Copyright © 2003 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2003, 55, 741–746 ISSN 1230-6002 DEFICIT OF ENDOGENOUS KYNURENIC ACID IN THE FRONTAL CORTEX OF RATS WITH A GENETIC FORM OF ABSENCE EPILEPSY Rafa³ M. Kamiñski1,*, El¿bieta Zieliñska1, Andrzej Dekundy1,†, Gilles van Luijtelaar2, Waldemar A. Turski1,3,# Department of Toxicology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland, Department of Biological Psychology, Nijmegen Institute for Cognition and Information, University of Nijmegen, P.O. Box 9104, 6500 HE Nijmegen, The Netherlands, !Department of Pharmacology and Toxicology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland *Present address: Epilepsy Research Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892, USA; VPresent address: Department of Pharmacology, Merz Pharmaceuticals, Frankfurt am Main, D-60318, Germany  Deficit of endogenous kynurenic acid in the frontal cortex of rats with a genetic form of absence epilepsy. R.M. KAMIÑSKI, E. ZIELIÑSKA, A. DEKUNDY, G. VAN LUIJTELAAR, W.A. TURSKI. Pol. J. Pharmacol., 2003, 55, 741–746. The present studies sought to determine the concentrations of endogenous kynurenic acid (KYNA) and to measure the activity of kynurenine aminotransferases (KAT) I and II in the discrete brain regions of 3- and 6-month old WAG/Rij rats, a genetic model of absence epilepsy. Analogues experiments were performed using age-matched ACI rats, which served as a nonepileptic control. The age-dependent increase in KYNA concentration in the frontal cortex of WAG/Rij rats was considerably reduced in comparison to what was found in ACI rats. Consequently, the concentration of KYNA in the frontal cortex of epileptic rats was significantly lower than in non-epileptic controls. There were no such strain differences in other brain regions. The activities of KAT I and II also showed age-dependent increase with an exception for KAT II in the frontal cortex. Our data suggest that selective deficits of endogenous KYNA may account for increased excitability in the frontal cortex, which in turn may lead to the development of spontaneous spike-wave discharges in WAG/Rij rats. Key words: kynurenic acid, aminotransferase, WAG/Rij rats, ACI rats, absence epilepsy, genetic models  correspondence; e-mail: turskiwa@asklepios.am.lublin.pl R.M. Kamiñski, E. Zieliñska, A. Dekundy, G. van Luijtelaar, W.A. Turski INTRODUCTION Kynurenic acid (KYNA) is endogenously synthesized from tryptophan via the kynurenine pathway by kynurenine aminotransferases (KAT) I and II. KYNA is a broad spectrum antagonist of glutamate receptors and it may bind to the glycine site of the N-methyl-D-aspartate (NMDA) receptor with relatively high affinity. It also binds with similar affinity to the nicotinic receptors containing the a7 subunit (for reviews see [30, 34–36]). Glutamatergic neurotransmission plays a pivotal role in the pathogenesis of epilepsy and antagonists of glutamate receptors are powerful anticonvulsants [3, 37]. In line with this, KYNA and its synthetic analogues are generally efficacious anticonvulsants in a variety of models of experimental epilepsy [9, 17, 39, 41]. Moreover, reduction of KYNA levels increases vulnerability to excitotoxic insults, whereas elevation of KYNA content has an opposite effect [29, 45, 46]. A significant role of glutamate receptors has also been postulated for non-convulsive epilepsy [4, 5, 16, 25, 26]. In WAG/Rij rats, serving as a well-validated, genetic model of absence epilepsy [42, 43], spontaneous spike-wave discharges (SWD) can be inhibited by KYNA administration. Moreover, KYNA blocks NMDA-induced potentiation of SWD in these rats [25, 26]. The above-mentioned findings prompted us to examine the concentrations of endogenous KYNA and the activity of KAT in the discrete brain regions of WAG/Rij rats. MATERIALS and METHODS Animals The analyses were performed in the parts of the brain where SWD are either evoked [23] or modulated [6]. The measurements were carried out at the age when WAG/Rij rats (i) begin to develop SWD (3 months) and (ii) display hundreds of SWD per day (6 months) [2]. Analogous experiments were performed in age-matched ACI rats, which are virtually devoid of SWD [15]. Such experimental design allows studying not only the age- and straindependent changes but also their interaction. Since only the 6 months old WAG/Rij rats show SWD, while the other groups do not, the interaction can be tentatively interpreted as that the biochemical changes are related to the presence of SWD. This paradigm has been previously employed in other studies [19, 20]. The number of animals in each 742 strain/age-matched group ranged from 3 to 6. Experimental procedures were in agreement with European Communities Council Directive (86/609/EEC) and were approved by the local Ethics Committee. Determination of KYNA concentration Animals were sacrificed by decapitation and their brains were rapidly removed from the scull. The frontal cortex, striatum and thalamus were dissected and immediately frozen for further studies. KYNA levels were estimated according to the modified method of Turski et al. [40]. Tissue material was homogenized with 1 ml of water by ultrasonication and centrifuged (20000 rpm, 10 min). Subsequently, 10 ml of 50% trichloroacatic acid and 100 ml of 1 M HCl were added to each sample of the obtained supernatant. Denatured protein was removed by centrifugation and the resulting supernatant was applied to the columns containing cation-exchange resin (Dowex 50 W+: 200–400 mesh) prewashed with 0.1 M HCl. Then, the columns were washed with 1ml of 0.1 M HCl and 1ml of water, and the fraction containing KYNA was eluted with 2 ml of water. Eluate was subjected to HPLC and KYNA was detected fluorometrically (Hewlett Packard 1050 HPLC system: ESA catecholamine HR-80, 3 mm, C18 reversed-phase column, flow rate of 1.0 ml/min, excitation 344 nm, emission 398 nm) according to the method of Shibata [32]. Determination of KAT I and KAT II activities KAT I and KAT II activities were assayed according to the method of Guidetti et al. [12] with modification. Tissue was homogenized (1:10 w/v) in 5 mM Tris-acetate buffer, pH 8.0, containing 50 mM pyridoxal-phosphate and 10 mM 2-mercaptoethanol. The resulting homogenate was dialyzed overnight at 8°C, using cellulose membrane dialysis tubing, against 4 l of the same buffer. Active samples contained the enzyme preparation with reaction mixture containing 2 mM L-kynurenine, 1 mM pyruvate, 70 mM pyridoxal-5’-phosphate, 150 mM Tris-acetate buffer pH of 7.0 (KAT II) or 150 mM Tris-acetate buffer pH 9.5 (KAT I) in a total volume of 0.2 ml. In KAT II assay a KAT I inhibitor, glutamine (final concentration: 2 mM), was added to each sample. Blank samples containing heat-deactivated enzyme (100°C for 10 min) and active samples were incubated at 37°C for 24 h. Transfer of the samples to an ice-bath and the addition of 10 ml of 50% trichloroacetic acid and 100 ml of 1 M HCl terminated the reaction. The denatured proteins were removed by Pol. J. Pharmacol., 2003, 55, 741–746 =>'*62') )
' ?;@6 ( 6 7 RESULTS The content of KYNA in the frontal cortex (F..0 = 20.3, p < 0.001), striatum (F..< = 15.4, p < 0.01) and thalamus (F..0 = 33.3, p < 0.001) increased in an age-dependent manner (Fig. 1A, B, C). Only in the frontal cortex a significant strain difference (F..0 = 9.5, p < 0.01) and strain times age interaction (F..0 = 7.4, p < 0.05) were observed (Fig. 1A). Outcomes of the post hoc tests showed that there was only a significant increase in the content of KYNA in the frontal cortex from 3 to 6 months in ACI rats, not in WAG/Rij rats (Fig. 1A, B, C). Moreover, the 6-month-old ACI rats had higher levels of KYNA (p < 0.01) than all other groups, including the agematched WAG/Rij rats (Fig. 1A). The activities of KAT I and KAT II are presented in Table 1. The two-way ANOVA