neurons (open arrows) do not contain PLK (B). SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar⫽100 ␮m.

matography and G-100 gel filtration. Enzymatic activity was measured by following change in absorbance at 388 nm, at which PLP has an absorption coefficient of 4900 cm⫺1 M⫺1 at pH 7.0. Initial rate measurements were carried out by monitoring the change in absorbance at 388 nm for at least 3 min in a double beam spectrophotometer. The standard assay mixture was to measure PLK activity contained 0.07 M potassium phosphate (pH 6.5), pyridoxal (0.1 mM), ATP (0.1 mM) and ZnCl2 (0.05 mM).

Quantification of data and statistical analysis Sections (10 sections per animal) were viewed through a microscope connected via CCD camera to a PC monitor. At a magnification of 25–50⫻, the regions were outlined on the monitor and measured their areas. Each studied field in each tissue was selected within the midpoint of hippocampal CA1-3 regions and the dentate gyrus containing all layers. Images of PLK immunoreactivity in each region of each animal were captured with an Applescanner. The brightness and contrast of each image file were calibrated by Adobe Photoshop version 2.4.1, and analyzed using NIH Image 1.59 software. Values of background staining were obtained and subtracted from the immunoreactive intensities. The intensity of PLK immunoreactivity was evaluated by means of a relative optical density (ROD) value. ROD value was obtained after transformation of mean gray values into ROD using the formula. Also the results of Western blot study were scanned and ROD value was obtained using Scion Image software (NIH). All data obtained from the quantitative data were analyzed using one-way ANOVA to determine statistical significance. Bonferroni’s test was used for post hoc comparisons. P value below 0.05 or 0.01 was considered statistically significant.

RESULTS PLK immunohistochemistry In the sham-operated group, PLK immunoreactivity was weakly detected in pyramidal and non-pyramidal cells in the hippocampal CA1-3 regions (Fig. 1A) and the granule cell layer of the dentate gyrus (data not shown). Ischemia-related changes of PLK immunoreactivity were significant in the hippocampal CA1 region, not in the CA2/3 region and the dentate gyrus (Fig. 2). In the CA1 region, some non-pyramidal cells show PLK immunoreactivity at 30 min after 5 min transient forebrain ischemia (Fig. 1B). At 3 h after ischemia-reperfusion, the immu-

nodensity and number of PLK-immunoreactive neurons was slightly increased (Figs. 1C and 2). At this time after ischemia, well-stained processes were distributed in the strata radiatum and oriens (Fig. 1C). At 12 h after ischemic insult, PLK immunoreactivity in the CA1 region showed significant differences after ischemic injury (Figs. 1D). In this group, strong PLK immunoreactivity was shown in the pyramidal cells in the stratum pyramidale. At this time point after ischemic insult, non-pyramidal cells were decreased in number as compared with 3 h post-ischemic group (Fig. 1D). At this time point, the number of NeuN-immunoreactive neurons was not altered in the hippocampal CA1 region (Figs. 1F, 1G). Compared with 3 h after ischemic insult, PLK immunoreactivity in the CA1 region was significantly elevated (Figs. 1D and 2). Thereafter, PLK immunoreactivity and PLK-immunoreactive cells were decreased in the CA1 region (Fig. 2). This immunoreactive pattern was maintained up to 2 days after ischemic insult. Three to 4 days after ischemic insult, PLK immunoreactivity disappeared in neurons in the CA1 region, and PLK immunoreactivity was low as compared with 2 days postischemic group (Figs. 1E and 2). In this time periods, PLK immunoreactivity was intensified in glial components because the delayed neuronal death of CA1 pyramidal cells occurred in the CA1 region (Figs. 1E and 2). Double immunofluorescence study From 30 min to 3 h after ischemic insult, PLK immunoreactivity was detected in non-pyramidal cells in the CA1 region, and its immunoreactivity was increased timedependently (Figs. 1B and 1C). Based on morphology and double immunofluorescence study, PLK-immunoreactive neurons were GABAergic (Fig. 3). Three to 5 days after ischemic insult, almost pyramidal cells in the CA1 region were lost because the delayed neuronal death occurred (data not shown). In this time period, PLK immunoreactivity was intensified in glial cells (Fig. 1E). In this study, based on the morphology and double immunofluorescent staining, the glial cells were

