Extracellular Kynurenic Acid Levels Research Articles

0298 Kynurenic Acid Synthesis Inhibitor Promotes Enhanced Sleep Recovery Following Acute Sleep Deprivation in Adult Wistar Rats

Abstract Introduction Sleep disorders and cognitive dysfunction often afflict the general population and are common amongst patients with neuropsychiatric disorders like schizophrenia. Sleep deprivation (SD) disrupts cognitive function, yet little is known about underlying mechanisms. The tryptophan metabolite kynurenic acid (KYNA), an endogenous a7nACh and NMDA receptor antagonist, is synthesized by kynurenine aminotransferase II (KAT II). KYNA is increased in the brain of patients with schizophrenia and our translational studies demonstrate that elevated KYNA disrupts hippocampal learning and sleep architecture in rodents (Pocivavsek et al. 2017 Sleep). We hypothesize a mechanistic link between KYNA, sleep, and cognitive dysfunction. Methods In vivo microdialysis in the dorsal hippocampus and simultaneous EEG/EMG telemetry was conducted in adult male Wistar rats (N=3-5 per group). Using a within-subjects experimental design, rats underwent a control and SD day. Animals received either vehicle or KAT II inhibitor PF-04859989 (PF), 30mg/kg s.c., on both days at zeitgeber time (ZT) 0 or ZT6. SD occurred from ZT0-6 by gentle handling. KYNA levels were evaluated in the microdialysate. Results SD effectively eliminated REM sleep (100%) and significantly reduced NREM (94%) during ZT0-6. Extracellular KYNA levels in the hippocampus significantly increased with SD (2-way ANOVA, time x SD: **P<0.0001) and PF readily prevented this accumulation. Initial sleep recovery (ZT6-12) did not significantly differ between treatment groups. During the dark phase (ZT12-24), PF treatment of SD animals promoted REM sleep parameters, including total REM duration (2-way ANOVA, SD x treatment ZT: P<0.05). PF treatment enhanced theta spectral power determined by Discrete Fourier transform during REM sleep recovery (ZT12-24). PF alone during the control day enhanced NREM delta power (P<0.05) during the late light phase (ZT6-12). Conclusion Importantly, the KAT II inhibitor PF promoted sleep recovery following acute SD, supporting our hypothesis that the accumulation of KYNA may exacerbate sleep disruptions. Changes in sleep parameters elicited by PF, a potential therapeutic avenue, may be indicative of mild somnolence. The present and future complementary experiments with cognitive behavioral tasks in rodents support our understanding of the role of KYNA in modulating sleep and cognition. Support (If Any) National Institutes of Health Grant No. NIH R01 NS102209

Stress-induced impairment in fear discrimination is causally related to increased kynurenic acid formation in the prefrontal cortex.

Stress is related to cognitive impairments which are observed in most major brain diseases. Prior studies showed that the brain concentration of the tryptophan metabolite kynurenic acid (KYNA) is modulated by stress, and that changes in cerebral KYNA levels impact cognition. However, the link between these phenomena has not been tested directly so far. To investigate a possible causal relationship between acute stress, KYNA, and fear discrimination. Adult rats were exposed to one of three acute stressors-predator odor, restraint, or inescapable foot shocks (ISS)-and KYNA in the prefrontal cortex was measured using microdialysis. Corticosterone was analyzed in a subset of rats. Another cohort underwent a fear discrimination procedure immediately after experiencing stress. Different auditory conditioned stimuli (CSs) were either paired with foot shock (CS+) or were non-reinforced (CS-). One week later, fear was assessed by re-exposing rats to each CS. Finally, to test whether stress-induced changes in KYNA causally impacted fear discrimination, a group of rats that received ISS were pre-treated with the selective KYNA synthesis inhibitor PF-04859989. ISS caused the greatest increase in circulating corticosterone levels and raised extracellular KYNA levels by ~ 85%. The two other stressors affected KYNA much less (< 25% increase). Moreover, only rats that received ISS were unable to discriminate between CS+ and CS-. PF-04859989 abolished the stress-induced KYNA increase and also prevented the impairment in fear discrimination in animals that experienced ISS. These findings demonstrate a causal connection between stress-induced KYNA increases and cognitive deficits. Pharmacological manipulation of KYNA synthesis therefore offers a novel approach to modulate cognitive processes in stress-related disorders.

