The Attenuation of Kainate-Induced Neurotoxicity by Chlormethiazole and Its Enhancement by Dizocilpine, Muscimol, and Adenosine Receptor Agonists

EXPERIMENTAL NEUROLOGY ARTICLE NO.

148, 110–123 (1997)

EN976625

The Attenuation of Kainate-Induced Neurotoxicity by Chlormethiazole and Its Enhancement by Dizocilpine, Muscimol, and Adenosine Receptor Agonists D. G. MacGregor,* D. I. Graham,† and T. W. Stone* *Division of Neuroscience and Biomedical Systems, West Medical Building, and †Department of Neuropathology, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom

Systemically administered kainate (10 mg · kg21) caused neuronal loss in both the hippocampus and the entorhinal regions of the rat brain. This resulted in a loss of 68.3 6 13.8 and 53.3 6 12.8% of pyramidal neurones in the hippocampal CA1 and CA3a regions, respectively. Chlormethiazole attenuated the loss of neurones in the hippocampal cell layers CA1 (cell loss 10 6 3.2%) and CA3a (cell loss 10 6 7.7%). The neuroprotective activity of chlormethiazole was apparent in the presence or absence of a low dose of clonazepam (200 mg · kg21 ip). The kainate-induced damage could also be measured by the increase in binding of the peripheral benzodiazepine ligand ([3H]PK11195) in the hippocampus. In kainate-treated rats there was a 350–500% increase in binding indicative of reactive gliosis. Chlormethiazole prevented this elevation in a dose- and time-dependent manner, with an ED50 of 10.64 mg · kg21 and an effective therapeutic window from 1 to 4 h posttreatment. Dizocilpine also attenuated damage significantly. The GABAA agonist muscimol was also able to attenuate the increase in [3H]PK11195 binding in a dose-dependent manner, with an ED50 of approximately 0.1 mg · kg21. If muscimol, dizocilpine, or the adenosine A1 receptor agonist R-N 6-phenylisopropyladenosine were administered together with chlormethiazole at their respective ED25 doses, a potentiation was apparent in the degree of neuroprotection. It is concluded that the combination of neuroprotective agents with different mechanisms of action can lead to a synergistic protection against excitotoxicity. r 1997 Academic Press

INTRODUCTION

Kainic acid is a glutamate receptor agonist that causes neuronal loss following either central or peripheral administration (54, 64, 86, 95, 96). Although the penetration of kainate across the blood–brain barrier is poor, estimated to be less than 1% of a parenterally administered dose (16), it is sufficiently able to cross to cause widespread damage in a characteristic pattern 0014-4886/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

(54, 64, 86, 96). Indeed, its ability to cross the blood– brain barrier makes it a very useful tool in the study of excitotoxicity. Both kainate and the closely related compound domoic acid cause tonic–clonic seizures after systemic administration and produce a pattern of brain damage that is similar to that induced by repeated epileptic seizures (11, 13). This form of excitotoxicity may therefore be relevant to an understanding of the etiology and prevention of seizure-induced brain damage in humans. GABAergic neurones appear to be sensitive to kainate toxicity, in that levels of the GABA-synthesizing enzyme glutamic acid decarboxylase decrease after injection (54), and there is a loss of GABA-mediated ipsps (42, 73, 76). This and other evidence suggest that part of the kainate neurotoxic mechanism is the inhibition and loss of GABAergic transmission (12, 57, 58, 93, 94). A similar deficit may be involved in ischemiainduced neuronal damage: although the GABAergic neurones are relatively resistant to ischemia (72, 80, 100), there is a loss of GABAA receptors (5, 6). Conversely, partial restoration of inhibition by several types of GABAergic modulator has been reported to be neuroprotective (21, 56, 66, 67, 71, 80, 89, 98, 100). Chlormethiazole has been shown to potentiate [3H]muscimol, but not [3H]diazepam binding, although it also appears to have some barbiturate-like properties (36, 51, 52, 62, 78, 102). Unlike other GABA modulators, chlormethiazole can activate the GABAA receptor in the absence of GABA (50, 51, 78). These properties have resulted in chlormethiazole being tested for neuroprotection in several neurotoxic models, including focal ischemia (middle cerebral artery occlusion and the photochemical-induced thrombosis) and global ischemia (bilateral occlusion of the common carotid artery). In all the models tested, chlormethiazole could attenuate the amount of neuronal damage in a dose- and time-dependent manner (5, 33, 34, 48, 99). The present study was therefore undertaken to determine whether chlormethiazole could attenuate neurotoxicity induced by systemically administered kainate. We have also sought to determine whether any interac-

110

CHLORMETHIAZOLE NEUROPROTECTION

tion would occur between chlormethiazole and muscimol, the NMDA antagonist dizocilpine, or an agonist producing neuroprotection via activation of A1 receptors. METHODS

In all experiments 8- to 10-week-old male Wistar rats, 190–280 g, were used. Animals were either bred in house or purchased from Harlan Olac and kept under the same conditions, 12-h light/dark regime, with food and water available ad libitum. Animals were injected ip with drugs in a volume not normally exceeding 1 ml · kg21. Kainic acid and chlormethiazole were dissolved in saline. Animals were pretreated, 10 min prior to kainate or vehicle injections, with clonazepam (200 µg · kg21) ip, which was used as Rivotril for injection and was diluted with the commercial diluent provided. In all cases vehicles were used as control injections. The animals were allowed to recover and were killed 7 days later. P2 Preparation The method used was that for the preparation of a mitochondrial P2 membrane fraction as described by Eshleman and Murray (38) with slight modifications (68). On the 7th day of the experiment the animals were killed by stunning followed by cervical dislocation and decapitation. The skull was opened and the brain removed and placed in 30 ml of ice-cold 0.27 M sucrose, pH 7.4. The cerebellum was then dissected away and the brain bisected along the corpus callosum. The hippocampi were then removed and placed in 5 ml of ice-cold sucrose. The hippocampi were then homogenized with a Braun homogenizer, 10 3 500 rpm, and the volume was brought to 20 ml with three washes of the homogenizer vessel and pestle. This was then stored at 220°C for 2–4 h. After completing the preparation of tissue from a group of animals the samples were defrosted at room temperature and centrifuged for 10 min at 4°C, 1000g (IEC DPR 6000 centrifuge), the pellet was discarded, and the supernatant was centrifuged for 20 min at 4°C at 23,000g (Sorval RC 5B Refrigerated Superspeed Centrifuge, SS 34 Rotor). After this step the supernatant was discarded and the pellet was resuspended in ice-cold 50 mM Tris–HCl buffer, pH 7.8 (50 mM Tris salt, brought to pH 7.8 with 11 M HCl), 20 ml, and then centrifuged for 20 min at 4°C at 43,000g (Sorval RC 5B). The new pellet was resuspended in 5 ml Tris–HCl buffer and stored at 220°C. On the day of the assay the samples were defrosted at room temperature, brought to 20 ml with Tris–HCl buffer, and centrifuged for 20 min at 4°C at 43,000g, and the supernatant was discarded. The pellet was

