- Email: [email protected]
Determination of extracellular kynurenic acid in the striatum of unanesthetized rats: Effect of aminooxyacetic acid
Neuroscienee Letters, 116 (1990) 198 203 Elsevier Scientific Publishers Ireland Ltd.
198
NSL 07052
Determination of extracellular kynurenic acid in the striatum of unanesthetized rats: effect of aminooxyacetic acid Carmela Speciale, Hui-Qiu Wu, Jan Bert P. Gramsbergen, Waldemar A. Turski, Urban Ungerstedt and Robert Schwarcz Maryland Psychiatric Research Center, University of Maryland School o[Medicine Baltimore, MD 21228 (U.S.A.) (Received 12 March 1990; Accepted 18 April 19903
Key words." Excitotoxin; Kynurenic acid; Kynurenine; Microdialysis; Neurodegenerative disease; Striaturn Kynurenic acid (KYNA) production from its bioprecursor L-kynurenine (KYN) was assessed in vivo by intrastriatal microdialysis in freely moving rats. In the absence of KYN, the extracellular concentration of KYNA was below the limit of assay sensitivity (i.e. < 8 pmol/30 Id). In the presence of KYN (5(~2000 I~M), KYNA concentration in the dialysate increased continuously to reach steady-state levels after 2 h of perfusion. Introduction of the unspecific transaminase inhibitor aminooxyacetic acid (AOAA) through the dialysis probe caused a progressive decrease of extracellular KYNA, which reached dose-dependent minimal levels within 2 h. One mM AOAA caused an almost complete depletion of KYNA in the dialysate. These data demonstrate that extracellular KYNA can be assessed by microdialysis and that AOAA can be used as a tool to examine the neurobiology of KYNA in awake, freely moving animals.
Following the discovery of its neuroinhibitory [7] and neuroprotective [1] properties, kynurenic acid (KYNA) has recently received substantial interest from neurobiologists. It is now clear that KYNA can interact competitively with all three classical ionotropic excitatory amino acids receptors in the brain [17], and in addition shows a particularly high affinity to the glycine site associated with the N-methyl-Daspartate receptor complex [3]. KYNA is an established tryptophan metabolite in the periphery [9] but can not penetrate the blood-brain barrier under physiological conditions (Fukui et al., submitted for publication). It is found in mammalian brain [5, 14] as a product of kynurenine aminotransferase (KAT), which has been described in rat and human brain tis-
Correspondence." R. Schwarcz, Maryland Psychiatric Research Center, P.O. Box 21247, Baltimore, MD 21228, U.S.A. 0304-3940/'90/$ 03.50 ((~, 1990 Elsevier Scientific Publishers Ireland Ltd.
199
sue [4, 6]. Experiments with rat tissue slices have demonstrated that KYNA's bioprecursor L-kynurenine (KYN) can be actively transported into brain cells [10] for subsequent transamination by KAT. Newly synthesized K Y N A is then rapidly liberated into the extracellular compartment by non-Ca 2+-dependent mechanisms [13]. Immunohistochemical studies using specific anti-rat KAT antibodies have very recently provided evidence for a predominantly astroglial localization of K Y N A biosynthesis in the rat brain (Okuno et al., submitted for publication). In order to extend the study of K Y N A neurobiology to the in vivo situation, we have now assessed the conversion of KYN to K Y N A by striatal microdialysis in awake, freely moving rats. Moreover, in a first attempt to modulate K Y N A production in vivo, we have tested the effects of aminooxyacetic acid (AOAA), an unspecific transaminase inhibitor which blocks KYNA production in brain slices with an IC50 of 25 FtM [13]. The present data have been in part published in abstract form [12]. Male Sprague-Dawley rats (180-230 g), kept at a 12h light-dark cycle with free access to food and water, were used in all experiments. All equipment used for the performance of intrastriatal microdialysis in unanesthetized rats was obtained from Carnegie Medicin (Stockholm, Sweden). L-kynurenine sulfate (KYN), K Y N A and AOAA were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Other chemicals were obtained from various commercial suppliers and were of the highest purity available. For microdialysis, rats were prepared as described previously [11]. Briefly, a 20 gauge guide cannula was positioned on the top of the striatum using a Kopf stereotaxic instrument (nosebar: - 2 . 