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Seizure activity causes elevation of endogenous extracellular kynurenic acid in the rat brain
BrainResearchBulletin,Voh 39, No. 3, pp. 155 162,1996 Copyright © 1996ElsevierScienceInc. Printedin the USA.All rightsreserved 0361-9230/96$15.00 + .00 ELSEVIER
SSDI 0361-9230 (95)02087-X
Seizure Activity Causes Elevation of Endogenous Extracellular Kynurenic Acid in the Rat Brain HUI-QIU WU 1 AND ROBERT SCHWARCZ
Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, MD 21228, USA [Received 30 May 1995; Revised 19 September 1995; Accepted 23 September 1995] Although the endogenous mechanisms by which the extracellular concentrations of KYNA in the mammalian brain (normal levels: approximately 20 nM) are regulated have not been clarified, it has been shown that KYNA levels can be manipulated experimentally. For example, KYNA concentrations decrease substantially when the brain is exposed to depolarizing agents such as veratridine or excitatory amino acids such as glutamate or L-~-aminoadipate [48,53]. Extracellular KYNA levels are also reduced in response to aminooxyacetic acid (AOAA), a potent but nonspecific inhibitor of kynurenine aminotransferase ( K A T ) , the enzyme responsible for the biosynthesis of KYNA from L-kynurenine [35,41,48]. Conversely, extracellular KYNA levels increase when KYNA synthesis is favored by the administration of indolepyruvic acid [27] or by pharmacological agents that block the competing degradative pathway of L-kynurenine to quinolinic acid [QUIN; 5,19], when the elimination of KYNA from the brain is prevented by probenecid [45 ], or when the precursor L-kynurenine is administered either systemically or intracerebrally [35,41,52]. Notably, several reports indicate that these pharmacologically induced fluctuations in extracellular KYNA in the brain have functional consequences. Thus, KYNA elevations appear to be anticonvulsant and neuroprotective [6,22], and KYNA decrements enhance excitotoxic vulnerability [ 17,18 ]. In a previous study using exogenously applied L-kynurenine to drive KYNA production, we concluded that the disposition of KYNA remains unchanged during seizures [51 ], but no assessment of endogenous KYNA levels was made. We therefore designed a series of experiments to examine the fate of endogenous KYNA in the brain following the administration of several classic chemoconvulsants. Some of these data, which were obtained in vivo by microdialysis in freely moving rats, have been published in abstract form [50].
ABSTRACT: This study was designed to examine the effects of several classic convulsants on the extracellular concentration of the anticonvulsant and neuroprotective brain metabolite kynurenic acid (KYNA) in the rat brain. Drug effects were investigated in vivo, mostly by unilateral microdialysis in the dorsal hippocampus. Systemic administration of pentylenetetrazole (60 mg/kg, SC), pilocarpine (325 mg/kg, SC), bicuculline (6 mg/kg, SC), or kainic acid (10 mg/kg, SC) caused characteristic clonic and/or tonic convulsions. In all seizure paradigms, KYNA levels in the dialysate began to rise within 1 h and gradually reached a plateau approximately 4 h after administration of the convulsants. Peak increases were 1.5-3-fold over basal levels. The duration of the elevation in KYNA levels was significantly prolonged following kainic acid application. In the kainic acid model, extracellular KYNA was also measured and found to be increased in the ventral hippocampus, piriform cortex, and striaturn. Moreover, temporary intrahippocampal infusion of the KYN synthesis inhibitor aminooxyacetic acid (1 mM) in the kainic acid- and pentylenetetrazole models attenuated the increase in extracellular KYNA levels, demonstrating that de novo production of KYNA in the brain accounts for the seizure-induced KYNA overflow. A separate group of animals received a unilateral intrahippocampal injection of the endogenous convulsant excitotoxin quinolinic acid (120 nmol) and showed long-lasting ( > 24 h) bilateral increases in extracellular KYNA levels. Taken together, these data indicate that an increase in extracellular KYNA may constitute a common occurrence in response to seizures and that KYNA elevations may signify the brain's attempt to counteract seizure activity. KEY WORDS: Convulsants, Epilepsy, Hippocampus, Kainic acid, Microdialysis.
INTRODUCTION Kynurenic acid (KYNA) is a broad spectrum antagonist of excitatory amino acid receptors and has a particularly high affinity for the glycine coagonist site of the N-methyl-D-aspartate (NMDA) receptor [ 15,40]. Because of its presence in the brain [40J and its anticonvulsant [11,42] and neuroprotective [1,14] properties in animals, it has been suggested that endogenous KYNA plays a role in the initiation and propagation of seizures and in seizure-related neurodegeneration [ 32,45 ]. More generally, we and others have proposed that fluctuations in the levels of endogenous KYNA may control neuronal susceptibility to excitotoxic insults [19,31,32,40].
MATERIALS AND METHODS Chemica&
KYNA, QUIN, kainic acid ( K A ), pentylenetetrazole (PTZ), bicuculline (BIC), scopolamine methylnitrate, pilocarpine hydrochloride (PIL), and AOAA were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of the highest commercially available purity.
Requests for reprints should be addressed to Hui-Qiu Wu, Maryland Psychiatric Research Center, P. O. Box 21247, Baltimore, MD 21228, USA. 155
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Animals Male Sprague-Dawley rats ( 2 2 0 - 2 5 0 g) were used in all experiments. The animals were housed under standard laboratory conditions with a 12 h / 1 2 h light/dark cycle and free access to food and water. All experiments were performed in accordance with the principles outlined in the " N I H Guide for the Care and Use of Laboratory A n i m a l s " and with the approval of the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Microdialysis Under chloral hydrate anesthesia (360 mg/kg, IP), the animals were mounted in a Kopf stereotaxic frame, and a guide cannula (outer diameter: 0.65 mm) was positioned, in most cases unilaterally, on top of the dorsal hippocampus (A: 3.4 mm posterior to bregma, L: 2.3 turn from the midline, V: 1 mm below the dura) and secured to the skull with anchor screws and acrylic dental cement. In three groups of animals (treated with K A + AOAA, PTZ + A O A A , or Q U I N ) , guide cannulae were implanted bilaterally over the dorsal hippocampus. For microdialysis in other areas, guide cannulae were implanted unilaterally over the following brain areas: piriform cortex (A: 2.4 mm posterior to bregma, L: 6.4 mm from the midline, V: 2.0 mm below dura, at a 20 ° angle to the vertical axis in the coronal plane); striatum (A: 1.0 mm anterior to bregma, L: 2.5 mm from the midline, V: 3.5 mm below dura); and ventral hippocampus (A: 2.5 mm anterior to lambda, L: 4.7 mm from the midline, V: 4.0 mm below dura). On the next day, a microdialysis probe ( C M A / 1 0 ; membrane length: 2 ram, Carnegie Medicin, Stockholm, Sweden) was inserted through the guide cannula, extending vertically through the targeted brain area. The probe was connected to a microperfusion pump ( C M A / 1 0 0 , Carnegie Medicin), and Ringer solution (in mM: NaCI, 144; KC1, 4.8; MgSO4, 1.2; CaCI2, 1.7; pH 6.7) was perfused at a speed of 1 #l/rain. Dialysate was collected every 30 rain. Routinely, the first fraction was discarded and the next four fractions were used to establish baseline conditions. Basal levels of K Y N A were computed from the last three consecutive baseline samples. Following the application of convulsant agents or respective vehicles (see below), animals were continuously perfused, and microdialysis fractions were collected every 30 min for an additional 6 h. In the groups treated with K A or QUIN, a single 12-h fraction (720 #1) was collected during the following perfusion overnight, and three consecutive 30-min fractions were collected on the morning of the second day. In controls (perfused with Ringer solution only) and in animals treated with PTZ, PIL, or BIC, overnight collections were made hourly for 12 h using a microfraction collector ( C M A / 1 4 2 , Carnegie Medicin). Only animals that showed typical tonic/clonic seizures within the first hour after the administration of convulsant agents were included in the study. For bilateral microdialysis, probes were inserted simultaneously into both hippocampi. The effect of A O A A in KA- or PTZtreated animals was examined by including the compound (at 1 mM) for 3 or 4 h in the perfusion solution using a C M A 110 Liquid Switch (Carnegie Medicin).