111

then homogenized in 5 ml Tris–HCl buffer and then centrifuged at 7 3 1500 rpm, the homogenate was kept on ice, and the homogenizer chamber and pestle were washed thrice with 5 ml Tris–HCl buffer. The washes were pooled with the homogenate, this being the P2 membrane fraction, and stored on ice until needed. [3H]PK11195 Binding Assay All assays were performed at 4°C (on ice) and samples were incubated for 60 min. The assay was performed in duplicate. The volume of the assay chamber was 2 ml and contained 5 µl [3H]PK11195, 5 µl cold ligand, and 1490 µl P2 membranes. The volume was brought to 2 ml with Tris–HCl buffer. Final assay conditions were 1.75 nM [3H]PK11195 in ethanol (0.25% final), 0.25% DMSO 6 10 µM PK11195, and 100–150 µg protein. The assay samples were vortexed at the start of their incubation and approximately every 20 min before filtration. The incubation was terminated by vacuum filtration, with all of the sample being filtered through prewetted Whatman GF/C glass filters using a Millipore 12-well 1225 sampling manifold. Filters were washed twice with 12 ml ice-cold Tris–HCl buffer and vacuum dried, before being placed in scintillation vials. Ecoscint scintillation fluid (5 ml) was then added and then the samples were left overnight and counted using a Packard 2000 scintillation counter. Quenching and efficiency of counting were calculated as being 32% using external standards. Protein concentrations were measured using the Lowry method (65), following solubilization with 0.25 M NaOH and with bovine serum albumin as the standard. Tissue Fixation After 7 days animals to be used for histological analysis were given an overdose of sodium pentobarbitone (5 ml of 60 mg/ml) and perfusion fixed with 40% formaldehyde, glacial acetic acid, methanol in the ratio 1:1:8 (v/v/v) (FAM medium) (22). Briefly, animals were placed in the supine position and a thoracotomy was performed to introduce a cannula into the ascending aorta via the left ventricle. The animal was then heparinized (1000 IU · kg21). Physiological saline was infused into the animal from a gravity feed system for 5–10 s after incising the right atrium. This was followed immediately by 200 ml of FAM fixative at the same pressure and a flow rate of approximately 10 ml · min21. After infusion the rats were decapitated and the head stored in FAM at 4°C for at least 12 h. The cerebral hemispheres were cut into five coronal slices, each 2 mm thick. The brain stem was cut at right angles to its long axis into 2-mm slices and the cerebellum was cut into two slices perpendicular to the folia on

112

MACGREGOR, GRAHAM, AND STONE

the dorsal surface. Bilateral blocks of brain were embedded in paraffin wax and sectioned at 7–8 µm. Sections were stained by hematoxylin and eosin and a method combining cresyl violet and Luxol fast blue and then examined by light microscopy at 4003 magnification, at the level of the mid-dorsal hippocampus, by two observers (D.G.M. and T.W.S.) unaware of treatment received. The left hippocampus was scored on a scale of 0 to 10: zero damage, but disruption of the cell layer was scored as 1, while complete loss of the cell layer in the field of vision was scored as 10. The hippocampus was examined at five sites, corresponding to the CA1, CA2, CA3a, CA3b, and CA4 regions, as reported previously (43), the area selected for assessment being a full field of view at 4003 magnification. The CA1 and CA3 areas were examined at the apex of curvature of the cell layer; the CA4 region was examined at the level of the dentate hilus. Extrahippocampal regions were examined as the entorhinal cortex (interaural position 13.4 mm), the pyriform cortex (111.7 mm), and the amygdaloid complex at the level of the posterior cortical amygdaloid nuclei (13.4 mm). The same 0 to 10 scale was used as for the assessment of hippocampal damage. Temperature Measurements As the kainate/chlormethiazole-treated animals were sedated for approximately 30 min after treatment, their rectal temperatures were recorded to determine whether there was any change in the core body temperature. Data Analysis

campal [3H]PK11195 binding compared to control (260.45 6 28.45 (n 5 10) and 77.51 6 8.23 fmol · mg protein21 (n 5 10), respectively, P , 0.001). This elevation was attenuated in a dose-dependent manner by chlormethiazole with maximal suppression being seen at a dose of 50 mg · kg21 (binding of 56.18 6 9.66 fmol · mg protein21 (n 5 6)). From the dose range tested of 3.6 to 100 mg · kg21, the ED50 was estimated as approximately 10.64 mg · kg21 (Fig. 1). Chlormethiazole (50 mg · kg21) inhibited gliosis in a time-dependent manner. Maximal activity was achieved when chlormethiazole was injected simultaneously with kainate. Significant suppression was also afforded by the injection of chlormethiazole 1 h prior to, or 1, 2, or 4 hours following, the administration of kainate. There was no significant effect when administered 2 h before, or more than 4 h after, kainate (Fig. 2). Effect of Kainate and Chlormethiazole on Rectal Temperature In those cases where rectal temperature was monitored, there was a significant decrease in temperature at 30 min, compared with Time zero, in both the kainate and the kainate/chlormethiazole-treated animals (Students’ paired t test). However, at no time point was there any significant difference among the three groups of animals. Effect of Clonazepam on Chlormethiazole-Induced Suppression of Gliosis All experiments were performed in animals that had been pretreated with a low dose of the anticonvulsant

Specific binding was calculated by the normal method. Specific binding was also calculated as percentage of same day control to minimize day to day variations. All values are means 6 SEM. Statistical significance was assessed by analysis of variance followed by the Student–Newman–Keuls test for multiple comparisons. The analysis of histological damage was performed using a Mann–Whitney U test. In all cases significance was considered as P , 0.05. RESULTS

Effects of Chlormethiazole on the Kainate-Induced Elevation of [3H]PK11195 Binding Chlormethiazole administration resulted in sedation of the animal, the duration depending on the dose, but once the animals had recovered from this, those which had received a simultaneous injection of kainate displayed classical kainate-induced behavioral disturbances (wet dog shakes, forelimb elevation, Straub tail, salivation, head bobbing), without any clonic–tonic seizures. Kainate produced a significant increase in hippo-

FIG. 1. The effect of chlormethiazole on kainate-induced neurotoxicity in the hippocampus. The histogram shows the dose–response relationship between chlormethiazole and the degree of prevention of reactive gliosis in the hippocampus of kainate-treated animals. The ordinate indicates the amount of [3H]PK11195 binding to hippocampal P2 mitochondrial membranes as femtomoles per milligram of protein. Animals were injected (ip) with 0.2 mg · kg21 clonazepam 10 min prior to 10 mg · kg21 kainate and saline/chlormethiazole injections. Columns indicate means 6 SEM. V, vehicle (saline) injection; KA, kainate; CMZ, chlormethiazole. *P # 0.05, **P # 0.01, ***P # 0.001 versus vehicle control; 1P # 0.05, 11P # 0.01, 111P # 0.001 versus kainate control.

CHLORMETHIAZOLE NEUROPROTECTION

clonazepam (0.2 mg · kg21 ip) in order to eliminate the complication introduced into assessing kainate toxicity by the occurrence of seizures. Although this dose attenuated clonic–tonic seizures and reduced animal mortality from 15 to 3%, it had no effect on either the level of [3H]PK11195 binding (69) or the histologically assessed hippocampal damage (MacGregor et al., unpublished observations). However, in order to examine the possibility that clonazepam and chlormethiazole might act synergistically to protect against kainate-induced damage, some animals were given kainate and chlormethiazole at moderate doses without clonazepam pretreatment. In this experiment chlormethiazole at its estimated ED50 (10.64 mg · kg21 ip) reduced kainateinduced binding from 284.6 6 49.9 to 117.6 6 28.46 fmol · mg protein21, giving a mean protection of 66% against kainate (compared with a 33% reduction from 261 6 29.7 to 174 6 15.4 fmol · mg protein21) and indicating a lack of potentiation between chlormethiazole and clonazepam at these doses.

113

FIG. 3. The effect of 50 mg · kg21 chlormethiazole on kainateinduced hippocampal damage as assessed by histology. The histogram summarizes the distribution of kainate-induced damage within the hippocampus and the protection by chlormethiazole. Ordinate indicates percentage of cell loss within each area of assessment. Columns indicate means 6 SEM of the damage assessed in layers CA1, CA2, CA3a, CA3b, and CA4. Shaded columns, kainate, n 5 6; filled columns, chlormethiazole, n 5 5. *P # 0.05, **P # 0.01 versus kainate damage in same cell layer.