3 , A: 1.0 mm anterior to bregma, L: 2.5 mm from the midline, V: 3.5 mm below the skull surface). On the next day, the dialysis probe (4 mm opening of the membrane) was inserted through the guide to extend vertically throughout the striatum. Ringer solution (150 mM NaCI, 4 mM KC1, 3 mM CaCI2, pH 6.0) was then delivered at a flow rate of 1 /A/min, and perfusion continued for 1 h to establish stable conditions for dialysis. Freshly prepared KYN, dissolved in Ringer solution, was then perfused for up to 5 h and fractions collected every 30 min. Under these conditions, approximately 25 % of the applied KYN diffuses through the membrane [11]. In experiments designed to examine the effect of AOAA on extracellular KYNA, animals were first perfused with a solution containing 500/~M KYN for 3 h to establish steady-state conditions (see below). Subsequently, various amounts of AOAA were added to the perfusion solution containing KYN, and dialysis was continued for an additional 4 h. K Y N A content of dialysate samples was measured in principle according to the method of Turski et al. [13]. Briefly, 30 ~1 perfusates were diluted (1:1, v:v) with 1 M HCl and applied to pre-washed Dowex 50 (H+-form) cation-exchange columns. The columns were then washed with 1 ml 0.1 M HC1 and 1 ml distilled water. K Y N A was eluted with 2 ml of distilled water and the eluate containing K Y N A was evaporated to dryness. The samples were resuspended in 300/~1 distilled water and a 200 /tl aliquot was applied to a 3/tm C18 HPLC column (100 × 3.2 mm i.d., Bioanalytical Systems, West Lafayette, IN, U.S.A.). K Y N A was eluted isocratically at a flow rate
200 400-
"
2ram
300.
Z
200-
I00" 5oopM -
200//.M 50/.LM
i
0 "~ Kyn
i
i
1
i
60
120
180
240
30o
T I M E (rain)
Fig. 1. Extracellular levels of KYNA in rat striatal dialysate following perfusion with KYN. The indicated KYN concentrations were present in the perfusion solution (i.e. only approximately 25 % reached the tissue, cf. text). Data represent the means _+ S.E.M. of 5 ~ rats per dose.
of 0.5 ml/min with a 50 mM ammonium acetate solution containing 5% methanol, and detected spectrophotometrically at 340 nm. Experimental data were corrected for KYNA recovery from the dialysis probe (established for every fiber in vitro prior to the experiment) and from the separation procedure (using appropriate standard KYNA). The average percentage of recovery for the entire procedure thus determined was 22.1 + 0.7 %. Following microdialysis, rats were perfused transcardially with a solution of 4% paraformaldehyde and 1% glutaraldehyde, and the position of the microdialysis probe in the striatum was examined in thionin-stained 30/Lm cryostat sections. Only data from animals with proper placement of the dialysis fiber were used for experimental analysis. KYNA concentrations in striatal dialysates obtained in the absence of KYN were below the limit of assay sensitivity (i.e. < 8 pmol/30 #1 fraction). Perfusion with KYN led to the appearance of extracellular KYNA. At all bioprecursor concentrations tested, KYNA levels in the dialysate increased during the initial 60-120 min of perfusion to reach dose-dependent steady-state concentrations lasting at least for an additional 3 h (Fig. 1). During the perfusion, animals did not display abnormal behavior.
201
IZO -
I00
3 0 p.M
"'8 ,oo
300/~ I mM !
0
t
I
60
!
=
fzo
i
I
Ioo
!