Determination of Kynurenic Acid in Dialysate The measurement of K Y N A in dialysate samples was performed as reported previously [48]. Briefly, samples were applied directly to a high performance liquid chromatography system coupled with a fluorescence detector (Perkin Elmer LC 240)
WU AND S C H W A R C Z
set at an excitation wavelength of 344 nm and an emission wavelength of 398 nm. A mobile phase consisting of 50 mM sodium acetate, 0.25 M zinc acetate, and 4% acetonitrile (pH 6.3) was pumped through an 8-cm HR-80, C~ 3-#m reverse-phase column (ESA Inc., Bedford, MA, U S A ) at a flow rate of I ml/min.
Chemically Induced Seizures All convulsant agents were dissolved in saline and adjusted to physiological pH. At time 0 (i.e., after the collection of baseline samples; cf. above), animals were injected subcutaneously (SC) with one of the following convulsants in a volume of 100 #1/100 g body weight: K A ( 10 m g / k g ) , PTZ (60 m g / k g ) , BIC (6 m g / k g ) , and PIL (325 mg/kg, 30 min after 1 m g / k g scopolamine). A control group received a SC injection of saline. A separate group of animals had their microdialysis probe removed following the collection of baseline samples, received a unilateral intrahippocampal injection of QUIN ( 120 nmol/1 /zl ) through a 30-gauge injection cannula, and had the microdialysis probe reinserted within 10 rain for continuing sample collection. All animals were observed for at least 6 h following drug administration.
Histology Four days after the termination of microdialysis, the animals were perfused transcardially under deep chloral hydrate (400 mg/kg, IP) anesthesia. The brains were removed and processed for routine histological analysis. Coronal cryostat sections (30#m-thick) were stained with thionin and were inspected by light microscopy for the correct placement of dialysis probes and apparent signs of neurodegeneration. Only animals where the fiber track was limited to the targeted brain area were included in the data analysis.
Data Presentation The extracellular levels of endogenous KYNA, mostly expressed as a percentage of baseline levels, were not corrected for recovery through the dialysis probes (cf. [53]). All data were analyzed by two-way analysis of variance ( A N O V A ) followed either by Scheffe's post hoc test (statistical significance set at p < 0.05) or by a Bonferroni-corrected paired t-test. RESULTS
Chemically induced seizures KA-induced seizures. A dose of 10 m g / k g K A (SC) reliably produced a complex spectrum of behavioral seizures, as described in detail previously [2,20,36]. Within 30 min, animals (N = 30) showed sporadic wet-dog shakes and increased locomotor activity. Subsequently, the frequency of wet-dog shakes increased, and seizures were often accompanied by rearing and forelimb clonus. Between 1 and 4 h after the K A injection, all animals developed severe limbic seizures characterized by generalized convulsions, rearing and falling with strong salivation. The convulsions gradually subsided after 5 h, and no animal died as a late consequence of seizure activity. PTZ-induced seizures. PTZ (60 mg/kg, SC) reliably produced typical motor seizures, characterized by tonic/clonic convulsions. The rats (N = 7) first displayed a short period (several seconds) of running, flexion, and/or extension of fore- and/or hindlimbs, and proceeded to the loss of righting reflex and myoclonic jerks with a latency of about 2 min. The total time spent in behavioral seizures was always less than 10 min, and no deaths occurred as a consequence of the seizures.
S E I Z U R E S A N D E X T R A C E L L U L A R K Y N U R E N I C ACID
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FIG. 1. Extracellular KYNA levels in the dorsal hippocampus of KA-treated rats: Effect of AOAA. KA was injected (10 mg/kg, SC) at time 0 (N = 6). Microdialysis was performed bilaterally as described in the text. In one hippocampus, AOAA (1 mM) was delivered for 3 h through the dialysis probe, starting 3 h after KA administration (11). The bar indicates the period of AOAA infusion. The other hippocampus was continuously perfused with Ringer solution alone (E]). Data (mean _+ SEM) are expressed as a percentage of basal levels obtained from the last three samples prior to time 0 (74.3 ± 6.3 fmol KYNA/30 #1 dialysate). See text for statistical analysis of the effect of KA. *p < 0.05 as compared to the same timepoint in the absence of AOAA.
BIC-induced seizures. In preliminary experiments, a dose of > 8 m g / k g BIC resulted in tonic/clonic convulsions and loss of the righting reflex followed by death in most animals. Thus, 6 m g / k g BIC ( S C ) was selected as a dose at which animals consistently showed behavioral seizure p h e n o m e n a without mortality. O f the six animals used in this group, two had tonic/clonic seizure episodes and displayed short periods of loss of righting reflex and limb extension. The other four rats showed running, jumping, and severe fore- a n d / o r hindlimb clonus, which appeared within 2 min after BIC administration. Behavioral abnormalities subsided within 30 min. PlL-induced seizures. The treatment regimen chosen for testing the effect of PIL-induced seizures consisted of an injection of scopolamine ( 1 m g / k g , SC) 30 min prior to PIL (325 m g / k g , SC). Animals receiving this treatment ( N = 6) developed behavioral seizures with an average latency of approximately 30 min. Animals showed wet dog shakes, head bobbing followed by intermittent forepaw myoclonus, rearing and falling, and severe salivation. These behavioral abnormalities lasted approximately 2 h. No mortality was observed. QUlN-Induced Seizures. An intrahippocampal injection of 120 nmol QUIN, which reliably induces EEG seizures lasting approximately 3 h [ 30,46 ], resulted in stereotypic behaviors such as sniffing, gnawing, and rearing in all rats ( N = 6) following a latency period of approximately 30 min. These changes, which were accompanied by " f r o z e n a p p e a r a n c e " and wet dog shakes, lasted about 1 h. Wild running, jumping, and ipsilateral turning occurred sporadically, and all behavioral indications of seizure activity subsided within 3 h after the injection. No seizure-related mortality was observed as a consequence of the QUIN injection. Seizure-Induced Changes in Endogenous KYNA In all five seizure paradigms, extracellular K Y N A began to rise from baseline levels (71.1 ± 2.6 fmol / 30 #1; total of 61 rats ) within the first hour after drug administration and gradually
reached a plateau of different duration. Thus, a peak of 1 . 5 - 3 times baseline concentrations was attained in the dorsal hippocampus after the administration of the chemoconvulsants. KA. Systemic K A administration caused the quantitatively largest increase in extracellular KYNA levels in the present series of experiments, reaching statistical significance at 2.5 h and a peak of approximately three times control levels after 5 h. This maximal elevation was sustained for another 4 h, and was followed by a slow decline over the course of the subsequent 12 h time period. Twenty-four hours after KA, KYNA levels were still twice as high as controls (Fig. 1 ). To examine if the increase in brain KYNA levels was caused by a seizure-related temporary opening of the b l o o d - b r a i n barrier to blood-borne KYNA, the nonspecific K Y N A synthesis inhibitor A O A A was used in a separate set of experiments. The drug ( 1 m M in the perfusion solution) was introduced 3 h after KA, i.e., at a time when KYNA levels had reached 60% of plateau values. In pilot experiments ( N = 6 ) , this treatment with A O A A caused neither behavioral abnormalities nor apparent neurodegenerative changes (data not shown). As depicted in Fig. 1, A O A A caused a rapid return to baseline KYNA levels within the 3 h perfusion period, but K Y N A concentration rebounded immediately after removal of the synthesis inhibitor. There was a significant interaction between treatment groups ( K A alone, K A plus A O A A , and controls) and posttreatment times [ F ( 3 6 , 2 7 0 ) = 12.94, P < 0.001]. This experiment demonstrated that cerebral de novo synthesis of K Y N A is primarily responsible for the overflow of extracellular KYNA after systemic K A administration. Quantitatively similar KA-induced increases in KYNA levels, here shown during the plateau period (5 h after K A ; Fig. 2), were observed in the dorsal and ventral hippocampus, the piriform cortex, and the striatum. Across regions, there was a significant difference between basal KYNA levels and KA-induced increases [ F ( 3 , 2 0 ) = 3.30, P < 0.05].