Histological Evidence of Chlormethiazole Neuroprotection As previously reported, kainate damaged both the CA1 and the CA3a regions of the hippocampus, with no significant difference between the two regions. The CA1 regions lost 68.3 6 13.8% of their pyramidal neurones, with a resulting loss of cell layer architecture and organization (see Figs. 3 and 4A), with a similar degree of damage being recorded in the CA3a region as well

(53.3 6 12.8%, see Fig. 3). Both the CA1 and the CA3a regions were significantly damaged compared to the CA2 and CA4 regions (8.3 6 4.0 and 10 6 6.4%, respectively). The mean damage in the CA3b region (15.0 6 5.0%) was significantly lower than that in the CA3a region (Figs. 3 and 4A). Chlormethiazole attenuated the kainate-induced damage in both the CA1 (see Fig. 4A) and the CA3a regions (see Fig. 4B) and to approximately the same extent (Figs. 3 and 4B). The CA1 area now showed only 10 6 3.2% neuronal loss, and the CA3a showed only 10 6 7.8% (P # 0.001 versus kainate-treated animals). Both the CA3b and the CA4 areas were completely spared of damage, and the CA2 only displayed cell layer disruption with little evidence of dying, pinkly stained cells (Fig. 4B). In extrahippocampal areas, protection was obtained in both the pyriform cortex and the amygdaloid nuclei, but not in the entorhinal cortex (Fig. 5). Effect of Muscimol on Kainate-Induced Elevation of [3H]PK11195

FIG. 2. The effect of time of administration of chlormethiazole on kainate-induced gliosis in the rat hippocampus. The histogram shows the time relationship between a single injection of 50 mg · kg21 chlormethiazole and the degree of prevention of ligand binding in the hippocampus of kainate-treated animals. The ordinate indicates the amount of [3H]PK11195 binding as femtomoles per milligram of protein. Animals were injected (ip) with 10 mg · kg21 kainate at Time zero. The hippocampal P2 membranes were prepared as discussed under Methods. Columns indicate means 6 SEM. V, vehicle (saline) injection; K, kainate; CMZ, chlormethiazole. *P # 0.05, **P # 0.01, ***P # 0.001 versus kainate control. 1P # 0.05, 11P # 0.01, 111P # 0.001 versus vehicle control.

In a dose range of 0.027–3 mg · kg21 muscimol was able to induce a dose-dependent reduction in the kainate-induced elevation in [3H]PK11195 binding (Fig. 6). At 0.1 mg · kg21 ip, muscimol produced approximately 50% protection against the kainate control (160.72 6 20.5 (n 5 6) and 253.13 6 40.37 (n 5 6) fmol · mg protein21, respectively, P , 0.01). This degree of damage was also significantly different from the vehicle control (57.26 6 9.83 fmol · mg protein21 (n 5 6), P , 0.01). Higher doses of muscimol produced a dose-dependent increase in the protection that become more significant

114

MACGREGOR, GRAHAM, AND STONE

against the kainate group, and nonsignificant against the vehicle control group, with maximal protection being achieved at 3.0 mg · kg21 ip (38.67 6 4.37 (n 5 5) fmol · mg protein21, P , 0.001 versus the kainate group). These findings resulted in an estimated ED25 and ED50 of 27 and 100 µg · kg21 ip, respectively. Combination of Chlormethiazole and Muscimol The previous experiments indicated that both muscimol and chlormethiazole were able to attenuate kainatemediated neurotoxicity in a dose-dependent manner. Harrison and Simmonds (51) reported that chlormethiazole was able to potentiate [3H]muscimol binding in vitro, and in the present experiment chlormethiazole and muscimol were coadministered at their estimated ED25 doses of 3.6 and 27 µg · kg21, respectively. Kainate (10 mg · kg21) caused a significant elevation in [3H]PK11195 binding compared to control (251.01 6 76.01 (n 5 5) and 38.89 6 6.19 (n 5 5) fmol · mg protein21, respectively, P , 0.001). Muscimol (27 µg · kg21) produced a 22% decrease in the mean degree of kainate-induced [3H]PK11195 binding values, while chlormethiazole (3.6 mg · kg21) resulted in a mean 33% decrease in the kainate effect, although neither of these changes was significantly different from the kainate group (Fig. 7). The coadministration of chlormethiazole and muscimol together resulted in a 73% decrease in the mean kainate induced effect (97.22 6 25.63 fmol · mg protein21, P , 0.01). This value was significantly different from the kainate group, but not from either of the single drug treatments, or from the amount of damage predicted to result from a simple additive effect of the two agents (Fig. 7). Effect of Chlormethiazole or Muscimol in Conjunction with Adenosine Receptor Ligands Previous studies have indicated that R-N 6-phenylisopropyladenosine (R-PIA) is able to attenuate kainatemediated elevation in [3H]PK11195 binding in a dosedependent manner (68), with an estimated ED25 and ED50 of 0.65 and 4.65 µg · kg21 ip, respectively. In one series of experiments R-PIA and chlormethiazole were both administered at their ED25 doses (0.65 and 3.6 mg · kg21, respectively) at T0 relative to kainate administration. Neither of these agents on their own produced significant neuroprotection (147.6 6 38.22 (n 5 6) and 168.93 6 20.62 (n 5 6) fmol · mg protein21, respectively), but when administered together there

was almost complete abolition of neurotoxicity (54.66 6 4.40 fmol · mg protein21 (n 5 6), P # 0.001 compared to kainate 191.25 6 13.18 fmol · mg protein21, n 5 5). This effect was also significantly different from the effect mediated by either chlormethiazole or R-PIA alone in the kainate-treated animals and from the effect predicted to result from a simple additive effect of the two agents (expected mean 125.28 fmol · mg protein21) (Fig. 8). A further experiment was performed with R-PIA and muscimol to determine whether a similar protection could also be seen with this GABAA agonist. Again ED25 doses were used (0.65 µg · kg21 R-PIA and 27 µg · kg21 muscimol ip). Neither of these single treatments produced significant protection (176.77 6 37.09 (n 5 6) and 171.96 6 51.21 (n 5 6) fmol · mg protein21, respectively). In this case the cotreatment resulted in a significant attenuation of the kainate effect (cotreatment 77.65 6 20.32 (n 5 6) and kainate alone 207.28 6 15.13 (n 5 5) fmol · mg protein21, respectively, P , 0.05). This combined treatment was also significantly different from either the kainate/R-PIA or the kainate/ muscimol treatments or from the predicted additive mean (141.45 fmol · mg protein21) (Fig. 9). A final experiment was performed to determine whether chlormethiazole was acting to produce neuroprotection indirectly by a release of adenosine which could then act upon A1 receptors. Animals were cotreated with chlormethiazole (35 mg · kg21, estimated ED75) and the selective adenosine A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), at doses that have previously been shown to block A1 receptors in the CNS (50 and 250 µg · kg21 ip; Ref. 69). DPCPX was unable to alter the neuroprotective effect of 35 mg · kg21 chlormethiazole at either of the doses used (Fig. 10). Effect of Dizocilpine and Combination with Chlormethiazole Dizocilpine was also able to attenuate the kainateinduced elevation with maximal protection seen at 3 mg · kg21 and a calculated ED50 of 0.12 and an ED25 of 0.056 mg · kg21. In a separate series of experiments kainate raised the level of [3H]PK11195 binding significantly from the control levels of 81.5 6 9.6 (n 5 4) to 358.0 6 33.5 fmol · mg protein21 (n 5 4). Administration of dizocilpine or chlormethiazole at their ED25 doses significantly attenuated the kainate-induced elevation. The simultaneous coadministration of dizocil-

FIG. 4. Kainate-induced neuronal loss in the rat hippocampus and protection by chlormethiazole. (A) Photomicrographs of hippocampal regions of a rat treated with 10 mg · kg21 kainic acid. a, CA1; b, CA2; c, CA3a; d, CA3b; e, CA4. There is severe disruption of the CA1 and CA3a cell layers with shrunken, darkly staining cells with condensed medium. Remaining layers within this animal show lesser amounts of damage. (B) Photomicrographs of hippocampal regions of a rat treated with 10 mg · kg21 kainic acid and 50 mg · kg21 chlormethiazole. a, CA1; b, CA2; c, CA3a; d, CA3b; e, CA4. There is very little evidence of damage in this animal, with all the layers showing an abundance of healthy cells with clear cytoplasm. Scale bars, 100 µm.