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

515

ischemia (Figs. 5). The PLK protein content was highest at 12 h after ischemic insult, thereafter the content decreased by 4 days after ischemia. The results of the quantitative data obtained from image analysis and Western blot study were consistent with the immunohistochemical study. Three to 4 days after ischemic insult, however, PLK immunoreactivity was elevated as compared with 2 days after ischemic insult, because of increased PLK immunoreactivity in astrocytes, presumably. Analysis of enzyme activity As for the result of the enzyme activity assay, the specific PLK activity (0.0027– 0.0031 unit/mg protein) was not altered following ischemic insult up to 2 days and thereafter the specific PLK activity decreased time-dependently (Figs. 6). However, the total activity of PLK was significantly increased 12–24 h after ischemic insult and thereafter, its immunoreactivity decreased (Fig. 6).

DISCUSSION

Fig. 4. Double immunofluorescent staining for PLK (green, A) and GFAP (red, B), and a merged image (C) for PLK and GFAP in the hippocampal CA1 region 4 days after ischemic insult. PLK immunoreactivity (green) is shown in the CA1 region (A). GFAP (red) representing astrocytes is shown in the CA1 region (B). Note that some astrocytes (arrows) show PLK immunoreactivity (C). SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Scale bar⫽50 ␮m.

identified as the astrocytes (Figs. 4). Some astrocytes contained PLK after occurrence of the delayed neuronal death in the CA1 region. Western bolt study In this study, we examined that the result of Western bolt study showed the pattern of PLK expression similar to that of immunohistochemistry after 5 min transient forebrain

In brain tissue, pyridoxal phosphate is essential for the synthesis of some neurotransmitters such as dopamine, norepinephrine, serotonin and GABA, which are important in the maintenance of physiological homeostasis in the CNS (Fabri and Manzoni, 2004; Grabbet et al., 2002; Marshall et al., 2001; Nagy and Hiripi, 2002; Won et al., 2001). Because the formation of pyridoxal phosphate from ATP, pyridoxal and divalent metal ions are catalyzed by PLK, the enzyme seems to play a pivotal role in pyridoxal phosphate synthesis (Costa et al., 1998; Geng et al., 1997; Nurgali et al., 2003; Yamashima et al., 2001). In the present study, we focused upon the chronological alterations of PLK expression concerning with GABA metabolism in the hippocampus after 5 min transient forebrain ischemia. In the present study, we observed that the PLK immunoreactivity, protein content in and total activity of this enzyme in the hippocampal CA1 region was increased by 12 h after ischemic insult, although the specific activity of this enzyme was not altered. In addition, accumulated PLK immunoreactivity is predominantly detected in the pyramidal (glutamtergic) neurons, while its immunoreactivity was significantly decreased in the non-pyramidal (GABAergic) neurons. In the previous study, we reported that GAD67 immunoreactivity was significantly increased in the hippocampal CA1 region at 12–24 h after ischemic insult (Kang et al., 2000, 2002b). In the present study, we also found that the PLK immunoreactivity was significantly increased in this time. The increase of PLK in this time point strongly supports our previous study that the GAD67 immunoreactivity was significantly increased at 12 h after ischemic insult (Kang et al., 2002a). We also found in the previous study that the temporal changes in glutamic acid transporter-1 (GAT-1) expression were similar to those of GAD67, but not GAD65 expression, at least prior to 12 h after ischemic insult. GAT-1 immunoreactivity was increased in the CA1 region at 30 min after ischemia and recovered at 3 h post-ischemia

516

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

Fig. 5. Western blot analysis of PLK in the gerbil hippocampus derived from sham-operated and operated groups. The ROD of immunoblot band is also represented. The bars indicate the means⫾S.E.