revealed significant age-dependent increases of KAT I activity in the frontal cortex (F..: = 5.2, p < 0.05) and thalamus (F..: = 10.1, p < 0.01); no strain effects were found. Likewise, the in vitro activity of KAT II measured in samples from frontal cortex and thalamus did not differ between the WAG/Rij and ACI rats. There was, however, an age-dependent increase in KAT II activity in the thalamus (F../ = 12.5, p < 0.01), but not in the frontal cortex. DISCUSSION The present study demonstrates an age-dependent increase in the content of KYNA in the discrete brain regions of epileptic WAG/Rij and non-epileptic ACI rats. These results are consistent with pre- 77' .: !0  KYNA (pmol/g of tissue) The results obtained from the assays were compared separately for each brain region with the use of two-way analysis of variance (ANOVA), where age and strain of rats were used as independent factors. The two-way ANOVA was followed by the Bonferroni t-test for group comparisons. 150 *, u 3-month-old 6-month-old 100 50 0 – ACI – – WAG – B) Striatum KYNA (pmol/g of tissue) Statistics A) Frontal cortex 200 3-month-old * 6-month-old 150 * 100 50 0 – ACI – – WAG – C) Thalamus KYNA (pmol/g of tissue) centrifugation and the supernatant was applied to a Dowex 50 W+ column and quantified by HPLC. Protein content was determined spectrophotometrically in small aliquots of the respective enzyme preparations according to the method of Lowry et al. [21]. The final results were expressed as the amount of KYNA (pmol) synthesized by 1 mg of protein during 1 h of incubation (pmol/h/mg). 250 3-month-old 200 * * 6-month-old 150 100 50 0 – ACI – – WAG – Fig. 1. The levels of endogenous kynurenic acid (KYNA) in the frontal cortex (A), striatum (B) and thalamus (C) of WAG/Rij and ACI rats. Each bar represents the mean ± SEM (pmol/g of wet weight). Number of animals used in these experiments was as follows: 3- and 6-month-old ACI rats (n = 3 per group), 3- and 6-month-old WAG/Rij rats (n = 6 per group). * p < 0.05 vs. 3-month-old same strain rats, K p < 0.05 vs. vs. 6-month-old WAG/Rij rats (Bonferroni t-test) 743 R.M. Kamiñski, E. Zieliñska, A. Dekundy, G. van Luijtelaar, W.A. Turski Table 1. In vitro activity of kynurenine aminotransferase (KAT) I and II obtained from the frontal cortex and thalamus of WAG/Rij and ACI rats. The results are expressed as means ± SEM and represent the amount of KYNA (pmol) synthesized by 1 mg of protein during 1 h of incubation (pmol/h/mg). The increase in KAT I activity in the frontal cortex (F1,13 = 5.2, p < 0.05) and thalamus (F1,13 = 10.1, p < 0.01) was age-dependent (two-way ANOVA). KAT II showed an age-dependent increase, but it reached statistical significance only in the thalamus (F1,14 = 12.5, p < 0.01). * p < 0.05 vs. 3-month-old the same strain rats (Bonferroni t-test) Age and strain KAT I (pmol/h/mg) KAT II (pmol/h/mg) Frontal cortex Thalamus Frontal cortex Thalamus 3-month-old ACI (n = 3) 7.9 ± 0.7 15.8 ± 2.1 12.4 ± 1.1 31.6 ± 2.4 6-month-old ACI (n = 3) 17.2 ± 2.7 20.7 ± 4.3 21.2 ± 3.0 62.7 ± 11.6* 3-month-old WAG/Rij (n = 4) 13.5 ± 0.9 12.1 ± 1.6 17.2 ± 0.5 37.5 ± 4.9 6-month-old WAG/Rij (n = 5) 21.2 ± 5.9 23.5 ± 2.3* 27.6 ± 9.1 55.9 ± 7.0 viously reported data from other rat strains [11, 24], thus, such age-depended increase in KYNA concentration could be considered as a more general feature. Interestingly, selectively in the frontal cortex of 6 months old WAG/Rij rats, this increase was absent. Therefore, it seems that genetically epileptic rats have lower levels of KYNA in the frontal cortex than age-matched ACI rats, whereas no difference was found in 3-month-old rats of both strains. It is important to underline that only 6 months old WAG/Rij rats display several hundreds of spontaneous SWD per day, while agematched ACI rats and 3 months old WAG/Rij rats have no or much less SWD [2, 15]. Ample data show that neuronal excitability and seizure activity can be