CHLORMETHIAZOLE NEUROPROTECTION

115

MACGREGOR, GRAHAM, AND STONE

Fig. 4–Continued

116

CHLORMETHIAZOLE NEUROPROTECTION

FIG. 5. The effect of 50 mg · kg21 chlormethiazole on kainateinduced extrahippocampal damage as assessed by histology. The histogram summarizes the extent of kainate-induced damage within the pyriform cortex (PC), entorhinal cortex (EC), and amygdala (AN) and the protection by chlormethiazole. The ordinate indicates percentage of damage within each area of assessment. Columns indicate means 6 SEM of the damage. Shaded columns, kainate, n 5 6; filled columns, chlormethiazole, n 5 5. *P # 0.05 versus kainate damage in the same region.

pine and chlormethiazole at these same doses, together with kainate, depressed binding further to 146.65 6 12.1 fmol · mg protein21 (n 5 6). This effect of the combined drug administration was also significantly greater than the predicted additive effect estimated as the sum of their individual activities (Fig. 11). DISCUSSION

The neuroprotective activity of chlormethiazole has not previously been studied using the systemic kainate model of excitotoxicity. The two methods that have been

FIG. 6. The histogram shows the dose–response relationship between muscimol and the degree of neuroprotection in kainateinduced neuronal damage. The ordinate indicates femtomoles of [3H]PK11195 bound per milligram of protein. Columns indicate means 6 SEM. V, vehicle (saline) injection. *P # 0.05, **P # 0.01, ***P # 0.001 versus vehicle control. 1P # 0.05, 11P # 0.01, 111P # 0.001 versus kainate control.

117

FIG. 7. Effect of muscimol on chlormethiazole attenuation of kainate-induced neurotoxicity in the hippocampus. The ordinate indicates femtomoles of [3H]PK11195 bound per milligram of protein. Columns indicate means 6 SEM. CMZ, chlormethiazole; p.a.e., predicted additive effect. *P # 0.05, **P # 0.01, ***P # 0.001 versus vehicle control. 11P # 0.01, 111P # 0.001 versus kainate control.

used to assess kainate neurotoxicity in this study are complementary. Neuronal damage is accompanied by a reactive gliosis, and the presence of binding sites for the peripheral benzodiazepine site ligands and their antagonists such as PK11195 allows the sensitivity of radioligand binding methods to be used in quantifying the gliosis. Indeed, [3H]PK11195 binding has now been used by several groups to quantify neurodegeneration after a variety of insults (3, 20, 14, 37, 84). Although it is difficult to compare results between the different studies due to variations in species, brain region, or the manner of expressing results, the present data seem to be broadly comparable with earlier work. Thus, the basal values of [3H]PK11195 binding found here are of the same order as binding in rat cortex (59) or dentate

FIG. 8. Synergistic neuroprotective effects of R-phenylisopropyladenosine (R-PIA) and chlormethiazole (CMZ) on kainate-induced neurotoxicity in the hippocampus. The ordinate indicates femtomoles of [3H]PK11195 bound per milligram of protein. Columns indicate means 6 SEM. CMZ, chlormethiazole; PIA, R-phenylisopropyladenosine; p.a.e., predicted additive protection. **P # 0.01, ***P , 0.001 versus vehicle control. 11P # 0.01, 111P # 0.001 versus kainate control. XXXP # 0.001 versus predicted additive neuroprotection (p.a.e.).

118

MACGREGOR, GRAHAM, AND STONE

FIG. 9. Neuroprotective effects of R-PIA and muscimol on kainate induced neurotoxicity in the hippocampus. The ordinate indicates femtomoles of [3H]PK11195 bound per milligram of protein. Columns indicate means 6 SEM. PIA, R-phenylisopropyladenosine; p.a.e., predicted additive protection. **P # 0.01 versus vehicle control. 1P # 0.05, 11P # 0.01 versus kainate control. XP # 0.05 versus predicted additive neuroprotection (p.a.e.).

gyrus (3). Similarly, the increase seen in response to kainate or ischemic damage is around two- to fivefold, comparable with the three- to fourfold changes seen here. Where studied specifically, an excellent correlation has been demonstrated between the distribution of PK11195 binding seen autoradiographically and the presence of neuronal damage visualized by conventional histology (37). Nevertheless, while the occurrence of damage in the present work has been confirmed directly using histology, it is always possible that the two techniques do not reflect neuronal damage per se to the same quantitative extent: any procedure which inhibits gliosis, for example, will suppress PK11195 binding quite independently of any neuronal damage or protection.

FIG. 10. Effect of DPCPX on the neuroprotective action of CMZ against kainate-induced neurotoxicity in the hippocampus. The ordinate indicates femtomoles of [3H]PK11195 bound per milligram of protein. Columns indicate means 6 SEM. CMZ, chlormethiazole; DPCPX, 1,3-dipropyl-8-cyclopentylxanthine. *P # 0.05 versus kainate control.

FIG. 11. Effect of dizocilpine on chlormethiazole’s attenuation of kainate-induced neurotoxicity in the hippocampus. The ordinate indicates binding of [3H]PK11195 (fentomoles per milligram of protein). Columns indicate mean 6 SEM. CMZ, chlormethiazole; p.a.e., predicted additive effect. *P # 0.05, ***P # 0.001 versus kainate alone. 111P # 0.001 versus vehicles control group. XXXP # 0.001 versus predicted additive effect (p.a.e.) of chlormethiazole plus dizocilpine.

Glutamate has been implicated in the mechanisms of excitotoxic damage. During ischemia there is a 1400– 3000% increase in the extracellular concentrations of glutamate in the brain (5, 15, 27) and antagonists at the NMDA and non-NMDA subtypes of glutamate receptor are able to attenuate brain damage (19, 24, 25, 44, 61, 87). Kainic acid provides a valuable tool with which to model some features of ischemic damage and of the damage induced by repeated epileptic seizures. It is effective after either central or peripheral administration (54, 64, 86, 95, 96) since it is sufficiently able to cross the blood–brain barrier to cause widespread damage in a characteristic pattern (54, 64, 86, 96) similar to that induced by repeated epileptic seizures (11, 13). Kainate-induced damage is believed to result partly from direct agonist activity at glutamate receptors and partly from the release of endogenous glutamate (28, 30, 40, 75, 81, 82). The present experiments were performed to determine whether chlormethiazole showed neuroprotective activity against kainate-induced excitoxicity. At present this drug has been shown to provide protection against ischemically induced brain damage at doses less than those which are used in the treatment of alcohol dependency (see Ref. 48 for review), and is effective when administered up to 6 h after an ischemic insult (5, 7, 33). When the drug was tested against intrahippocampally injected NMDA, a focal ischemic model, it was found to be less effective and have a shorter therapeutic window (33) than against ischemia. Chlormethiazole at 100 mg · kg21 would provide virtually complete protection against focal ischemia and in the present study it produced complete protection

CHLORMETHIAZOLE NEUROPROTECTION

against the effects of kainate. The 30 mg · kg21 dose gave approximately 75% protection, while the dose of 10 mg · kg21 was close to the estimated ED50 (10.64 mg · kg21). This ED50 value for chlormethiazole is lower than that reported by Cross and Green (personal communication) of 35 mg · kg21 in a focal ischemia model, suggesting that chlormethiazole may be more effective against some forms of neurotoxicity (e.g., epileptic or kainate-receptor-mediated brain damage) than others (e.g., ischemic-induced brain damage). The prevention by chlormethiazole of kainate-induced neurotoxicity or gliosis was not dependent on the routine treatment of the animals with clonazepam, nor did the low dose of clonazepam used here itself prevent excitotoxicity. Previous experience had indicated that the present dose of clonazepam reduced the incidence of tonic–clonic seizures almost to zero and similarly almost prevented animal fatalities due to the kainate injections. Clonazepam thus eliminated neuronal damage due to seizures and not directly attributable to kainate excitotoxicity. This dose of clonazepam did not, however, significantly modify kainate-induced hippocampal damage (69). Similarly there was no significant difference in the degree of protection afforded by chlormethiazole in the presence or absence of clonazepam, although the reduction in PK11195 binding from 284 to 117 fmol/mg protein by chlomethiazole alone was greater than that of chlormethiazole plus clonazepam. This might indicate a degree of occlusion between the two agents. It has been reported previously that kainate-induced neuronal damage did not parallel exactly the degree of seizure activity (54). Excitotoxic damage in the hippocampus seems to be particularly resistant to benzodiazepines (12, 53), probably reflecting the view that it is extrahippocampal damage by kainate which results from seizure activity, and Koh and Choi (60) reported that diazepam did not prevent glutamate toxicity in neuronal culture. There is some prior evidence, therefore, that kainate-induced seizures and toxicity can be separated. In cases where both have been suppressed apparently in parallel, experiments have usually employed diazepam at relatively high doses of 1–10 mg · kg21. Our use of low doses of the more centrally selective and specific anticonvulsant clonazepam appears to optimize this separation. There have been several reports that changes of body temperature can affect the degree of neuronal damage or protection occurring in the CNS after excitotoxic or ischemic insults (45, 46). In the present study, although kainate or kainate with chlormethiazole induced a small but significant decrease in core temperature after 30 min with respect to the zero time points, there was no significant difference between treated and control animals at any individual time point. It is unlikely,