(Kang et al., 2002b). At 12 h after ischemic insult, GAT-1 immunoreactivity was re-enhanced in the CA1 region, and maintained by 24 h after ischemia-reperfusion. These results and our present study indicate that the temporal alterations of GAT-1 expression could be closely related to the turnover of GABA release or synthesis prior to 12 h after ischemia. This is because the temporal alterations of GAT-1, GAD and PLK expressions showed the proportionality. At 12 h after ischemia, PLK immunoreactivity was significantly elevated in the CA1 region. This finding postulates that GABA release or synthesis is elevated in the CA1 region at this time point after ischemia. This is because an overactive inhibitory system has been observed in the CA1 region at 12 h after ischemia-reperfusion (Kang et al., 2000). Transient cerebral ischemia stimulates GABA synthesis via GAD67 and at the same time increases GABA breakdown by GABA transaminase (GABA-T) in the hippocampal CA1 region (Kang et al., 2000, 2002b). GAD67 and GABA-T immunoreactivities were significantly increased in pyramidal cells, and the increased GAD67 and GABA-T immunoreactivities may be associated with the destruction of hyperactive glutamatergic pyramidal cells in the hippocampal CA1 region. It has

been reported that drugs, which potentiate GABA levels or have GABAergic properties, provide significant neuronal protection when used before or after ischemic insult in global or focal ischemic animal stroke models (Manev et al., 1989; Shuaib et al., 1992). Increased GAD67 and GABA-T immunoreactivities in the non-pyramidal cells would also impede the entry of carbon atoms from GABA into TCA cycle via the succinic acid. Hence these GABAergic neurons in the hippocampus are considered to be particularly resistant to ischemia and N-methyl-D-aspartate excitotoxicity (Gagliardi, 2000; Schousboe et al., 1973; Won et al., 2001). This result is contradictory to previous study of lateral caudoputamen and lower parietal cortex using middle cerebral artery occlusion model (Håberg et al., 2001). They reported that GABAergic neurons in the lateral caudoputamen and lower parietal cortex were more sensitive to ischemia than glutamatergic neurons in the same region. This discrepancy may be the differences in whether the GABA shunt pathway is excitation or inhibition. In the caudoputamen and lower parietal cortex, cerebral ischemia stimulates GABA synthesis via GAD and the same time inhibits GABA breakdown by GABA-T (Sloviter et al., 1996). These changes consequently in-

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

517

Fig. 6. The enzyme activity of PLK in the hippocampus after ischemic insult. There is no statistical difference in the specific activity between groups until 2 days after ischemic insult. Thereafter, the specific activity of PLK is significantly decreased. However, the total activity is increased 12–24 h after ischemic insult, and thereafter the total activity is decreased. (* P⬍0.05 significant differences from adjacent group.)

crease the GABA content (Erecinska et al., 1984). Our previous report describes the decrease of the GABA-T immunoreactivity in the CA1 region at the same time point (Kang et al., 2002b). In fact, the inhibition of GABA-T increases GABA efflux via reverse transport (Kang et al., 2002b). Therefore, these findings also suggest that extracellular GABA concentration may elevate to attenuate the excitability of pyramidal cells in the CA1 region. At 24 h after ischemic insult, enhanced neuronal types of GAT-1, GAD67 and PLK-immunoreactive cells in the CA1 area were pyramidal cells and non-pyramidal GABAergic neurons. These PLK and GAD67 expressions in CA1 pyramidal cells may be related to the GABA metabolism, not neurotransmitter. In the previous study, we also observed that GABA-T and succinic semialdehyde dehydrogenase expressions were highly elevated in CA1 pyramidal cells at this time point after ischemic insult (Kang et al., 2000, 2002b). Hence, these results support that in this time period the increased PLK and GAD immunoreactivity in pyramidal neurons may be used in GABA shunt metabolism to form the succinic acid, and this material metabolizes into ATP. In conclusion, therefore, our findings suggest that, at least 24 h after transient ischemic damage, an increase of PLK expression in the hippocampal CA1 region may be associated with cellular energy metabolism from ischemic damage via the GABA shunt enzymes. Acknowledgments—This study was supported by a grant of the Korean Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (02-PJ1-PG10-20706-0002).