=
240
TIME (rain)
Fig. 2. Effectof different concentrations of AOAA on newlysynthesizedextracellular KYNA. AOAAwas added to the perfusion solution containing 500 aM KYN 3 h after perfusion with KYN alone. Results are expressed as a percentage of the mean KYNA levels measured in the last three 30 min dialysates obtained prior to the introduction of AOAA (average of all rats used: 83.8+ 4. I pmol KYNA/30~1). Data are the means + S.E.M. of 5 rats per dose of AOAA.
Introduction of A O A A through the dialysate probe after perfusion for three hours with K Y N (500 pM) alone caused a progressive decline of extracellular K Y N A concentrations. The action of A O A A was clearly dose-dependent, with 30 p M being inactive and 1 m M of the drug resulting in an almost quantitative abolition of measurable K Y N A in the dialysate. At all doses used, A O A A exerted its maximal effect within 2-2.5 h (Fig. 2). The present study confirms and extends to the in vivo situation the conclusions reached from our previous work with brain slices [13]. Thus, under steady-state conditions 1-2 % of the applied K Y N was converted to extracellular K Y N A , no saturation of the process was noted when up to 2 m M K Y N were included in the perfusion solution (i.e. approximately 500 p M can be expected to reach the brain), and A O A A was found to be a potent inhibitor of K Y N A production. Notably, assessment by microdialysis did not reveal any evidence for a down-regulation of K Y N A production over time even at the highest K Y N concentration used. Clearly, the precise mechanisms involved in the regulation of extracellular steady-state concentrations of K Y N A remain to be examined, and can be expected to involve modulation at the level of K Y N uptake [10], K A T [4, 6] and diffusion out of the brain [15]. In addition, as yet only vaguely defined neuron-glia interactions appear to have the capacity for modulating extracellular K Y N A concentrations [2], whereas K Y N A re-
202 uptake mechanisms and enzymatic degradation do not seem to exist in cerebral tissue [15]. Although the diffusion of A O A A through the dialysis membrane was not determined in quantitative terms in this study, the dose-effect relationship for A O A A shown here is reasonably close to that determined in brain tissue slices [13]. Work with a pure K A T preparation indicates that the A O A A effect shown in vitro and now in the in vivo situation is due to a direct drug effect on the enzyme (unpublished data). Importantly, the present data demonstrate that AOAA, in spite of its promiscuous qualities as an unspecific transaminase inhibitor and its well-established effects on the GABAergic system [18, 19], can be used as a tool to examine the neurobiology of K Y N A in vivo. For example, A O A A could be employed to investigate if a depletion of brain K Y N A causes a relative over-activation of endogenous excitatory amino acid receptors; if verified experimentally, this would indicate that K Y N A ' s presence in the extracellular compartment is instrumental for the maintenance of tonic inhibition of those receptors under physiological conditions. As a pharmacological probe capable of interfering with the disposition of brain K Y N A in vivo, A O A A should also prove to be of value for examining the long-proposed [1, 8] metabolic relationship between K Y N A and the excitotoxin quinolinic acid in the brain. Thus, it will now be possible to test experimentally if a decrease in K Y N A causes a rise in the concentration of brain quinolinic acid and, as discussed previously [1, 8], if such a metabolic shift might result in quinolinate-mediated neuronal damage. Preliminary experiments employing intrahippocampal (McMaster et