119

therefore, that a change of core temperature could have significantly affected the results obtained. An important characteristic of the efficacy of chlormethiazole is that it is apparent even when the drug is administered up to 4 h after the injection of kainate. A similar, long-lasting window of efficacy has been reported by other groups using chlormethiazole against ischemically induced brain damage (33, 88). It is unlikely that this could be explained on pharmacokinetic grounds, as chlormethiazole has a half-life of approximately 40 min (48). Other protective agents such as dizocilpine, for example, are protective for several hours after a cerebral insult, and it has been suggested that this results from the blockade of receptors for glutamate which would normally induce depolarization and thus stimulate further release of glutamate in a protracted positive feedback cycle. Kainate toxicity is likely to involve the release of neuronal or glial glutamate: kainate causes the release of radiolabeled and endogenous excitatory amino acids from intact neurones or synaptosomes (30, 40, 81), and this may contribute substantially to its excitotoxic properties (75). It is known that chlormethiazole does not interfere directly with the N-methyl-Daspartate (NMDA)-sensitive population of glutamate receptor (35), but it has been shown to reduce the ischemia-induced extracellular accumulation of glutamate (7). It is possible therefore that chlormethiazole breaks the cycle of release/depolarization/release by suppressing presynaptic glutamate release. Chlormethiazole is known to potentiate the action of GABA and prolong the opening of the GABAA receptorassociated ion channel (36, 51, 78). The present results clearly indicate that the direct activation of GABAA receptors by muscimol is sufficient to provide substantial protection against kainate. This demonstration is in line with reports that activation of GABA receptors can also protect against ischemically induced damage (21, 67, 68, 90, 98) and would be consistent with chlormethiazole acting purely as a GABAA receptor agonist. The combination of chlormethiazole with muscimol produced a degree of neuroprotection which was not significantly different from that produced by the individual agents, nor from the predicted additive effect of the combination, but was different from the kainate group. This suggests that the two agents are acting via a similar mechanism and provides strong support for the view that the activation of GABA receptors or their associated ion channels are importantly involved in the mechanism of neuroprotection by chlormethiazole. Like GABA, the endogenous purine adenosine is able to cause hyperpolarization and inhibition of glutamate release (26, 32, 39, 63, 74, 79, 83, 91, 92, 101). A high proportion of A1 receptors is believed to be located on the terminals of excitatory neurones, including those

120

MACGREGOR, GRAHAM, AND STONE

releasing acetylcholine and glutamate (39, 47, 97). The activation of adenosine receptors has been found to induce neuroprotection, mediated by A1 receptors (4, 17, 18, 31, 68, 69, 77, 85). Since kainate causes the release of excitatory amino acids from neurones (30, 40, 81), this action may contribute substantially to its excitotoxic properties (75). It is therefore possible that part of the neuroprotective activity of adenosine results from a suppression of kainate-induced glutamate release. Consistent with this, Heron et al. (55) have reported that ip R-PIA (20 µg · kg21 given 30 min before and 10 µg · kg21 30 min after) significantly decreased glutamate release following a 20-min period of ischemia. Assuming the even distribution of R-PIA around the body, the ED25 dose used here of 0.65 µg · kg21 would yield a mean tissue concentration of approximately 1.6 nM. This is close to the Kd value for the A1 receptor obtained using ex vivo binding (1.5 6 0.2 nM; Ref. 10) and would be entirely consistent with an action of R-PIA which involved the selective activation of these sites. This would in turn be consistent with an inhibition of transmitter release via A1 receptors. The data of Baumgold et al. (10) also confirm that R-PIA is able to cross the blood–brain barrier. The results indicate that R-PIA and chlormethiazole exert a potentiative effect against kainate, yielding a greater degree of protection than either agent alone or their predicted additive effect. The mechanism of this interaction cannot be determined from the present results, but the finding is consistent with the view that R-PIA and chlormethiazole are acting by independent mechanisms. Since chlormethiazole may work partly through GABAA receptors, it is relevant that there have recently been two reports of synergism between GABA and adenosine receptors in the brain. In the report by Fern et al. (41) the GABAB and adenosine A1/A2 receptors were able to act synergistically to reduce an in vitro hypoxic insult to an isolated optic nerve. This interaction appeared to be at the level of protein kinase C and may involve the downregulation of the Na1/Ca21 exchanger. In the second report, by Akhondzadeh and Stone (2), GABA and adenosine were able to inhibit population spike size in the in vitro hippocampal slice preparation. When present together at submaximal levels these two neuroactive agents were able to act synergistically to reduce population spike size, a result believed to be due to GABAA and adenosine A1 receptors interacting at the level of chloride and potassium ion channels. DPCPX is a highly selective A1 antagonist (23) which has low nanomolar affinity for the receptor. A recent report by Baumgold et al. (10) indicates that the xanthine can cross the blood–brain barrier readily. A parenteral dose of 250 µg · kg21 DPCPX would, according to Baumgold et al. (10), yield an intracranial

concentration of 0.34 µM and should completely block all A1 receptors. At this dose, as well as at the lower dose of 50 µg · kg21, the present results demonstrate a failure to modify the neuroprotective activity of chlormethiazole. This indicates that the protective activity against kainate is not mediated indirectly by a release of endogenous adenosine. This result also indicates that, despite the potentiative interaction between chlormethiazole and R-PIA, the former is not inducing protection by potentiating the activation of A1 receptors (or probably A2 receptors) by endogenous adenosine present at basal levels or the higher levels resulting from the actions of kainate. Receptors for NMDA may be involved in the damage induced by systemic kainate, since Wolfe et al. (103) and Guarnieri et al. (49) found that magnesium sulfate inhibited the damage induced by systemically administered kainate, while several groups have reported that the noncompetitive NMDA receptor channel blocker dizocilpine was also able to attenuate the kainate effect (8, 29, 49). Both of these agents bind to the open state of the NMDA receptor. In the present study we report that dizocilpine is extremely effective as a neuroprotectant against damage induced by kainic acid, with an ED50 of 0.12 mg · kg21. This finding supports the view that kainate induced neurotoxicity involves the overstimulation of NMDA receptors. Furthermore, the coadministration of chlormethiazole and dizocilpine induced an increase in neuroprotection. The protection achieved was greater than the predicted sum of the individual agents, indicating that a degree of synergism was involved. This synergism is consistent with the view that chlormethiazole does not interact directly with either kainate or NMDA-mediated responses in vivo (1) nor does it alter dizocilpine binding in vitro (35), indicating that chlormethiazole is probably acting by mechanisms that are independent of NMDA receptors. It is interesting to note that the degree of potentiation between the ED25 doses of chlormethiazole and dizocilpine (combined protection of 78.7 6 3.9%) was less than that reported previously with the combined administration of chlormethiazole and the adenosine A1 agonist R-PIA (70) (combined protection 5 94.8 6 3.1%, P # 0.01). The chlormethiazole/R-PIA effect may be due to the synergism between the adenosine A1 and GABAA receptors in the hippocampus (2). The report by Bartrup and Stone (9) of the dependence of adenosine A1 receptor activation on NMDA receptor inactivation may explain the chlormethiazole/dizocilpine response, in that there may be a small but significant enhancement of adenosine-mediated inhibition in this model. ACKNOWLEDGMENTS The project was supported in part by Astra Arcus, Sweden. The authors are grateful to Mrs. P. Davidson and the late Mr. J.