REFERENCES Choi SY, Kwok F, Bahn JH, Jeon SG, Ahn YK, Yoon BH, Lee BR, Choi KS, Gao GZ (1999) Production and characterization of monoclonal antibodies to porcine brain pyridoxal kinase. Biofactors 10:35– 42. Costa B, Giusti L, Martini C, Lucacchini A (1998) Chemical modification of the dihydropyridines binding sites by lysine reagent, pyridoxal 5=-phosphate. Neurochem Int 32:361–364. Erecinska M, Nelson D, Wilson DF, Silver IA (1984) Neurotransmitter amino acids in the CNS. I: Regional changes in amino acid levels in rat brain during ischemia and reperfusion. Brain Res 304:9 –22. Erlander MG, Tillakaratne NJ, Feldblum S, Patel N, Tobin AJ (1991) Two genes encode distinct glutamate decarboxylases. Neuron 7:91–100. Erlander MG, Tobin AJ (1991) The structural and functional heterogeneity of glutamic acid decarboxylase: a review. Neurochem Res 16:215–226. Esclapez M, Houser CR (1999) Up-regulation of GAD65 and GAD67 in remaining hippocampal GABA neurons in a model of temporal lobe epilepsy. J Comp Neurol 412:488 –505. Esclapez M, Tillakaratne NJ, Kaufman DL, Tobin AJ, Houser CR (1994) Comparative localization of two forms of glutamic acid decarboxylase and their mRNAs in rat brain supports the concept of functional differences between the forms. J Neurosci 14:1834 – 1855. Fabri M, Manzoni T (2004) Glutamic acid decarboxylase immunoreactivity in callosal projecting neurons of cat and rat somatic sensory areas. Neuroscience 123:557–566. Fukuda T, Aika Y, Heizmann CW, Kosaka T (1998) GABAergic axon terminals at perisomatic and dendritic inhibitory sites show different immunoreactivities against two GAD isoforms, GAD67 and GAD65, in the mouse hippocampus: a digitized quantitative analysis. J Comp Neurol 395:177–194. Gagliardi RJ (2000) Neuroprotection, excitotoxicity and NMDA antagonists. Ar Qneuropsiquiatr 58:583–588.

518

I. K. Hwang et al. / Neuroscience 128 (2004) 511–518

Geng MY, Saito H, Nishiyama N (1997) Protective effects of pyridoxal phosphate against glucose deprivation-induced damage in cultured hippocampal neurons. J Neurochem 68:2500 –2506. Grabb MC, Lobner D, Turetsky DM, Choi DW (2002) Preconditioned resistance to oxygen-glucose deprivation-induced cortical neuronal death: alterations in vesicular GABA and glutamate release. Neuroscience 115:173–183. Håberg A, Qu H, Sæther O, Unsgård G, Haraldseth O, Sonnewald U (2001) Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival. J Cereb Blood Flow Metab 21: 1451– 1463. Houser CR, Esclapez M (1994) Localization of mRNAs encoding two forms of glutamic acid decarboxylase in the rat hippocampal formation. Hippocampus 4:530 –545. Kang TC, Park SK, Bahn JH, Chang JS, Koh WS, Jo SM, Cho SW, Choi SY, Won MH (2000) Elevation of the gamma-aminobutyric acid transaminase expression in the gerbil CA1 area after ischemia-reperfusion damage. Neurosci Lett 294:33–36. Kang TC, Park SK, Hwang IK, An SJ, Bahn JH, Kim DW, Choi SY, Kwon OS, Baek NI, Lee HY, Won MH (2002a) Changes in pyridoxal kinase immunoreactivity in the gerbil hippocampus following spontaneous seizure. Brain Res 957:242–250. Kang TC, Park SK, Hwang IK, An SJ, Choi SY, Cho SW, Won MH (2002b) Spatial and temporal alterations in the GABA shunt in the gerbil hippocampus following transient ischemia. Brain Res 944:10 –18. Kang TC, Park SK, Hwang IK, An SJ, Choi SY, Kwon OS, Baek NI, Lee HY, Won MH (2003) The altered expression of GABA shunt enzymes in the gerbil hippocampus before and after seizure generation. Neurochem Int 42:239 –249. Kerry JA, Rohde M, Kwok F (1986) Brain pyridoxal kinase. Eur J Biochem 158:581–585. Kouyoumdjian JC, Ebadi M (1981) Anticonvulsant activity of muscimol and gamma-aminobutyric acid against pyridoxal phosphate-induced epileptic seizures. J Neurochem 36:251–257. Laming PR, Elwood RW, Best PM (1989) Epileptic tendencies in relation to behavioral responses to a novel environment in the Mongolian gerbil. Behav Neural Biol 51:92–101. Marshall JF, Henry BL, Billings LM, Hoover BR (2001) The role of the globus pallidus D2 subfamily of dopamine receptors in pallidal immediate early gene expression. Neuroscience 105:365–378.