al., manuscript in preparation) or intrastriatal [16] injections of A O A A in the rat have shown that the drug can indeed cause quinolinate-like neurodegeneration and seizures, thus providing the first evidence that excitotoxic damage may be inflicted by an agent which indirectly influences excitatory amino acid receptor function. We thank Owen McMaster and Deborah Parks for their contributions and Mrs. Joyce Burgess for excellent assistance with the preparation of the manuscript. This work was supported by a Fogarty fellowship (to C.S.) and, in part, by U S P H S Grants NS 28236 and M H 44211. 1 Foster, A.C., Vezzani. A., French, E.D. and Schwarcz, R., Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid, Neurosci. Len., 48 (1984) 273 278. 2 Gramsbergen, J.B.P., Turski, W.A. and Schwarcz, R., Neuronal activityaffectskynurenicacid production in rat brain slices, Soc. Neurosci. Abstr., 14 (1988) 479.12. 3 Kessler, M., Terramani, T., Lynch, G. and Baudry, M., A glycine site associated with N-methyl-t)aspartic acid receptors: Characterization and identification of a new class of antagonists, J. Neurochem., 52 (1989) 1319-1328. 4 Minatogawa, Y., Noguchi, T. and Kido, R., Kynurenine pyruvate transaminase in rat brain, J. Neurochem., 47 (1974) 27l 272. 5 Moroni, F., Russi, P., Lombardi, G., Beni, M. and Carla, V., Presence of kynurenic acid in the mammalian brain, J. Neurochem., 51 (1988) 177-180. 6 Nakamura, M., Okuno, E., Whetsell, W.O. Jr. and Schwarcz, R., Kynurenine aminotransferasein human brain, Soc, Neurosci. Abstr., 14 (1988) 479.10.
203 7 Perkins, M.N. and Stone, T.W., An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid, Brain Res., 247 (1982) 183187. 8 Schwarcz, R., Foster, A.C., French, E.D., Whetsell, W.O. Jr. and K6hler, C., Excitotoxic models for neurodegenerative disorders, Life Sci., 35 (1984) 19-32. 9 Schwarcz, R., Young, S.N. and Brown, R.R. (Eds.), Kynurenine and Serotonin Pathways: Progress in Tryptophan Research, Plenum, New York, 1990. 10 Speciale, C. and Schwarcz, R., Uptake of kynurenine into rat brain slices, J. Neurochem., 54 (1990) 156-163. I 1 Speciale, C., Ungerstedt, U. and Schwarcz, R., Production of extracellular quinolinic acid in the striaturn studied by microdialysis in unanesthetized rats, Neurosci. Lett., 104 (1989) 345-350. 12 Turski, W.A., Gramsbergen, J.B.P., Speciale, C., Ungerstedt, U. and Schwarcz, R., Synthesis ofkynurenic acid from its bioprecursor L-kynurenine in rat brain in vitro and in vivo, Soc. Neurosci. Abstr., 14 (1988) 479.11. 13 Turski, W.A., Gramsbergen, J.B.P., Traitler, H. and Schwarcz, R., Rat brain slices produce and liberate kynurenic acid upon exposure to L-kynurenine, J. Neurochem., 52 (1989) 1629-1636. 14 Turski, W.A., Nakamura, M., Todd, W.P., Carpenter, B.K., Whetsell, W.O. Jr. and Schwarcz, R., Identification and quantification of kynurenic acid in human brain tissue, Brain Res., 454 (1988) 164169. 15 Turski, W.A. and Schwarcz, R., On the disposition of intrahippocampally injected kynurenic acid in the rat, Exp. Brain Res., 71 (1988) 563-567. 16 Urbanska, E., Ikonomidou, C., Sieklucka, M. and Turski, W.A., Aminooxyacetic acid produces excitotoxic lesions in the rat striatum, Soc. Neurosci. Abstr., 15 (1989) 306.1. 17 Watkins, J.C., Krogsgaard-Larsen, P. and Honor6, T., Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists, Trends Pharmacol. Sci., 11 (1990) 26-33. 18 Wood, J.D., Kurylo, E. and Newstead, J.D., Aminooxyacetic acid induced changes in y-aminobutyrate metabolism on the subcellular level, Can. J. Biochem., 56 (1978) 667~72. 19 Wood, J.D. and Peesker, S.J., The role of GABA metabolism in the convulsant and anti-convulsant actions of aminooxyacetic acid, J. Neurochem., 20 (I 973) 379-387.