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FIG. 2. KA-induced elevation of extracellular KYNA levels in different brain areas. KA was injected (10 mg/kg, SC) at time 0 (N 6 rats per group). Open bars represent basal KYNA levels in the four brain regions, obtained from the last three samples prior to time 0. Hatched bars represent KYNA levels in dialysates during the fifth hour after KA treatment (plateau value; cf. Fig. I). *p < 0.05, **p < 0.01 as compared to baseline values (Bonferroni corrected t-test).
PTZ, BIC. and PIL. These three compounds caused transient increases in extracellular KYNA, peaking between 3 and 5 h and returning to baseline levels approximately 8 h later (Fig. 3 ). The elevation in K Y N A concentrations reached statistical significance at 1 h ( P T Z ) or 2.5 b (BIC and PIL) after the administration of the convulsants; i.e., the increase followed the occurrence of behavioral seizures. Peak values were reached at 3.5 h (PTZ; BIC) or 4 h ( P I L ) . Statistical significance was lost 6 h ( P T Z ) , 5 h ( B I C ) , or 11 h ( P I L ) following drug application. There was a significant interaction between treatment groups (PTZ, BIC, PIL, and controls) and posttreatment times [ F ( 6 0 , 4 0 0 ) = 8.30, p < 0.001]. In separate animals, A O A A ( 1 raM) was infused unilaterally during the first 4 h after PTZ administration ( N = 4 ) . In contrast to the contralateral hippocampus, which showed the expected increase in extracellular KYNA levels, no KYNA elevation was observed on the AOAA-treated side (data not shown). QUIN. As illustrated in Fig. 4, a unilateral intrahippocampal QUIN injection caused bilateral increases in extracellular KYNA. At 1.5 h, the increases reached statistical significance. Plateau levels ( l . 8 - f o l d control concentrations on both sides) were attained simultaneously in both hippocampi 3 - 4 h after the QUIN injection. Notably, similar to data obtained with KA, KYNA concentrations remained elevated bilaterally for at least 24 h following the application of QUIN. There was a significant interaction between treatment groups (ipsi- and contralateral sides and controls) and posttreatment times [ F ( 3 4 , 2 5 5 ) - 4.85, p < 0.001]. In an additional experiment, K Y N A levels in the injected hippocampus were found to remain elevated during 7 days of continuous perfusion, whereas KYNA levels on the contralateral side returned to baseline values within 48 h ( N = 4; data not s h o w n ) . Neuropathology Following Chemoconvulsant Administration Neuropathological changes were e x a m i n e d in all five seizure models. In Nissl-stained tissue sections, no apparent nerve cell loss was observed in the h i p p o c a m p u s or in other brain regions
in PTZ- or BIC-treated rats. Animals that received PIL or K A showed the well-documented, typical pattern of seizure-related neurodegeneration in the h i p p o c a m p u s and extrahippocampal brain areas [33,37,43]. As described previously, intrahippocampal QUIN application resulted in a circumscribed neuronal lesion that was limited to the h i p p o c a m p u s on the injected hemisphere [ 30]. DISCUSSION Using five well-established animal models of epilepsy, the present results demonstrate that chemoconvulsant-induced seizures are associated with an elevation of extracellular KYNA levels. KYNA levels began to rise within the first hour after drug administration, i.e., at the same time as behavioral signs of seizure activity were noted. Although the limited time resolution of microdialysis methodology and the absence of EEG recordings did not allow us to perform the desirable tight correlative analysis of neurochemical changes and seizure activity, the data clearly demonstrate that a spectrum of pharmacologically distinct convulsive triggers elicit similar responses of the endogenous KYNA system. It therefore appears reasonable to assume that the increase in extracellular KYNA described here constitutes a rapid yet rather nonspecific tissue reaction to seizure activity. The present study does not directly address the question of the cellular source of KYNA or of the mechanisms that are responsible for its elevation in the extracellular compartment following seizures. Both immunohistochemical and biochemical studies, the former using antibodies against K Y N A ' s biosynthetic enzyme KAT, have demonstrated that in the rat brain KYNA is predominantly produced in and liberated from astrocytes. Thus, the normal hippocampus harbors an abundance of KAT-containing astrocytes with only sporadic immunopositive neurons [ 9 ] , and extracellular baseline levels of KYNA are substantially elevated in the neuron-depleted, astrogliotic hippocampus in vivo [48]. Astrocytes are known to react rapidly to seizures and, as major determinants of the composition of the extracellular milieu, may play an important role in epileptic
SEIZURES AND E X T R A C E L L U L A R K Y N U R E N I C ACID
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FIG. 3. Extracellular KYNA levels in the dorsal hippocampus of PTZ (60 mg/kg; e)-, PIL (325 mg/kg + 1 mg/kg scopolamine; A)-, and BIC (6 mg/kg; II)-treated rats (N = 6 - 7 rats per group). Drugs were administered and experiments were performed as described in the text. Convulsants were administered at time 0 (2 h after perfusion with Ringer solution alone). Data (mean +_ SEM) are expressed as a percentage of basal levels obtained from the last three samples prior to time 0. Saline-injected controls (C)) contained 76.9 _+ 1.9 fmol KYNA/30 #1 dialysate, compiled over 20 h (N = 6). See text for statistical analyses.
pathophysiology [ 4,38,47 ]. It is therefore likely that the increase in extracellular KYNA observed in the present set o f experiments can be traced to hippocampal astrocytes. In this context, it is noteworthy that greatly hypertrophied astrocytes containing KAT-immunoreactivity have been described in the hippocampus in a rat model of chronic epilepsy [10]. This has led to the suggestion that an increased release o f KYNA from astrocytes into
the extracellular compartment may be involved in recurrent seizure phenomena. Very little is known about the mechanisms that govern the entry of K Y N A into the extracellular milieu and, in particular, how these mechanisms might be affected in pathologic situations. In the normal brain, KYNA release is Ca 2* -independent, and the bioavailability of K Y N A ' s precursor L-kynurenine constitutes an
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FIG. 4. Extracellular KYNA levels in the dorsal hippocampus of rats receiving a unilateral intrahippocampal injection of QUIN. QUIN (I 20 nmol/1 #1) was infused at time 0 (N = 6). Microdialysis was performed in the ipsilateral (11) and contralateral ([3) hippocampus for an additional 24 h as described in the text. Data (mean -+ SEM) are expressed as a percentage of basal levels obtained from the last three samples prior to time 0 (77.8 -+ 6.9 fmol KYNA/30 #1 dialysate). See text for statistical analyses.