Martin DL, Liu H, Martin SB, Wu SJ (2000) Structural features and regulatory properties of the brain glutamate decarboxylases. Neurochem Int 37:111–119. Manev H, Favaron M, Guidotti A, Costa E (1989) Delayed increase of Ca2⫹ influx by glutamate: role in neuronal death. Mol Pharmacol 36:106 –112. Nagy L, Hiripi L (2002) Role of tyrosine, DOPA and decarboxylase enzymes in the synthesis of monoamines in the brain of the locust. Neurochem Int 41:9 –16. Nurgali K, Furness JB, Stebbing MJ (2003) Analysis of purinergic and cholinergic fast synaptic transmission to identified myenteric neurons. Neuroscience 116:335–347. Qu M, Mittmann T, Luhmann HJ, Schleicher A, Zilles K (1998) Longterm changes of ionotropic glutamate and GABA receptors after unilateral permanent focal cerebral ischemia in the mouse brain. Neuroscience 85:29 – 43. Robin LN, Kalloniatis M (1992) Interrelationship between retinal ischaemic damage and turnover and metabolism of putative amino acid neurotransmitters, glutamate and GABA. Doc Ophthalmol 80:273–300. Schousboe A, Yu JY, Roberts E (1973) Purification and characterization of the 4-aminobutyrate-2-ketoglutarate transaminase from mouse brain. Biochemistry 12:2868 –2873. Shuaib A, Ijaz S, Hasan S, Kalra J (1992) Gamma-vinyl GABA prevents hippocampal and substantia nigra reticulata damage in repetitive transient ischemia in gerbils. Brain Res 590:13–17. Sloviter RS, Dichter MA, Rachinsky TL, Dean E, Goodman JH, Sollas AL, Martin DL (1996) Basal expression and induction of glutamate decarboxylase and GABA in excitatory granule cells of the rat and monkey hippocampal dentate gyrus. J Comp Neurol 373:593– 618. Weiner N, Molinoff PB (1994) Catecholamines. In: Basic neurochemistry: molecular, cellular and medical aspects, 4th ed. (Siegel JG, Agranoff B, Albers RW, Molinoff PB, eds), pp 233–269. New York: Raven Press. Won MH, Kang T, Park S, Jeon G, Kim Y, Seo JH, Choi E, Chung M, Cho SS (2001) The alterations of N-methyl-D-aspartate receptor expressions and oxidative DNA damage in the CA1 area at the early time after ischemia-reperfusion insult. Neurosci Lett 301:139 –142. Yamashima T, Zhao L, Wang XD, Tsukada T, Tonchev AB (2001) Neuroprotective effects of pyridoxal phosphate and pyridoxal against ischemia in monkeys. Nutr Neurosci 4:389 –397.

(Accepted 26 June 2004) (Available online 8 September 2004)