198
NSL 07052
Determination of extracellular kynurenic acid in the striatum of unanesthetized rats: effect of aminooxyacetic acid Carmela Speciale, Hui-Qiu Wu, Jan Bert P. Gramsbergen, Waldemar A. Turski, Urban Ungerstedt and Robert Schwarcz Maryland Psychiatric Research Center, University of Maryland School o[Medicine Baltimore, MD 21228 (U.S.A.) (Received 12 March 1990; Accepted 18 April 19903
Key words." Excitotoxin; Kynurenic acid; Kynurenine; Microdialysis; Neurodegenerative disease; Striaturn Kynurenic acid (KYNA) production from its bioprecursor L-kynurenine (KYN) was assessed in vivo by intrastriatal microdialysis in freely moving rats. In the absence of KYN, the extracellular concentration of KYNA was below the limit of assay sensitivity (i.e. < 8 pmol/30 Id). In the presence of KYN (5(~2000 I~M), KYNA concentration in the dialysate increased continuously to reach steady-state levels after 2 h of perfusion. Introduction of the unspecific transaminase inhibitor aminooxyacetic acid (AOAA) through the dialysis probe caused a progressive decrease of extracellular KYNA, which reached dose-dependent minimal levels within 2 h. One mM AOAA caused an almost complete depletion of KYNA in the dialysate. These data demonstrate that extracellular KYNA can be assessed by microdialysis and that AOAA can be used as a tool to examine the neurobiology of KYNA in awake, freely moving animals.
Following the discovery of its neuroinhibitory [7] and neuroprotective [1] properties, kynurenic acid (KYNA) has recently received substantial interest from neurobiologists. It is now clear that KYNA can interact competitively with all three classical ionotropic excitatory amino acids receptors in the brain [17], and in addition shows a particularly high affinity to the glycine site associated with the N-methyl-Daspartate receptor complex [3]. KYNA is an established tryptophan metabolite in the periphery [9] but can not penetrate the blood-brain barrier under physiological conditions (Fukui et al., submitted for publication). It is found in mammalian brain [5, 14] as a product of kynurenine aminotransferase (KAT), which has been described in rat and human brain tis-
Correspondence." R. Schwarcz, Maryland Psychiatric Research Center, P.O. Box 21247, Baltimore, MD 21228, U.S.A. 0304-3940/'90/$ 03.50 ((~, 1990 Elsevier Scientific Publishers Ireland Ltd.
199
sue [4, 6]. Experiments with rat tissue slices have demonstrated that KYNA's bioprecursor L-kynurenine (KYN) can be actively transported into brain cells [10] for subsequent transamination by KAT. Newly synthesized K Y N A is then rapidly liberated into the extracellular compartment by non-Ca 2+-dependent mechanisms [13]. Immunohistochemical studies using specific anti-rat KAT antibodies have very recently provided evidence for a predominantly astroglial localization of K Y N A biosynthesis in the rat brain (Okuno et al., submitted for publication). In order to extend the study of K Y N A neurobiology to the in vivo situation, we have now assessed the conversion of KYN to K Y N A by striatal microdialysis in awake, freely moving rats. Moreover, in a first attempt to modulate K Y N A production in vivo, we have tested the effects of aminooxyacetic acid (AOAA), an unspecific transaminase inhibitor which blocks KYNA production in brain slices with an IC50 of 25 FtM [13]. The present data have been in part published in abstract form [12]. Male Sprague-Dawley rats (180-230 g), kept at a 12h light-dark cycle with free access to food and water, were used in all experiments. All equipment used for the performance of intrastriatal microdialysis in unanesthetized rats was obtained from Carnegie Medicin (Stockholm, Sweden). L-kynurenine sulfate (KYN), K Y N A and AOAA were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Other chemicals were obtained from various commercial suppliers and were of the highest purity available. For microdialysis, rats were prepared as described previously [11]. Briefly, a 20 gauge guide cannula was positioned on the top of the striatum using a Kopf stereotaxic instrument (nosebar: - 2 . 