160
important determinant of both KYNA production and efflux [44]. Because astrocytes avidly accumulate kynurenine [34], it is also possible that a seizure-induced activation of glial uptake underlies the enhanced production of KYNA observed in the present study. Thus, seizure-induced increases in the permeability of the blood-brain barrier to glutamine [24] may cause a secondary augmentation of kynurenine transport into astrocytes [3]. Alternatively, the increases in the extracellular levels of KYNA reported here could be a consequence of enhanced K A T activity, increased synthesis of kynurenine in the brain, or enhanced influx of kynurenine from the periphery into the brain [12]. An increase in K A T activity is unlikely for two reasons. First, it is questionable if the speed of the seizure-induced KYNA elevation would allow sufficient time for the increased expression of KAT. Second, an increase in the activity of K A T should cause elevations in extracellular K Y N A also when exogenous kynurenine is used to elicit KYNA release. However, acute KAinduced seizures were shown previously not to result in elevated extracellular K Y N A levels when 200 pM kynurenine was employed to drive KYNA production [ 51 ]. The experiment using transient in vivo perfusion with the nonspecific K A T inhibitor A O A A demonstrates that de novo production of KYNA in the brain must be responsible for the enhanced overflow of KYNA after convulsive treatments (cf. Fig. 1). Thus, A O A A effectively reverted extracellular KYNA to baseline levels in K A - and PTZ-treated animals. Because the enzymatic machinery for kynurenine synthesis in the rodent brain is very inefficient [7,28], an increased influx of kynurenine from the periphery, perhaps accentuated by a temporary, seizure-related impairment of the blood-brain barrier [8,21], is likely to be the main cause of the K Y N A elevations reported here. Notably, as judged from the K A model, increases in extracellular K Y N A were not limited to the hippocampus but were also detected in the piriform cortex and the striatum. It therefore appears that the increase in the amount of kynurenine available for transamination may be rather uniform throughout the brain. Clearly, the precise fate of kynurenine during seizure episodes warrants further study. As a preferential antagonist of the glycine coagonist site of the N M D A receptor, which appears to play a critical role in seizure phenomena [16,26], K Y N A has been conceptually linked to seizure mechanisms since its anticonvulsant properties were discovered a decade ago [ 11 ]. Although exogenously supplied K Y N A is effective in experimental seizure models [11,29,39,42], however, the doses needed to prevent seizure activity far exceed endogenous K Y N A levels. Effective systemic administration, in particular, requires large quantities of the compound due to the very limited penetration of KYNA through the b l o o d - b r a i n barrier [12]. Until recently, most investigators therefore considered K Y N A a useful pharmacological tool but did not contemplate an anticonvulsant role of endogenous KYNA. Recent studies suggest, however, that fluctuations in the levels of endogenous extracellular K Y N A have functional consequences that are likely related to changes in N M D A receptor function. For example, K A T inhibition by nonspecific aminotransferase blockers such as A O A A or yacetylenic G A B A is associated with seizures and excitotoxic neurodegeneration, which can be blocked by N M D A receptor antagonists [17,18]. Conversely, > 10-fold increases in extracellular K Y N A in the hippocampus, caused by pharmacologically shifting brain kynurenine metabolism toward enhanced K Y N A production, have pronounced anticonvulsant effects against sound-induced seizures in D B A / 2 mice and maximal electroshock-induced seizures in rats [5,19]. A recent ultrastructural study using anti-K AT antibodies provides an anatom-
WU AND S C H W A R C Z
ical basis for the apparent effectiveness of endogenous KYNA. Thus, KAT-containing astrocytic processes were shown to be closely apposed to excitatory asymmetric synapses, suggesting that the concentration of KYNA in the cleft (i.e., at the postsynaptic receptor) may be higher than hitherto assumed [25 ]. If increases in extracellular K Y N A concentrations are indeed functionally significant, it is tempting to interpret the present data teleologically as an attempt of the brain to protect itself against an overstimulation of excitatory amino acid receptors. Regardless of the nature of the primary convulsant stimulus, seizure-related neurodegeneration is clearly excitotoxic in nature, and its extent is dependent on seizure intensity and duration [23]. It is therefore possible that increases in KYNA levels were sufficient to prevent nerve cell loss following the relatively brief seizures evoked by PTZ and BIC, but failed to counteract the more enduring effects of PIC and KA, which result in extensive neuronal damage in several regions of the brain [ 27,28,31 ]. Notably, KYNA elevations were particularly prolonged following the administration of KA, which produced the longest lasting seizures of all systemically applied chemoconvulsants. It is also noteworthy that a unilateral intrahippocampal injection of the direct N M D A receptor agonist QUIN, which causes bilateral seizures but only ipsilateral neuronal damage [ 30], resulted in prolonged bilateral increases in KYNA concentrations. This finding supports the idea that endogenous KYNA can limit seizure-related damage, but indicates that its potency is insufficient for blocking excessive receptor stimulation in tissue exposed to large amounts of an excitatory amino acid receptor agonist. The present study, and the recent discovery that extracellular K Y N A concentrations are also elevated in kindled animals [49], may be of relevance for conceptualizing new treatment strategies for epilepsy. A trend toward higher interictal and postictal K Y N A levels was recently demonstrated to exist in the cerebrospinal fluid of patients with complex partial seizures [13], so that mechanisms that operate in the rat may resemble those in the human epileptic brain. Although cerebrospinal fluid kynurenine concentrations in epileptic patients are reportedly below control values [ 54], further studies using a medication-free population and, ideally, brain microdialysis are needed to reexamine the status of both kynurenine and KYNA. Should such data reveal further parallels to the animal studies described here, epilepsy therapy with KYNA-enhancing pharmacological agents may be indicated. ACKNOWI,EDGEMENTS
The authors thank Mrs. Joycc Burgess for excellent secretarial assistance and Drs. Deborah Medoff and Kevin O'Grady for expert advice with statistical analyses. This work was supported by USPHS grant NS 16102.
REFERENCES I. Andin6, P.; Lehmann, A.; Ellr6n, K.; Wennberg, E.; Kjellmer, I.; Nielsen, T.; Hagberg, H. The excitatory amino acid antagonist kynurenic acid administered after hypoxic ischemia in neonatal rats offers neuroprotection. Neurosci. Lett. 90:208-212; 1988. 2. Ben-Ari, Y.; Tremblay, E.; Riche, D.; Ghilini, G.; Naquet, R. Electrographic, clinical and pathological alterations following systemic administration of kainic acid, bicuculline or pentetrazolc: Metabolic mapping using the deoxyglucose method with special reference to the pathology of epilepsy. Neuroscience 6:1361 - 1391 ; 1981. 3. Brookes, N. Interaction between the glutamine cycle and the uptake of large neutral amino acids in astrocytes. J. Neurochem. 60:19231928; 1993.