CHLORMETHIAZOLE NEUROPROTECTION Thompson for invaluable technical assistance and Dr. W. L. Maxwell for help with the brain perfusions.

16.

REFERENCES 17. 1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Addae, J. I., and T. W. Stone. 1983. Effects of anticonvulsants on responses to excitatory amino acids applied topically to rat cerebral cortex. Gen. Pharmacol. 19: 455–462. Akhondzadeh, S., and T. W. Stone. 1994. Interaction between adenosine and GABA receptors on hippocampal neurones. Brain Res. 665: 229–236. Altar, C. A., and M. Baudry. 1990. Systemic injection of kainic acid: Gliosis in olfactory and limbic brain regions quantified with [3H]PK11195 binding autoradiography. Exp. Neurol. 109: 333–341. Arvin, B., L. F. Neville, J. Pan, and P. J. Roberts. 1989. 2-Chloroadenosine attenuates kainic acid-induced toxicity within the rat striatum: relationship to release of glutamate and Ca21 influx. Br. J. Pharmacol. 98: 225–235. Baldwin, H. A., J. A. Jones, A. J. Cross, and A. R. Green. 1993. Histological, biochemical and behavioural evidence for the neuroprotective action of chlormethiazole following prolonged carotid artery occlusion. Neurodegeneration 2: 139–146. Baldwin, H. A., M. F. Snape, J. L. Williams, A. Misra, J. A. Jones, M. Snares, A. J. Cross, and A. R. Green. 1993. The role of glutamate and GABA in a rat model of focal cerebral ischaemia: biochemical and pharmacological investigations. Neurodegeneration 2: 129–138. Baldwin, H. A., J. L. Williams, M. Snares, T. Ferreira, A. J. Cross, and A. R. Green. 1994. Attenuation by chlormethiazole administration of the rise in extracellular amino acids following focal ischaemia in the cerebral cortex of the rat. Br. J. Pharmacol. 112: 188–194. Baran, H., W. Loscher, and M. Mevissen. 1994. The glycine/ NMDA receptor partial agonist D-cycloserine blocks kainateinduced seizures in rats: Comparison with MK801 and diazepam. Brain Res. 652: 195–200. Bartrup, J. T., and T. W. Stone. 1990. Activation of NMDA receptor-coupled channels suppresses the inhibitory action of adenosine on hippocampal slices. Brain Res. 530: 330–334. Baumgold, J., O. Nikodijevic, and K. A. Jacobson. 1992. Penetration of adenosine antagonists into mouse brain as determined by ex vivo binding. Biochem. Pharmacol. 43: 889–894. Ben-Ari, Y. 1985. Limbic seizure and brain damage produced by kainic acid: Mechanism and relevence to human temporal lobe epilepsy. Neuroscience 14: 373–403. Ben-Ari, Y., E. Tremblay, O. P. Ottersen, and R. Naquet. 1979. Evidence suggesting secondary epileptogenic lesions after kainic acid: pretreatment with diazepam reduces distant but not local brain damage. Brain Res. 165: 362–365. Ben-Ari, Y., E. Tremblay, D. Richie, G. Ghilni, and R. Naquet. 1981. Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline and pentetrazole: Metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy. Neuroscience 6: 1361–1391. Benavides, J., D. Fage, C. Carter, and B. Scatton. 1987. Peripheral type benzodiazepine binding sites are a sensitive indirect index of neuronal damage. Brain Res. 421: 167–172. Benveniste, H., J. Drejer, A. Schousboe, and N. H. Deimer. 1984. Elevation of extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischaemia monitored by intracerebral microdialysis. J. Neurochem. 43: 1369–1374.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

121

Berger, M. L., J. M. Lefauconnier, E. Tremblay, and Y. Ben-Ari. 1986. Limbic seizures induced by systemically applied kainic acid: how much kainic acid reaches the brain? In Excitatory Amino Acids and Epilepsy (E. Schwarcz and Y. Ben-Ari, Eds.), pp. 199–209. Plenum, New York. Bielenberg, G. W. 1989. R-PIA protects cortical tissue in permanent MCA occlusion in the rat. J. Cereb. Blood Flow Metab. 9: 645. Block, G. A., and W. A. Pulsinelli. 1987. The adenosine agonist R-phenylisopropyl-adenosine attenuates ischemic neuronal damage. J. Cereb. Blood Flow Metab. 7: S258. Boast, C. A., S. C. Gerhardt, G. Pastor, J. Lehmann, P. E. Etienne, and J. M. Liebman. 1988. The N-methyl-D-aspartate antagonists CGS-19755 and CPP reduce ischemic brain damage in gerbils. Brain Res. 442: 345–348. Bourdiol, F., S. Toulmond, A. Serrano, J. Benavides, and B. Scatton. 1991. Increase in v 3 (peripheral type benzodiazepine) binding sites in the rat cortex and striatum after local injection of interleukin-1, tumour necrosis factor-a and lipopolysaccharide. Brain Res. 543: 194–200. Brailowsky, S., R. B. Knight, K. Blood, and D. Scabini. 1986. g-Aminobutyric acid-induced potentiation of cortical hemiplegia. Brain Res. 362: 322–330. Brown, A. W., and J. B. Brierley. 1972. Anoxic–ischaemic cell change in rat brain: Light microscopic and fine structural observations. J. Neurol. Sci. 16: 59–84. Bruns, R. F., J. H. Fergus, E. W. Badger, J. A. Bristol, L. A. Santay, J. D. Hartman, S. J. Hays, and C. C. Huang. 1987. Binding of the A1-selective adenosine antagonist 8-cyclopentyl1,3-dipropylxanthine to rat brain membranes. Arch. Pharmacol. 335: 59–63. Buchan, A. M., H. Li, S. Cho, and W. A. Pulsinelli. 1991. Blockade of the AMPA receptor prevents CA1 hippocampal injury following severe but transient forebrain ischemia in adult rats. Neurosci. Lett. 132: 255–258. Bullock, R., J. McCulloch, D. I. Graham, D. Lowe, M. H. Chen, and G. M. Teasdale. 1990. Focal ischemic damage is reduced by CPP-ene. Studies in two animal models. Stroke 21(Suppl. III)III-32–III-36. Burke, S. P., and J. V. Nadler. 1988. Regulation of glutamate and aspartate release from slices of hippocampal CA1 area: Effects of adenosine and baclofen. J. Neurochem. 51: 1541–1551. Butcher, S. P., R. Bullock, D. I. Graham, and J. McCulloch. 1990. Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion. Stroke 21: 1727–1733. Butcher, S. P., J. Lazarewicz, and A. Hamberger. 1987. In vivo microdialysis studies on the effects of decortication and excitotoxic lesions on kainic acid induced Ca21 fluxes, and endogenous amino acid release in the rat striatum. J. Neurochem. 49: 1355–1360. Clifford, D. B., J. W. Olney, A. M. Benz, T. A. Fuller, and C. F. Zorumski. 1990. Ketamine, phencyclidine and MK-801 protect against kainic acid-induced seizure-related brain damage. Epilepsia 31: 382–390. Connick, J. H., and T. W. Stone. 1986. The effect of kainic, quinolinic and b-kainic acids on the release of endogenous amino acids from rat brain slices. Biochem. Pharmacol. 35: 3631–3635. Connick, J. H., and T. W. Stone. 1989. Quinolinic acid neurotoxicity, protection by intracerebral PIA and potentiation by hypotension. Neurosci. Lett. 101: 191–196. Corradetti, R., G. Lo Conte, F. Moroni, B. Passani, and G. Pepeu. 1984. Adenosine decrease aspartate and glutamate

122

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48. 49.