3 , A: 1.0 mm anterior to bregma, L: 2.5 mm from the midline, V: 3.5 mm below the skull surface). On the next day, the dialysis probe (4 mm opening of the membrane) was inserted through the guide to extend vertically throughout the striatum. Ringer solution (150 mM NaCI, 4 mM KC1, 3 mM CaCI2, pH 6.0) was then delivered at a flow rate of 1 /A/min, and perfusion continued for 1 h to establish stable conditions for dialysis. Freshly prepared KYN, dissolved in Ringer solution, was then perfused for up to 5 h and fractions collected every 30 min. Under these conditions, approximately 25 % of the applied KYN diffuses through the membrane [11]. In experiments designed to examine the effect of AOAA on extracellular KYNA, animals were first perfused with a solution containing 500/~M KYN for 3 h to establish steady-state conditions (see below). Subsequently, various amounts of AOAA were added to the perfusion solution containing KYN, and dialysis was continued for an additional 4 h. K Y N A content of dialysate samples was measured in principle according to the method of Turski et al. [13]. Briefly, 30 ~1 perfusates were diluted (1:1, v:v) with 1 M HCl and applied to pre-washed Dowex 50 (H+-form) cation-exchange columns. The columns were then washed with 1 ml 0.1 M HC1 and 1 ml distilled water. K Y N A was eluted with 2 ml of distilled water and the eluate containing K Y N A was evaporated to dryness. The samples were resuspended in 300/~1 distilled water and a 200 /tl aliquot was applied to a 3/tm C18 HPLC column (100 × 3.2 mm i.d., Bioanalytical Systems, West Lafayette, IN, U.S.A.). K Y N A was eluted isocratically at a flow rate
200 400-
"
2ram
300.
Z
200-
I00" 5oopM -
200//.M 50/.LM
i
0 "~ Kyn
i
i
1
i
60
120
180
240
30o
T I M E (rain)
Fig. 1. Extracellular levels of KYNA in rat striatal dialysate following perfusion with KYN. The indicated KYN concentrations were present in the perfusion solution (i.e. only approximately 25 % reached the tissue, cf. text). Data represent the means _+ S.E.M. of 5 ~ rats per dose.
of 0.5 ml/min with a 50 mM ammonium acetate solution containing 5% methanol, and detected spectrophotometrically at 340 nm. Experimental data were corrected for KYNA recovery from the dialysis probe (established for every fiber in vitro prior to the experiment) and from the separation procedure (using appropriate standard KYNA). The average percentage of recovery for the entire procedure thus determined was 22.1 + 0.7 %. Following microdialysis, rats were perfused transcardially with a solution of 4% paraformaldehyde and 1% glutaraldehyde, and the position of the microdialysis probe in the striatum was examined in thionin-stained 30/Lm cryostat sections. Only data from animals with proper placement of the dialysis fiber were used for experimental analysis. KYNA concentrations in striatal dialysates obtained in the absence of KYN were below the limit of assay sensitivity (i.e. < 8 pmol/30 #1 fraction). Perfusion with KYN led to the appearance of extracellular KYNA. At all bioprecursor concentrations tested, KYNA levels in the dialysate increased during the initial 60-120 min of perfusion to reach dose-dependent steady-state concentrations lasting at least for an additional 3 h (Fig. 1). During the perfusion, animals did not display abnormal behavior.
201
IZO -
I00
3 0 p.M
"'8 ,oo
300/~ I mM !
0
t
I
60
!
=
fzo
i
I
Ioo
!