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ACID
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25. Roberts, R. C.; Du, F.; McCarthy, K. E.; Okuno, E.; Schwarcz, R. Immunocytochemical localization of kynurenine aminotransferase in the rat striatum: A light and electron microscopic study. J. Comp. Neurol. 326:82-90; 1992. 26. Rogawski, M. A. The NMDA receptor, NMDA antagonists and epilepsy therapy. Drugs 44:279-292; 1992. 27. Russi, P.; Carlh, V.; Moroni F. Indolepyruvic acid administration increases the brain content of kynurenic acid. Is this a new avenue to modulate excitatory amino acids in vivo? Biochem. Pharmacol. 38:2405-2409; 1989. 28. Saito, K.; Markey, S. P.; Heyes, M. P. Chronic effects of y-interferon on quinolinic acid and indoleamine-2,3-dioxygenase in brain of C57BL6 mice. Brain Res. 546:151-154; 1991. 29. Schneiderman, J. H.; MacDonald, J. F. Excitatory amino acid blockers differentially affect bursting of in vitro hippocampal neurons in two pharmacological models of epilepsy. Neuroscience 31:593603; 1989. 30. Schwarcz, R.; Brush, G. S.; Foster, A. C.; French, E. D. Seizure activity and lesions after intrahippocampal quinolinic acid injection. Exp. Neurol. 84:1-17; 1984. 31. Schwarcz, R.; Du, F.; Schmidt, W.; Turski, W, A.; Gramsbergen, J. B. P.; Okuno, E.; Roberts, R. C. Kynurenic acid: A potential pathogen in brain disorders. In: Langston, J. W.; Young, A., eds. Neurotoxins and neurodegenerative disease. New York: Ann. N. Y. Acad. Sci. 1992:648:140-153. 32. Schwarcz, R.; Foster, A. C.; French, E. D.; Whetsell, W. O. Jr.; K6hler, C. Excitotoxic models for neurodegenerative disorders. Life Sci. 35:19-32; 1984. 33. Schwob, J. E.; Fuller, T.; Price, J. L.; Olney, J. W. Widespread patterns of neuronal damage following systemic or intracerebral injections of kainic acid: A histological study. Neuroscience 5:9911014; 1980. 34. Speciale, C.; Hares, K.; Schwarcz, R.; Brookes, N. High-affinity uptake of L-kynurenine by a Na + -independent transporter of neutral amino acids in astrocytes. J. Neurosci. 9:2066-2072; 1989. 35. Speciale, C.; Wu, H.-Q.; Gramsbergen, J. B. P.; Turski, W. A.; Ungerstedt, U.; Schwarcz, R. Determination of extracellular kynurenic acid in the striatum of unanesthetized rats: Effect of aminooxyacetic acid. Neurosci. Lett. 116:198-203; 1990. 36. Sperk, G. Kainic acid seizures in the rat. Progr. Neurobiol. 42:132; 1994. 37. Sperk, G.; Lassmann, H.; Baran, H.; Kish, S. J.; Seitelberger, F.; Hornykiewicz, O. Kainic acid induced seizures: Neurochemical and histopathological changes. Neuroscience 10:1301 - 1315; 1983. 38. Steward, O.; Torre, E. R.; Tomasulo, R.; Lothman, E. Neuronal activity upregulates astroglial gene expression. Proc. Natl. Acad. Sci. USA 88:6819-6823; 1991. 39. Stone, T. W. Comparison of kynurenic acid and 2-APV suppression of epileptiform activity in rat hippocampal slices. Neurosci. Lett. 84:234-238; 1988. 40. Stone, T. W. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol. Rev. 45:309-379; 1993. 41. Swartz, K.; During, J.; Freese, M. J.; Beal, M. F. Cerebral synthesis and release of kynurenic acid: An endogenous antagonist of excitatory amino acid receptors. J. Neurosci. 10:2965-2973; 1990. 42. Thompson, J. L.; Holmes, G. L.; Taylor, G. W.; Feldman, D. R. Effect of kynurenic acid on amygdaloid kindling in the rat. Epilepsy Res. 2:302-308; 1988. 43. Turski, W. A.; Cavalheiro, E. A.; Schwarz, M.; Czuczwar, S. J.; Kleinrok, Z.; Turski, L. Limbic seizures produced by pilocarpine in rats: Behavioural, electroencephalographic and neuropathological study. Bebav. Brain. Res. 9:315-335; 1983. 44. Turski, W. A.; Gramsbergen, J. B. P.; Traitler, H.; Schwarcz, R. Rat brain slices produce and liberate kynurenic acid upon exposure to L-kynurenine. J. Neurochem. 52:1629-1636; 1989. 45. V6csei, L.; Miller, J.; MacGarvay, U.; Beal, M. F. Kynurenine and probenecid inhibit pentylenetetrazol- and NMDLA-induced seizures and increase kynurenic acid concentrations in the brain. Brain Res. Bull. 28:233-238; 1992. 46. Vezzani, A.; Wu, H.-Q.; Tullii, M.; Samanin, R. Anticonvulsant drugs effective against human temporal lobe epilepsy prevent seizures but not neurotoxicity induced in rats by quinolinic acid: Elec-
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SSDI 0361-9230 (95)02087-X
Seizure Activity Causes Elevation of Endogenous Extracellular Kynurenic Acid in the Rat Brain HUI-QIU WU 1 AND ROBERT SCHWARCZ
Maryland Psychiatric Research Center, University of Maryland School of Medicine, Baltimore, MD 21228, USA [Received 30 May 1995; Revised 19 September 1995; Accepted 23 September 1995] Although the endogenous mechanisms by which the extracellular concentrations of KYNA in the mammalian brain (normal levels: approximately 20 nM) are regulated have not been clarified, it has been shown that KYNA levels can be manipulated experimentally. For example, KYNA concentrations decrease substantially when the brain is exposed to depolarizing agents such as veratridine or excitatory amino acids such as glutamate or L-~-aminoadipate [48,53]. Extracellular KYNA levels are also reduced in response to aminooxyacetic acid (AOAA), a potent but nonspecific inhibitor of kynurenine aminotransferase ( K A T ) , the enzyme responsible for the biosynthesis of KYNA from L-kynurenine [35,41,48]. Conversely, extracellular KYNA levels increase when KYNA synthesis is favored by the administration of indolepyruvic acid [27] or by pharmacological agents that block the competing degradative pathway of L-kynurenine to quinolinic acid [QUIN; 5,19], when the elimination of KYNA from the brain is prevented by probenecid [45 ], or when the precursor L-kynurenine is administered either systemically or intracerebrally [35,41,52]. Notably, several reports indicate that these pharmacologically induced fluctuations in extracellular KYNA in the brain have functional consequences. Thus, KYNA elevations appear to be anticonvulsant and neuroprotective [6,22], and KYNA decrements enhance excitotoxic vulnerability [ 17,18 ]. In a previous study using exogenously applied L-kynurenine to drive KYNA production, we concluded that the disposition of KYNA remains unchanged during seizures [51 ], but no assessment of endogenous KYNA levels was made. We therefore designed a series of experiments to examine the fate of endogenous KYNA in the brain following the administration of several classic chemoconvulsants. Some of these data, which were obtained in vivo by microdialysis in freely moving rats, have been published in abstract form [50].
ABSTRACT: This study was designed to examine the effects of several classic convulsants on the extracellular concentration of the anticonvulsant and neuroprotective brain metabolite kynurenic acid (KYNA) in the rat brain. Drug effects were investigated in vivo, mostly by unilateral microdialysis in the dorsal hippocampus. Systemic administration of pentylenetetrazole (60 mg/kg, SC), pilocarpine (325 mg/kg, SC), bicuculline (6 mg/kg, SC), or kainic acid (10 mg/kg, SC) caused characteristic clonic and/or tonic convulsions. In all seizure paradigms, KYNA levels in the dialysate began to rise within 1 h and gradually reached a plateau approximately 4 h after administration of the convulsants. Peak increases were 1.5-3-fold over basal levels. The duration of the elevation in KYNA levels was significantly prolonged following kainic acid application. In the kainic acid model, extracellular KYNA was also measured and found to be increased in the ventral hippocampus, piriform cortex, and striaturn. Moreover, temporary intrahippocampal infusion of the KYN synthesis inhibitor aminooxyacetic acid (1 mM) in the kainic acid- and pentylenetetrazole models attenuated the increase in extracellular KYNA levels, demonstrating that de novo production of KYNA in the brain accounts for the seizure-induced KYNA overflow. A separate group of animals received a unilateral intrahippocampal injection of the endogenous convulsant excitotoxin quinolinic acid (120 nmol) and showed long-lasting ( > 24 h) bilateral increases in extracellular KYNA levels. Taken together, these data indicate that an increase in extracellular KYNA may constitute a common occurrence in response to seizures and that KYNA elevations may signify the brain's attempt to counteract seizure activity. KEY WORDS: Convulsants, Epilepsy, Hippocampus, Kainic acid, Microdialysis.
INTRODUCTION Kynurenic acid (KYNA) is a broad spectrum antagonist of excitatory amino acid receptors and has a particularly high affinity for the glycine coagonist site of the N-methyl-D-aspartate (NMDA) receptor [ 15,40]. Because of its presence in the brain [40J and its anticonvulsant [11,42] and neuroprotective [1,14] properties in animals, it has been suggested that endogenous KYNA plays a role in the initiation and propagation of seizures and in seizure-related neurodegeneration [ 32,45 ]. More generally, we and others have proposed that fluctuations in the levels of endogenous KYNA may control neuronal susceptibility to excitotoxic insults [19,31,32,40].
MATERIALS AND METHODS Chemica&
KYNA, QUIN, kainic acid ( K A ), pentylenetetrazole (PTZ), bicuculline (BIC), scopolamine methylnitrate, pilocarpine hydrochloride (PIL), and AOAA were purchased from Sigma (St. Louis, MO, USA). All other chemicals were of the highest commercially available purity.