MACGREGOR, GRAHAM, AND STONE release from rat hippocampal slices. Eur. J. Pharmacol. 104: 19–26. Cross, A. J., J. A. Jones, H. A. Baldwin, and A. R. Green. 1991. Neuroprotective activity of chloromethiazole following transient forebrain ischaemia in the gerbil. Br. J. Pharmacol. 104: 406–411. Cross, A. J., J. A. Jones, M. Snares, K.-G. Jostell, U. Bredberg, and A. R. Green. 1995. The protective action of chlormethiazole against ischaemia-induced neurodegeneration in gerbils when infused at doses having little sedative or anticonvulsant activity. Br. J. Pharmacol. 114: 1625–1630. Cross, A. J., M. F. Snape, and A. R. Green. 1993. Chlormethiazole antagonises seizures induced by N-methyl-D-aspartate without interacting with the NMDA receptor complex. Psychopharmacology 112: 403–406. Cross, A. J., J. M. Stirling, T. N. Robinson, D. M. Bowen, P. T. Francis, and A. R. Green. 1989. The modulation by chlormethiazole of the GABAA–receptor complex in rat brain. Br. J. Pharmacol. 98: 284–290. Dubois, A., J. Benavides, B. Peny, D. Duverger, D. Fage, B. Gotti, E. T. Mackenzie, and B. Scatton. 1988. Imaging of primary and remote excitotoxic brain lesions: An autoradiographic study of peripheral type benzodiazepine binding sites in the rat and cat. Brain Res. 445: 77–90. Eshleman, A. J., and T. F. Murray. 1989. Differential binding properties of the peripheral benzodiazepine ligands [3H]PK11195 and [3H]Ro 5-4864 in trout and mouse brain membranes. J. Neurosci. 53: 494–502. Fastbom, J., and B. B. Fredholm. 1985. Inhibition of [3H]glutamate from rat hippocampal slices by L-PIA. Acta Physiol. Scand. 125: 121–123. Ferkany, J. W., R. Zaczek, and J. T. Coyle. 1982. Kainic acid stimulates excitatory amino acid neurotransmitter release at presynaptic receptors. Nature 298: 757–759. Fern, R., S. G. Waxman, and B. R. Ransom. 1994. Modulation of anoxic injury in CNS white matter by adenosine and interaction between adenosine and GABA. J. Neurophysiol. 72: 2609–2616. Fisher, R. S., and B. E. Alger. 1984. Electrophysiological mechanisms of kainic acid-induced epileptiform activity in the rat hippocampal slice. J. Neurosci. 1312–1322. Franck, J. E., and P. A. Schwartzkroin. 1984. Immature rabbit hippocampus is damaged by systemic but not intraventricular kainic acid. Dev. Brain Res. 13: 219–227. Gill, R., A. C. Foster, and G. N. Woodruff. 1987. Systemic administration of MK-801 protects against ischemia induced hippocampal neurodegeneration in the gerbil. J. Neurosci. 7: 3343–3349. Ginsberg, M. D., MY. T. Globus, W. D. Dietrich, and R. Busto. 1993. Temperature modulation of ischemic brain injury—A synthesis of recent advances. Prog. Brain Res. 96: 13–22. Ginsberg, M. D., L. L. Sternau, M. Y. T. Globus, W. D. Dietrich, and R. Busto. 1992. Therapeutic modulation of brain temperature—Relevance to ischemic brain injury. Cereb. Brain Metab. Rev. 4: 189–225. Goodman, R. R., and S. H. Snyder. 1982. Autoradiographic localisation of adenosine receptors using [3H]cyclohexyladenosine. J. Neurosci. 2: 1230–1241. Green, A. R., and A. J. Cross. 1994. The neuroprotective actions of chlormethiazole. Prog. Neurobiol. 44: 463–484. Guarnieri, T., M. Virgili, L. Villani, F. Facchinetti, A. Contestabile, and P. Migani. 1993. Pharmacological manipulation of the NMDA receptor differentially protects from systemic kainic acid: Evaluation through ornithime decarboxylase induction,

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

67.

morphology and GFAP immunocytochemistry. Restor. Neurol. Neurosci. 5: 327–335. Hales, T. G., and J. J. Lambert. 1992. Modulation of GABAA and glycine receptors by chlormethiazole. Eur. J. Pharmacol. 210: 239–246. Harrison, N. L., and M. A. Simmonds. 1983. Two distinct interactions of barbiturates and chlormethiazole with the GABA receptor channel complex in rat cuneate nucleus in vitro. Br. J. Pharmacol. 80: 387–394. Hedlund, B., and S. O. Ogren. 1987. Chlormethiazole acts on chloride channels in cultured neurons. Neurosci. Lett. 78: 217–221. Heggli, D. A., and D. Malthe-Sorenssen. 1982. Systemic injection of kainic acid: Effect on neurotransmitter markers in pyriform cortex, amygdaloid complex and hippocampus and protection by cortical lesioning and anticovulsants. Neuroscience 7: 1257–1264. Heggli, D. A., A. Aamodt, and D. Malthe-Sorenssen. 1981. Kainic acid neurotoxicity. Effect of systemic injection on neurotransmitter markers in different brain regions. Brain Res. 230: 253–262. Heron, A., T. Lasbennes, and J. Seylaz. 1992. Effects of two different routes of administration of R-PIA on glutamate release during ischaemia. Neurosci. Lett. 147: 205–208. Johansen, F. F., and N. H. Diemer. 1991. Enhancement of GABA neurotransmission after cerebral ischemia in the rat reduces loss of hippocampal CA1 pyramidal cells. Acta Neurol. Scand. 84: 1–6. Kapur, J., and E. W. Lothman. 1989. Loss of inhibition proceeds delayed spontaneous seizures in the hippocampus after tetanic electrical stimulation. J. Neurophysiol. 61: 427–434. Kapur, J., J. L. Stringer, and E. W. Lothman. 1989. Evidence that repetitive seizures in the hippocampus causes lasting reduction of GABAergic inhibition. J. Neurophysiol. 61: 417–426. Kenny, B. A., A. C. Mackinnon, M. Spedding, and C. M. Brown. 1993. Changes in [3H]PK11195 and [3H]8-OH-DPAT binding following forebrain ischaemia in the gerbil. Br. J. Pharmacol. 109: 437–442. Koh, J.-J., and D. W. Choi. 1987. Effect of anticonvulsant drugs on glutamate neurotoxicity in cortical cell culture. Neurology 37: 319–322. Le Peillet, E., B. Arvin, C. Moncada, and B. S. Meldrum. 1992. The non-NMDA antagonists, NBQX and GYKI 52466, protect against cortical and striatal cell loss following transient global ischaemia in the rat. Brain Res. 571: 115–120. Leeb-Lundberg, F., A. Snowman, and R. W. Olsen. 1981. Interaction of anticonvulsants with the barbiturate–benzodiazepine– GABA receptor complex. Eur. J. Pharmacol. 72: 125–129. Lloyd, H. G. E., and G. Cleva. 1994. Enhancement of D[3H]aspartate release from rat hippocampal slices during combined hypoxia and glucose deprivation by the adenosine antagonist 1,3-dipropyl-8-p-sulfophenylxanthine. Drug Dev. Res. 34: 291. Lothman, E. W., and R. C. Collins. 1981. Kainic acid induced limbic seizures: metabolic, behavioural, electroencephalographic and neuropathological correlates. Brain Res. 218: 299–318. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. A. Randall. 1951. Protein measurements with the folin phenol reagent. J. Biol. Chem. 193: 265–275. Lyden, P. D., and L. Lonzo. 1994. Combination therapy protects ischemic brain in rats A glutamate antagonist plus a gaminobutyric acid agonist. Stroke 25: 189–196. Lyden, P. D., and B. Hedges. 1992. Protective effect of synaptic

CHLORMETHIAZOLE NEUROPROTECTION

68.

69.

70.

71. 72.

73.