=
240
TIME (rain)
Fig. 2. Effectof different concentrations of AOAA on newlysynthesizedextracellular KYNA. AOAAwas added to the perfusion solution containing 500 aM KYN 3 h after perfusion with KYN alone. Results are expressed as a percentage of the mean KYNA levels measured in the last three 30 min dialysates obtained prior to the introduction of AOAA (average of all rats used: 83.8+ 4. I pmol KYNA/30~1). Data are the means + S.E.M. of 5 rats per dose of AOAA.
Introduction of A O A A through the dialysate probe after perfusion for three hours with K Y N (500 pM) alone caused a progressive decline of extracellular K Y N A concentrations. The action of A O A A was clearly dose-dependent, with 30 p M being inactive and 1 m M of the drug resulting in an almost quantitative abolition of measurable K Y N A in the dialysate. At all doses used, A O A A exerted its maximal effect within 2-2.5 h (Fig. 2). The present study confirms and extends to the in vivo situation the conclusions reached from our previous work with brain slices [13]. Thus, under steady-state conditions 1-2 % of the applied K Y N was converted to extracellular K Y N A , no saturation of the process was noted when up to 2 m M K Y N were included in the perfusion solution (i.e. approximately 500 p M can be expected to reach the brain), and A O A A was found to be a potent inhibitor of K Y N A production. Notably, assessment by microdialysis did not reveal any evidence for a down-regulation of K Y N A production over time even at the highest K Y N concentration used. Clearly, the precise mechanisms involved in the regulation of extracellular steady-state concentrations of K Y N A remain to be examined, and can be expected to involve modulation at the level of K Y N uptake [10], K A T [4, 6] and diffusion out of the brain [15]. In addition, as yet only vaguely defined neuron-glia interactions appear to have the capacity for modulating extracellular K Y N A concentrations [2], whereas K Y N A re-
202 uptake mechanisms and enzymatic degradation do not seem to exist in cerebral tissue [15]. Although the diffusion of A O A A through the dialysis membrane was not determined in quantitative terms in this study, the dose-effect relationship for A O A A shown here is reasonably close to that determined in brain tissue slices [13]. Work with a pure K A T preparation indicates that the A O A A effect shown in vitro and now in the in vivo situation is due to a direct drug effect on the enzyme (unpublished data). Importantly, the present data demonstrate that AOAA, in spite of its promiscuous qualities as an unspecific transaminase inhibitor and its well-established effects on the GABAergic system [18, 19], can be used as a tool to examine the neurobiology of K Y N A in vivo. For example, A O A A could be employed to investigate if a depletion of brain K Y N A causes a relative over-activation of endogenous excitatory amino acid receptors; if verified experimentally, this would indicate that K Y N A ' s presence in the extracellular compartment is instrumental for the maintenance of tonic inhibition of those receptors under physiological conditions. As a pharmacological probe capable of interfering with the disposition of brain K Y N A in vivo, A O A A should also prove to be of value for examining the long-proposed [1, 8] metabolic relationship between K Y N A and the excitotoxin quinolinic acid in the brain. Thus, it will now be possible to test experimentally if a decrease in K Y N A causes a rise in the concentration of brain quinolinic acid and, as discussed previously [1, 8], if such a metabolic shift might result in quinolinate-mediated neuronal damage. Preliminary experiments employing intrahippocampal (McMaster et al., manuscript in preparation) or