Requests for reprints should be addressed to Hui-Qiu Wu, Maryland Psychiatric Research Center, P. O. Box 21247, Baltimore, MD 21228, USA. 155
156
Animals Male Sprague-Dawley rats ( 2 2 0 - 2 5 0 g) were used in all experiments. The animals were housed under standard laboratory conditions with a 12 h / 1 2 h light/dark cycle and free access to food and water. All experiments were performed in accordance with the principles outlined in the " N I H Guide for the Care and Use of Laboratory A n i m a l s " and with the approval of the Institutional Animal Care and Use Committee of the University of Maryland School of Medicine.
Microdialysis Under chloral hydrate anesthesia (360 mg/kg, IP), the animals were mounted in a Kopf stereotaxic frame, and a guide cannula (outer diameter: 0.65 mm) was positioned, in most cases unilaterally, on top of the dorsal hippocampus (A: 3.4 mm posterior to bregma, L: 2.3 turn from the midline, V: 1 mm below the dura) and secured to the skull with anchor screws and acrylic dental cement. In three groups of animals (treated with K A + AOAA, PTZ + A O A A , or Q U I N ) , guide cannulae were implanted bilaterally over the dorsal hippocampus. For microdialysis in other areas, guide cannulae were implanted unilaterally over the following brain areas: piriform cortex (A: 2.4 mm posterior to bregma, L: 6.4 mm from the midline, V: 2.0 mm below dura, at a 20 ° angle to the vertical axis in the coronal plane); striatum (A: 1.0 mm anterior to bregma, L: 2.5 mm from the midline, V: 3.5 mm below dura); and ventral hippocampus (A: 2.5 mm anterior to lambda, L: 4.7 mm from the midline, V: 4.0 mm below dura). On the next day, a microdialysis probe ( C M A / 1 0 ; membrane length: 2 ram, Carnegie Medicin, Stockholm, Sweden) was inserted through the guide cannula, extending vertically through the targeted brain area. The probe was connected to a microperfusion pump ( C M A / 1 0 0 , Carnegie Medicin), and Ringer solution (in mM: NaCI, 144; KC1, 4.8; MgSO4, 1.2; CaCI2, 1.7; pH 6.7) was perfused at a speed of 1 #l/rain. Dialysate was collected every 30 rain. Routinely, the first fraction was discarded and the next four fractions were used to establish baseline conditions. Basal levels of K Y N A were computed from the last three consecutive baseline samples. Following the application of convulsant agents or respective vehicles (see below), animals were continuously perfused, and microdialysis fractions were collected every 30 min for an additional 6 h. In the groups treated with K A or QUIN, a single 12-h fraction (720 #1) was collected during the following perfusion overnight, and three consecutive 30-min fractions were collected on the morning of the second day. In controls (perfused with Ringer solution only) and in animals treated with PTZ, PIL, or BIC, overnight collections were made hourly for 12 h using a microfraction collector ( C M A / 1 4 2 , Carnegie Medicin). Only animals that showed typical tonic/clonic seizures within the first hour after the administration of convulsant agents were included in the study. For bilateral microdialysis, probes were inserted simultaneously into both hippocampi. The effect of A O A A in KA- or PTZtreated animals was examined by including the compound (at 1 mM) for 3 or 4 h in the perfusion solution using a C M A 110 Liquid Switch (Carnegie Medicin).
Determination of Kynurenic Acid in Dialysate The measurement of K Y N A in dialysate samples was performed as reported previously [48]. Briefly, samples were applied directly to a high performance liquid chromatography system coupled with a fluorescence detector (Perkin Elmer LC 240)
WU AND S C H W A R C Z
set at an excitation wavelength of 344 nm and an emission wavelength of 398 nm. A mobile phase consisting of 50 mM sodium acetate, 0.25 M zinc acetate, and 4% acetonitrile (pH 6.3) was pumped through an 8-cm HR-80, C~ 3-#m reverse-phase column (ESA Inc., Bedford, MA, U S A ) at a flow rate of I ml/min.
Chemically Induced Seizures All convulsant agents were dissolved in saline and adjusted to physiological pH. At time 0 (i.e., after the collection of baseline samples; cf. above), animals were injected subcutaneously (SC) with one of the following convulsants in a volume of 100 #1/100 g body weight: K A ( 10 m g / k g ) , PTZ (60 m g / k g ) , BIC (6 m g / k g ) , and PIL (325 mg/kg, 30 min after 1 m g / k g scopolamine). A control group received a SC injection of saline. A separate group of animals had their microdialysis probe removed following the collection of baseline samples, received a unilateral intrahippocampal injection of QUIN ( 120 nmol/1 /zl ) through a 30-gauge injection cannula, and had the microdialysis probe reinserted within 10 rain for continuing sample collection. All animals were observed for at least 6 h following drug administration.
Histology Four days after the termination of microdialysis, the animals were perfused transcardially under deep chloral hydrate (400 mg/kg, IP) anesthesia. The brains were removed and processed for routine histological analysis. Coronal cryostat sections (30#m-thick) were stained with thionin and were inspected by light microscopy for the correct placement of dialysis probes and apparent signs of neurodegeneration. Only animals where the fiber track was limited to the targeted brain area were included in the data analysis.
Data Presentation The extracellular levels of endogenous KYNA, mostly expressed as a percentage of baseline levels, were not corrected for recovery through the dialysis probes (cf. [53]). All data were analyzed by two-way analysis of variance ( A N O V A ) followed either by Scheffe's post hoc test (statistical significance set at p < 0.05) or by a Bonferroni-corrected paired t-test. RESULTS
Chemically induced seizures KA-induced seizures. A dose of 10 m g / k g K A (SC) reliably produced a complex spectrum of behavioral seizures, as described in detail previously [2,20,36]. Within 30 min, animals (N = 30) showed sporadic wet-dog shakes and increased locomotor activity. Subsequently, the frequency of wet-dog shakes increased, and seizures were often accompanied by rearing and forelimb clonus. Between 1 and 4 h after the K A injection, all animals developed severe limbic seizures characterized by generalized convulsions, rearing and falling with strong salivation. The convulsions gradually subsided after 5 h, and no animal died as a late consequence of seizure activity. PTZ-induced seizures. PTZ (60 mg/kg, SC) reliably produced typical motor seizures, characterized by tonic/clonic convulsions. The rats (N = 7) first displayed a short period (several seconds) of running, flexion, and/or extension of fore- and/or hindlimbs, and proceeded to the loss of righting reflex and myoclonic jerks with a latency of about 2 min. The total time spent in behavioral seizures was always less than 10 min, and no deaths occurred as a consequence of the seizures.
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FIG. 1. Extracellular KYNA levels in the dorsal hippocampus of KA-treated rats: Effect of AOAA. KA was injected (10 mg/kg, SC) at time 0 (N = 6). Microdialysis was performed bilaterally as described in the text. In one hippocampus, AOAA (1 mM) was delivered for 3 h through the dialysis probe, starting 3 h after KA administration (11). The bar indicates the period of AOAA infusion. The other hippocampus was continuously perfused with Ringer solution alone (E]). Data (mean _+ SEM) are expressed as a percentage of basal levels obtained from the last three samples prior to time 0 (74.3 ± 6.3 fmol KYNA/30 #1 dialysate). See text for statistical analysis of the effect of KA. *p < 0.05 as compared to the same timepoint in the absence of AOAA.