dampened by increased concentration of KYNA, resulting from either exogenous application or enhanced endogenous production [9, 27, 39, 41, 45, 46]. KYNA (50–500 nmol) injected icv inhibits spontaneous [26] and NMDAenhanced SWD [25] in WAG/Rij rats. Local administration of KYNA (5 mM) to a slice preparation with intact thalamocortical circuitry also completely abolishes spindle-like oscillations, resembling those seen in electroencephalogram (EEG) during absence seizures [38]. More recent studies performed on similar preparations obtained from WAG/Rij rats also confirm the importance of NMDA-mediated events in the generation of rhythmic thalamocortical oscillations [5]. It should be noted, however, that the effective concentrations of KYNA in the above-mentioned experiments were much higher than those established in the present study. As such, the possible role of endogenous KYNA in controlling epileptic phenomena should be further scrutinized. Nevertheless, these findings suggest that KYNA could be an important factor involved in suppression of cortical excitability. 744 The frontal cortex, striatum and thalamus are key structures for the development and maintenance of SWD hallmarking absence epilepsy [1, 4, 23, 33]. The present study shows selective reduction of KYNA concentration in the frontal cortex of WAG/ Rij rats at an age when they display large number of spontaneous SWD [2]. In contrast, there was no such difference in the striatum and thalamus of either epileptic or non-epileptic rats. Several pieces of evidence suggest that neuronal hyperexcitability in the frontal cortex seems to have critical importance for the initiation of SWD [4, 5, 22, 33]. Indeed, using non-linear association analysis of the EEG signals recorded in vivo from multiple cortical and thalamic sites, it has recently been demonstrated that spontaneous SWD developing in WAG/Rij rats originate from the frontal cortex [23]. Moreover, stimulation of the sensorimotor cortex by NMDA application produces responses that spread much more widely in rats with a genetic form of absence epilepsy (GAERS) than those seen in control rats [28]. These observations correspond well with the theory developed by Steriade and Contreras [31], suggesting a dominant role of the neocortex in generation of SWD. Moreover, a hyperexcitable cortex is a prerequisite for the transformation from sleep spindles to SWD [18]. Therefore, it could be hypothesized that selective reduction of KYNA content in the frontal cortex may account, at least in part, for the development of SWD in WAG/Rij rats. Both strains of rats displayed age-dependent increase in KAT I activity in samples obtained from the frontal cortex and thalamus. Similar changes in KAT II were also found in the samples obtained from the thalamus. Finally, KAT II activity in the frontal cortex also showed an age-dependent in- Pol. J. Pharmacol., 2003, 55, 741–746 =>'*62') )
' ?;@6 ( 6 7 crease, however, this effect did not reach statistical significance. The results obtained in the KAT activity assays should be interpreted with caution, even though they showed some degree of correlation with the endogenous KYNA levels. In vitro assays of KAT reflect only the maximal capability of these enzymes to synthesize KYNA under artificial conditions. It should be underlined that in vivo activity of KAT enzymes is regulated by a number of factors [10, 12, 44] and that in vitro KAT activity does not necessarily correlate with in vivo KYNA levels [11]. Nevertheless, the lack of significant, age-dependent increase in KAT II activity in the frontal cortex is intriguing. It has been previously established that KAT II is primarily responsible for the synthesis of endogenous KYNA [12]. Therefore, we may suggest that the observed deficit of KYNA might be, at least partially, related to enzymatic dysfunction of KAT II in the frontal cortex. 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