74. 75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

inhibition during cerebral ischemia in rats and rabbits. Stroke 23: 1463–1470. MacGregor, D. G., and T. W. Stone. 1993. Inhibition by the adenosine analogue (R-)N6-phenylisopropyladenosine, of kainic acid neurotoxicity in rat hippocampus after systemic administration. Br. J. Pharmacol. 109: 316–321. MacGregor, D. G., W. J. Miller, and T. W. Stone. 1993. The neuroprotective action of (R-)-N6-phenylisopropyladenosine is mediated through a centrally located adenosine A1 receptor. Br. J. Pharmacol. 110: 470–476. MacGregor, D. G., H. V. Ogilvy, and T. W. Stone. 1994. Systemically administered chlormethiazole attenuates kainate neurotoxicity. Br. J. Pharmacol. 113: 55P. Madden, K. P. 1994. Effect of g-aminobutyric acid modulation of neuronal ischaemia in rabbits. Stroke 25: 2271–2275. Matsumoto, K., S. Ueda, T. Hashimoto, and K. Kuriyama. 1991. Ischemic neuronal injury in the rat hippocampus following transient forebrain ischemia: Evaluation using in vivo microdialysis. Brain Res. 543: 236–242. McCarren, M., and B. E. Alger. 1985. Use-dependent depression of IPSPs in the rat hippocampal pyramidal cells in vitro. J. Neurophysiol. 53: 557–571. McCormick, D. A. 1989. GABA as an inhibitory neurotransmitter in human cerebral cortex. J. Neurophysiol. 62: 1018–1027. McGeer, E. G., P. L. McGeer, and K. Singh. 1978. Kainate induced degeneration of neostriatal neurones: dependency upon Ca21. Brain Res. 139: 381–383. Milgram, N. W., T. Yearwood, M. Khurgel, G. O. Ivy, and R. Racine. 1991. Changes in inhibitory processes in the hippocampus following recurrent seizures induced by systemic administration of kainic acid. Brain Res. 551: 236–246. Miller, L. P., and C. Hsu. 1992. Therapeutic potential for adenosine receptor activation in ischemic brain injury. J. Neurotrauma 9(Suppl. 2): S563–S577. Moody, E. J., and P. Skolnick. 1989. Chlormethiazole: Neurochemical actions at the gamma-aminobutyric acid receptor complex. Eur. J. Pharmacol. 164: 153–158. Morrisett, R. A., D. D. Mott, D. V. Lewis, H. S. Swartzwelder, and W. A. Wilson. 1991. GABAb-receptor-mediated inhibition of the N-methyl-D-aspartate component of synaptic transmission in the rat hippocampus. J. Neurosci. 11: 203–209. Nitsch, C., G. Goping, and I. Klatzo. 1991. Preservation of GABAergic perikarya and boutons after transient ischemia in the gerbil hippocampal CA1 field. Brain Res. 495: 243–252. Notman, H., R. Whitney, and K. Jhamandas. 1984. Kainic acid evoked release of D-[3H]aspartate from rat striatum in vitro; characterization and pharmacological modulation. Can. J. Physiol. Pharmacol. 62: 1070–1077. Palmer, A. M., C. T. Reiter, and M. Botscheller. 1992. Comparison of the release of exogenous and endogenous excitatory amino acids from rat cerebral cortex. Ann. N.Y. Acad. Sci. 648: 361–364. Phillis, J. W., G. A. Walters, and R. E. Simpson. 1991. Release of purines and neurotransmitter amino acids into cerebral cortical perfusates during and following transient ischemia. Int. J. Purine Pyrimidine Res. 2: 41–48. Price, G. W., R. G. Ahier, S. P. Hume, R. Myers, L. Manjil, J. E. Creamer, S. K. Luthra, C. Pascali, V. Pike, and R. S. J. Frackowiak. 1990. In vivo binding to peripheral benzodiazepine binding sites in lesioned rat brain: comparison between [3H]PK11195 and [18F]PK14105 as markers for neuronal damage. J. Neurochem. 55: 175–185. Rudolphi, K. A., P. Schubert, F. E. Parkinson, and B. B. Fredholm. 1992. Neuroprotective role of adenosine in cerebral ischaemia. Trends Pharmacol. Sci. 13: 439–445.

123

86.

Schwob, J. E., T. Fuller, J. L. Price, and J. W. Olney. 1980. Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: A histological study. Neuroscience 5: 991–1014.

87.

Sheardown, M. J., E. O. Nielsen, A. J. Hansen, P. Jacobsen, and T. Honore. 1990. 2,3-Dihydoxy-6-nitro-7-sulfamoyl-benzo( f )quinoxaline: A neuroprotectant for cerebral ischemia. Science 247: 571–574.

88.

Shuaib, A., and S. Ijaz. 1994. Chlormethiazole protects the brain from transient forebrain ischemia when used up to four hours after the insult. Ann. Neurol. 36: 306. Shuaib, A., S. Ijaz, S. Hassan, and J. Kalra. 1992. Gammavinyl GABA prevents hippocampal and substantia nigra reticulata damage in repetitive transient forebrain ischemia. Brain Res. 590: 13–17. Shuaib, A., R. Mazagri, and S. Ijaz. 1993. GABA agonist muscimol is neuroprotective in repetitive transient forebrain ischemia in gerbils. Exp. Neurol. 123: 284–288. Simpson, R. E., M. H. O’Regan, L. M. Perkins, and J. W. Phillis. 1992. Excitatory transmitter amino acid release from the ischemic rat cerebral cortex: effects of adenosine receptor agonists and antagonists. J. Neurochem. 58: 1683–1680. Sivilotti, L., and A. Nistri. 1991. GABA receptor mechanisms in the central nervous system. Prog. Neurosci. 36: 35–92. Sloviter, R. S. 1987. Decreased hippocampal inhibition and a selective loss of interneurons in experimental epilepsy. Science 235: 73–76. Sloviter, R. S., and B. P. Damiano. 1981. Duplication of kainic acid-induced electrophysiological effects and hippocampal damage by electrical stimulation of perforant path. Fed. Proc. 40: 309. Sperk, G. 1994. Kainic acid seizures in the rat. Prog. Brain Res. 42: 1–32. Sperk, G., H. Lassman, H. Baran, F. Seitelberger, and O. Hornykiewicz. 1985. Kainic acid-induced seizures, doserelationship of behavioural, neurochemical and histopathological changes. Brain Res. 338: 289–295. Spignoli, G., F. Pedata, and G. Pepeu. 1984. A1 and A2 receptors modulate acetylcholine release from brain slices. Eur. J. Pharmacol. 97: 341–342. Sternau, L. L., D. Lust, A. J. Ricci, and R. Ratcheson. 1989. Role g-aminobutyric acid in selective vulnerability in gerbils. Stroke 20: 281–287. Sydserff, S. G., A. J. Cross, K. J. West, and A. R. Green. 1995. The effect of chlormethiazole on neuronal damage in a model of transient focal ischaemia. Br. J. Pharmacol. 114: 1631–1635.

89.

90.

91.

92. 93.

94.

95. 96.

97.

98.

99.

100.

Tecoma, E. S., and D. W. Choi. 1989. GABAergic neocortical neurons are resistant to NMDA receptor-mediated injury. Neurology 39: 676–682. 101. Thompson, S. M., H. L. Haas, and B. H. Gahwiler. 1992. Comparison of the action of adenosine at pre- and postsynaptic receptors in the rat hippocampus in vitro. J. Physiol. 451: 347–363. 102.

Vincens, M., A. Enjalabert, K. G. Lloyd, K. G. Paillard, J. J. Thuret, F. C. Kordon, and P. Lechat. 1989. Evidence that chlormethiazole interacts with the macromolecular GABAA– receptor complex in the central nervous system and in the anterior pituitary gland. Naunyn-Schmiedbergs Arch. Pharmacol. 339: 397–402.

103.

Wolfe, G., S. Fischer, P. Hass, K. Abicht, and G. Keilhoff. 1991. Magnesium sulphate subcutaneously injected protects against kainate-induced convulsions and neurodegenerations—In vivo study on the rat hippocampus. Neuroscience 43: 31–34.