BIC-induced seizures. In preliminary experiments, a dose of > 8 m g / k g BIC resulted in tonic/clonic convulsions and loss of the righting reflex followed by death in most animals. Thus, 6 m g / k g BIC ( S C ) was selected as a dose at which animals consistently showed behavioral seizure p h e n o m e n a without mortality. O f the six animals used in this group, two had tonic/clonic seizure episodes and displayed short periods of loss of righting reflex and limb extension. The other four rats showed running, jumping, and severe fore- a n d / o r hindlimb clonus, which appeared within 2 min after BIC administration. Behavioral abnormalities subsided within 30 min. PlL-induced seizures. The treatment regimen chosen for testing the effect of PIL-induced seizures consisted of an injection of scopolamine ( 1 m g / k g , SC) 30 min prior to PIL (325 m g / k g , SC). Animals receiving this treatment ( N = 6) developed behavioral seizures with an average latency of approximately 30 min. Animals showed wet dog shakes, head bobbing followed by intermittent forepaw myoclonus, rearing and falling, and severe salivation. These behavioral abnormalities lasted approximately 2 h. No mortality was observed. QUlN-Induced Seizures. An intrahippocampal injection of 120 nmol QUIN, which reliably induces EEG seizures lasting approximately 3 h [ 30,46 ], resulted in stereotypic behaviors such as sniffing, gnawing, and rearing in all rats ( N = 6) following a latency period of approximately 30 min. These changes, which were accompanied by " f r o z e n a p p e a r a n c e " and wet dog shakes, lasted about 1 h. Wild running, jumping, and ipsilateral turning occurred sporadically, and all behavioral indications of seizure activity subsided within 3 h after the injection. No seizure-related mortality was observed as a consequence of the QUIN injection. Seizure-Induced Changes in Endogenous KYNA In all five seizure paradigms, extracellular K Y N A began to rise from baseline levels (71.1 ± 2.6 fmol / 30 #1; total of 61 rats ) within the first hour after drug administration and gradually
reached a plateau of different duration. Thus, a peak of 1 . 5 - 3 times baseline concentrations was attained in the dorsal hippocampus after the administration of the chemoconvulsants. KA. Systemic K A administration caused the quantitatively largest increase in extracellular KYNA levels in the present series of experiments, reaching statistical significance at 2.5 h and a peak of approximately three times control levels after 5 h. This maximal elevation was sustained for another 4 h, and was followed by a slow decline over the course of the subsequent 12 h time period. Twenty-four hours after KA, KYNA levels were still twice as high as controls (Fig. 1 ). To examine if the increase in brain KYNA levels was caused by a seizure-related temporary opening of the b l o o d - b r a i n barrier to blood-borne KYNA, the nonspecific K Y N A synthesis inhibitor A O A A was used in a separate set of experiments. The drug ( 1 m M in the perfusion solution) was introduced 3 h after KA, i.e., at a time when KYNA levels had reached 60% of plateau values. In pilot experiments ( N = 6 ) , this treatment with A O A A caused neither behavioral abnormalities nor apparent neurodegenerative changes (data not shown). As depicted in Fig. 1, A O A A caused a rapid return to baseline KYNA levels within the 3 h perfusion period, but K Y N A concentration rebounded immediately after removal of the synthesis inhibitor. There was a significant interaction between treatment groups ( K A alone, K A plus A O A A , and controls) and posttreatment times [ F ( 3 6 , 2 7 0 ) = 12.94, P < 0.001]. This experiment demonstrated that cerebral de novo synthesis of K Y N A is primarily responsible for the overflow of extracellular KYNA after systemic K A administration. Quantitatively similar KA-induced increases in KYNA levels, here shown during the plateau period (5 h after K A ; Fig. 2), were observed in the dorsal and ventral hippocampus, the piriform cortex, and the striatum. Across regions, there was a significant difference between basal KYNA levels and KA-induced increases [ F ( 3 , 2 0 ) = 3.30, P < 0.05].
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FIG. 2. KA-induced elevation of extracellular KYNA levels in different brain areas. KA was injected (10 mg/kg, SC) at time 0 (N 6 rats per group). Open bars represent basal KYNA levels in the four brain regions, obtained from the last three samples prior to time 0. Hatched bars represent KYNA levels in dialysates during the fifth hour after KA treatment (plateau value; cf. Fig. I). *p < 0.05, **p < 0.01 as compared to baseline values (Bonferroni corrected t-test).
PTZ, BIC. and PIL. These three compounds caused transient increases in extracellular KYNA, peaking between 3 and 5 h and returning to baseline levels approximately 8 h later (Fig. 3 ). The elevation in K Y N A concentrations reached statistical significance at 1 h ( P T Z ) or 2.5 b (BIC and PIL) after the administration of the convulsants; i.e., the increase followed the occurrence of behavioral seizures. Peak values were reached at 3.5 h (PTZ; BIC) or 4 h ( P I L ) . Statistical significance was lost 6 h ( P T Z ) , 5 h ( B I C ) , or 11 h ( P I L ) following drug application. There was a significant interaction between treatment groups (PTZ, BIC, PIL, and controls) and posttreatment times [ F ( 6 0 , 4 0 0 ) = 8.30, p < 0.001]. In separate animals, A O A A ( 1 raM) was infused unilaterally during the first 4 h after PTZ administration ( N = 4 ) . In contrast to the contralateral hippocampus, which showed the expected increase in extracellular KYNA levels, no KYNA elevation was observed on the AOAA-treated side (data not shown). QUIN. As illustrated in Fig. 4, a unilateral intrahippocampal QUIN injection caused bilateral increases in extracellular KYNA. At 1.5 h, the increases reached statistical significance. Plateau levels ( l . 8 - f o l d control concentrations on both sides) were attained simultaneously in both hippocampi 3 - 4 h after the QUIN injection. Notably, similar to data obtained with KA, KYNA concentrations remained elevated bilaterally for at least 24 h following the application of QUIN. There was a significant interaction between treatment groups (ipsi- and contralateral sides and controls) and posttreatment times [ F ( 3 4 , 2 5 5 ) - 4.85, p < 0.001]. In an additional experiment, K Y N A levels in the injected hippocampus were found to remain elevated during 7 days of continuous perfusion, whereas KYNA levels on the contralateral side returned to baseline values within 48 h ( N = 4; data not s h o w n ) . Neuropathology Following Chemoconvulsant Administration Neuropathological changes were e x a m i n e d in all five seizure models. In Nissl-stained tissue sections, no apparent nerve cell loss was observed in the h i p p o c a m p u s or in other brain regions
in PTZ- or BIC-treated rats. Animals that received PIL or K A showed the well-documented, typical pattern of seizure-related neurodegeneration in the h i p p o c a m p u s and extrahippocampal brain areas [33,37,43]. As described previously, intrahippocampal QUIN application resulted in a circumscribed neuronal lesion that was limited to the h i p p o c a m p u s on the injected hemisphere [ 30]. DISCUSSION Using five well-established animal models of epilepsy, the present results demonstrate that chemoconvulsant-induced seizures are associated with an elevation of extracellular KYNA levels. KYNA levels began to rise within the first hour after drug administration, i.e., at the same time as behavioral signs of seizure activity were noted. Although the limited time resolution of microdialysis methodology and the absence of EEG recordings did not allow us to perform the desirable tight correlative analysis of neurochemical changes and seizure activity, the data clearly demonstrate that a spectrum of pharmacologically distinct convulsive triggers elicit similar responses of the endogenous KYNA system. It therefore appears reasonable to assume that the increase in extracellular KYNA described here constitutes a rapid yet rather nonspecific tissue reaction to seizure activity. The present study does not directly address the question of the cellular source of KYNA or of the mechanisms that are responsible for its elevation in the extracellular compartment following seizures. Both immunohistochemical and biochemical studies, the former using antibodies against K Y N A ' s biosynthetic enzyme KAT, have demonstrated that in the rat brain KYNA is predominantly produced in and liberated from astrocytes. Thus, the normal hippocampus harbors an abundance of KAT-containing astrocytes with only sporadic immunopositive neurons [ 9 ] , and extracellular baseline levels of KYNA are substantially elevated in the neuron-depleted, astrogliotic hippocampus in vivo [48]. Astrocytes are known to react rapidly to seizures and, as major determinants of the composition of the extracellular milieu, may play an important role in epileptic
SEIZURES AND E X T R A C E L L U L A R K Y N U R E N I C ACID
159
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150
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V
100
0
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FIG. 3. Extracellular KYNA levels in the dorsal hippocampus of PTZ (60 mg/kg; e)-, PIL (325 mg/kg + 1 mg/kg scopolamine; A)-, and BIC (6 mg/kg; II)-treated rats (N = 6 - 7 rats per group). Drugs were administered and experiments were performed as described in the text. Convulsants were administered at time 0 (2 h after perfusion with Ringer solution alone). Data (mean +_ SEM) are expressed as a percentage of basal levels obtained from the last three samples prior to time 0. Saline-injected controls (C)) contained 76.9 _+ 1.9 fmol KYNA/30 #1 dialysate, compiled over 20 h (N = 6). See text for statistical analyses.