intrastriatal [16] injections of A O A A in the rat have shown that the drug can indeed cause quinolinate-like neurodegeneration and seizures, thus providing the first evidence that excitotoxic damage may be inflicted by an agent which indirectly influences excitatory amino acid receptor function. We thank Owen McMaster and Deborah Parks for their contributions and Mrs. Joyce Burgess for excellent assistance with the preparation of the manuscript. This work was supported by a Fogarty fellowship (to C.S.) and, in part, by U S P H S Grants NS 28236 and M H 44211. 1 Foster, A.C., Vezzani. A., French, E.D. and Schwarcz, R., Kynurenic acid blocks neurotoxicity and seizures induced in rats by the related brain metabolite quinolinic acid, Neurosci. Len., 48 (1984) 273 278. 2 Gramsbergen, J.B.P., Turski, W.A. and Schwarcz, R., Neuronal activityaffectskynurenicacid production in rat brain slices, Soc. Neurosci. Abstr., 14 (1988) 479.12. 3 Kessler, M., Terramani, T., Lynch, G. and Baudry, M., A glycine site associated with N-methyl-t)aspartic acid receptors: Characterization and identification of a new class of antagonists, J. Neurochem., 52 (1989) 1319-1328. 4 Minatogawa, Y., Noguchi, T. and Kido, R., Kynurenine pyruvate transaminase in rat brain, J. Neurochem., 47 (1974) 27l 272. 5 Moroni, F., Russi, P., Lombardi, G., Beni, M. and Carla, V., Presence of kynurenic acid in the mammalian brain, J. Neurochem., 51 (1988) 177-180. 6 Nakamura, M., Okuno, E., Whetsell, W.O. Jr. and Schwarcz, R., Kynurenine aminotransferasein human brain, Soc, Neurosci. Abstr., 14 (1988) 479.10.
203 7 Perkins, M.N. and Stone, T.W., An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid, Brain Res., 247 (1982) 183187. 8 Schwarcz, R., Foster, A.C., French, E.D., Whetsell, W.O. Jr. and K6hler, C., Excitotoxic models for neurodegenerative disorders, Life Sci., 35 (1984) 19-32. 9 Schwarcz, R., Young, S.N. and Brown, R.R. (Eds.), Kynurenine and Serotonin Pathways: Progress in Tryptophan Research, Plenum, New York, 1990. 10 Speciale, C. and Schwarcz, R., Uptake of kynurenine into rat brain slices, J. Neurochem., 54 (1990) 156-163. I 1 Speciale, C., Ungerstedt, U. and Schwarcz, R., Production of extracellular quinolinic acid in the striaturn studied by microdialysis in unanesthetized rats, Neurosci. Lett., 104 (1989) 345-350. 12 Turski, W.A., Gramsbergen, J.B.P., Speciale, C., Ungerstedt, U. and Schwarcz, R., Synthesis ofkynurenic acid from its bioprecursor L-kynurenine in rat brain in vitro and in vivo, Soc. Neurosci. Abstr., 14 (1988) 479.11. 13 Turski, W.A., Gramsbergen, J.B.P., Traitler, H. and Schwarcz, R., Rat brain slices produce and liberate kynurenic acid upon exposure to L-kynurenine, J. Neurochem., 52 (1989) 1629-1636. 14 Turski, W.A., Nakamura, M., Todd, W.P., Carpenter, B.K., Whetsell, W.O. Jr. and Schwarcz, R., Identification and quantification of kynurenic acid in human brain tissue, Brain Res., 454 (1988) 164169. 15 Turski, W.A. and Schwarcz, R., On the disposition of intrahippocampally injected kynurenic acid in the rat, Exp. Brain Res., 71 (1988) 563-567. 16 Urbanska, E., Ikonomidou, C., Sieklucka, M. and Turski, W.A., Aminooxyacetic acid produces excitotoxic lesions in the rat striatum, Soc. Neurosci. Abstr., 15 (1989) 306.1. 17 Watkins, J.C., Krogsgaard-Larsen, P. and Honor6, T., Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists, Trends Pharmacol. Sci., 11 (1990) 26-33. 18 Wood, J.D., Kurylo, E. and Newstead, J.D., Aminooxyacetic acid induced changes in y-aminobutyrate metabolism on the subcellular level, Can. J. Biochem., 56 (1978) 667~72. 19 Wood, J.D. and Peesker, S.J., The role of GABA metabolism in the convulsant and anti-convulsant actions of aminooxyacetic acid, J. Neurochem., 20 (I 973) 379-387.