pathophysiology [ 4,38,47 ]. It is therefore likely that the increase in extracellular KYNA observed in the present set o f experiments can be traced to hippocampal astrocytes. In this context, it is noteworthy that greatly hypertrophied astrocytes containing KAT-immunoreactivity have been described in the hippocampus in a rat model of chronic epilepsy [10]. This has led to the suggestion that an increased release o f KYNA from astrocytes into
the extracellular compartment may be involved in recurrent seizure phenomena. Very little is known about the mechanisms that govern the entry of K Y N A into the extracellular milieu and, in particular, how these mechanisms might be affected in pathologic situations. In the normal brain, KYNA release is Ca 2* -independent, and the bioavailability of K Y N A ' s precursor L-kynurenine constitutes an
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~ 8
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16
20
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FIG. 4. Extracellular KYNA levels in the dorsal hippocampus of rats receiving a unilateral intrahippocampal injection of QUIN. QUIN (I 20 nmol/1 #1) was infused at time 0 (N = 6). Microdialysis was performed in the ipsilateral (11) and contralateral ([3) hippocampus for an additional 24 h as described in the text. Data (mean -+ SEM) are expressed as a percentage of basal levels obtained from the last three samples prior to time 0 (77.8 -+ 6.9 fmol KYNA/30 #1 dialysate). See text for statistical analyses.
160
important determinant of both KYNA production and efflux [44]. Because astrocytes avidly accumulate kynurenine [34], it is also possible that a seizure-induced activation of glial uptake underlies the enhanced production of KYNA observed in the present study. Thus, seizure-induced increases in the permeability of the blood-brain barrier to glutamine [24] may cause a secondary augmentation of kynurenine transport into astrocytes [3]. Alternatively, the increases in the extracellular levels of KYNA reported here could be a consequence of enhanced K A T activity, increased synthesis of kynurenine in the brain, or enhanced influx of kynurenine from the periphery into the brain [12]. An increase in K A T activity is unlikely for two reasons. First, it is questionable if the speed of the seizure-induced KYNA elevation would allow sufficient time for the increased expression of KAT. Second, an increase in the activity of K A T should cause elevations in extracellular K Y N A also when exogenous kynurenine is used to elicit KYNA release. However, acute KAinduced seizures were shown previously not to result in elevated extracellular K Y N A levels when 200 pM kynurenine was employed to drive KYNA production [ 51 ]. The experiment using transient in vivo perfusion with the nonspecific K A T inhibitor A O A A demonstrates that de novo production of KYNA in the brain must be responsible for the enhanced overflow of KYNA after convulsive treatments (cf. Fig. 1). Thus, A O A A effectively reverted extracellular KYNA to baseline levels in K A - and PTZ-treated animals. Because the enzymatic machinery for kynurenine synthesis in the rodent brain is very inefficient [7,28], an increased influx of kynurenine from the periphery, perhaps accentuated by a temporary, seizure-related impairment of the blood-brain barrier [8,21], is likely to be the main cause of the K Y N A elevations reported here. Notably, as judged from the K A model, increases in extracellular K Y N A were not limited to the hippocampus but were also detected in the piriform cortex and the striatum. It therefore appears that the increase in the amount of kynurenine available for transamination may be rather uniform throughout the brain. Clearly, the precise fate of kynurenine during seizure episodes warrants further study. As a preferential antagonist of the glycine coagonist site of the N M D A receptor, which appears to play a critical role in seizure phenomena [16,26], K Y N A has been conceptually linked to seizure mechanisms since its anticonvulsant properties were discovered a decade ago [ 11 ]. Although exogenously supplied K Y N A is effective in experimental seizure models [11,29,39,42], however, the doses needed to prevent seizure activity far exceed endogenous K Y N A levels. Effective systemic administration, in particular, requires large quantities of the compound due to the very limited penetration of KYNA through the b l o o d - b r a i n barrier [12]. Until recently, most investigators therefore considered K Y N A a useful pharmacological tool but did not contemplate an anticonvulsant role of endogenous KYNA. Recent studies suggest, however, that fluctuations in the levels of endogenous extracellular K Y N A have functional consequences that are likely related to changes in N M D A receptor function. For example, K A T inhibition by nonspecific aminotransferase blockers such as A O A A or yacetylenic G A B A is associated with seizures and excitotoxic neurodegeneration, which can be blocked by N M D A receptor antagonists [17,18]. Conversely, > 10-fold increases in extracellular K Y N A in the hippocampus, caused by pharmacologically shifting brain kynurenine metabolism toward enhanced K Y N A production, have pronounced anticonvulsant effects against sound-induced seizures in D B A / 2 mice and maximal electroshock-induced seizures in rats [5,19]. A recent ultrastructural study using anti-K AT antibodies provides an anatom-
WU AND S C H W A R C Z
ical basis for the apparent effectiveness of endogenous KYNA. Thus, KAT-containing astrocytic processes were shown to be closely apposed to excitatory asymmetric synapses, suggesting that the concentration of KYNA in the cleft (i.e., at the postsynaptic receptor) may be higher than hitherto assumed [25 ]. If increases in extracellular K Y N A concentrations are indeed functionally significant, it is tempting to interpret the present data teleologically as an attempt of the brain to protect itself against an overstimulation of excitatory amino acid receptors. Regardless of the nature of the primary convulsant stimulus, seizure-related neurodegeneration is clearly excitotoxic in nature, and its extent is dependent on seizure intensity and duration [23]. It is therefore possible that increases in KYNA levels were sufficient to prevent nerve cell loss following the relatively brief seizures evoked by PTZ and BIC, but failed to counteract the more enduring effects of PIC and KA, which result in extensive neuronal damage in several regions of the brain [ 27,28,31 ]. Notably, KYNA elevations were particularly prolonged following the administration of KA, which produced the longest lasting seizures of all systemically applied chemoconvulsants. It is also noteworthy that a unilateral intrahippocampal injection of the direct N M D A receptor agonist QUIN, which causes bilateral seizures but only ipsilateral neuronal damage [ 30], resulted in prolonged bilateral increases in KYNA concentrations. This finding supports the idea that endogenous KYNA can limit seizure-related damage, but indicates that its potency is insufficient for blocking excessive receptor stimulation in tissue exposed to large amounts of an excitatory amino acid receptor agonist. The present study, and the recent discovery that extracellular K Y N A concentrations are also elevated in kindled animals [49], may be of relevance for conceptualizing new treatment strategies for epilepsy. A trend toward higher interictal and postictal K Y N A levels was recently demonstrated to exist in the cerebrospinal fluid of patients with complex partial seizures [13], so that mechanisms that operate in the rat may resemble those in the human epileptic brain. Although cerebrospinal fluid kynurenine concentrations in epileptic patients are reportedly below control values [ 54], further studies using a medication-free population and, ideally, brain microdialysis are needed to reexamine the status of both kynurenine and KYNA. Should such data reveal further parallels to the animal studies described here, epilepsy therapy with KYNA-enhancing pharmacological agents may be indicated. ACKNOWI,EDGEMENTS
The authors thank Mrs. Joycc Burgess for excellent secretarial assistance and Drs. Deborah Medoff and Kevin O'Grady for expert advice with statistical analyses. This work was supported by USPHS grant NS 16102.
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