Prenatal THC exposure raises kynurenic acid levels in the prefrontal cortex of adult rats

Cannabis remains one of the most widely used illicit drugs during pregnancy. The main psychoactive component of marijuana (Δ9-tetrahydrocannabinol, THC) is correlated with untoward physiological effects in the offspring. Neurobehavioral and cognitive impairments have been reported in longitudinal studies on children and adolescents prenatally exposed to marijuana, and a link to psychiatric disorders has been proposed. Interestingly, the deleterious effects of prenatal cannabis use are similar to those observed in adult rats prenatally exposed to (L)-kynurenine, the direct bioprecursor of the neuroactive metabolite kynurenic acid (KYNA). We therefore investigated whether alterations in KYNA levels in the rat brain might play a role in the long-term consequences of prenatal cannabinoid exposure. Pregnant Wistar rats were treated daily with THC [5 mg/kg, p.o.] from gestational day (GD)5 through GD20. Using in vivo microdialysis in the medial prefrontal cortex, adult animals were then used to determine the extracellular levels of KYNA and glutamate. Compared to controls, extracellular basal KYNA levels were higher, and basal glutamate levels were lower, in prenatally THC-exposed rats. These rats also showed abnormal short-term memory. Following an additional acute challenge with a low dose of kynurenine (5 mg/kg i.p.) in adulthood, the increase in extracellular KYNA levels in the mPFC was more pronounced in in prenatally THC-exposed rats. These effects could be causally related to the cognitive dysfunction seen in prenatally THC-exposed rats. In the translational realm, these experiments raise the prospect of prevention of KYNA neosynthesis as a promising novel approach to combat some of the detrimental long-term effects of prenatal cannabis use.

Astrocytic Mechanisms Involving Kynurenic Acid Control Δ9-Tetrahydrocannabinol-Induced Increases in Glutamate Release in Brain Reward-Processing Areas.

The reinforcing effects of Δ9-tetrahydrocannabinol (THC) in rats and monkeys, and the reinforcement-related dopamine-releasing effects of THC in rats, can be attenuated by increasing endogenous levels of kynurenic acid (KYNA) through systemic administration of the kynurenine 3-monooxygenase inhibitor, Ro 61-8048. KYNA is a negative allosteric modulator of α7 nicotinic acetylcholine receptors (α7nAChRs) and is synthesized and released by astroglia, which express functional α7nAChRs and cannabinoid CB1 receptors (CB1Rs). Here, we tested whether these presumed KYNA autoreceptors (α7nAChRs) and CB1Rs regulate glutamate release. We used in vivo microdialysis and electrophysiology in rats, RNAscope in situ hybridization in brain slices, and primary culture of rat cortical astrocytes. Acute systemic administration of THC increased extracellular levels of glutamate in the nucleus accumbens shell (NAcS), ventral tegmental area (VTA), and medial prefrontal cortex (mPFC). THC also reduced extracellular levels of KYNA in the NAcS. These THC effects were prevented by administration of Ro 61-8048 or the CB1R antagonist, rimonabant. THC increased the firing activity of glutamatergic pyramidal neurons projecting from the mPFC to the NAcS or to the VTA in vivo. These effects were averted by pretreatment with Ro 61-8048. In vitro, THC elicited glutamate release from cortical astrocytes (on which we demonstrated co-localization of the CB1Rs and α7nAChR mRNAs), and this effect was prevented by KYNA and rimonabant. These results suggest a key role of astrocytes in interactions between the endocannabinoid system, kynurenine pathway, and glutamatergic neurotransmission, with ramifications for the pathophysiology and treatment of psychiatric and neurodegenerative diseases.

Alternative kynurenic acid synthesis routes studied in the rat cerebellum.

Kynurenic acid (KYNA), an astrocyte-derived, endogenous antagonist of α7 nicotinic acetylcholine and excitatory amino acid receptors, regulates glutamatergic, GABAergic, cholinergic and dopaminergic neurotransmission in several regions of the rodent brain. Synthesis of KYNA in the brain and elsewhere is generally attributed to the enzymatic conversion of L-kynurenine (L-KYN) by kynurenine aminotransferases (KATs). However, alternative routes, including KYNA formation from D-kynurenine (D-KYN) by D-amino acid oxidase (DAAO) and the direct transformation of kynurenine to KYNA by reactive oxygen species (ROS), have been demonstrated in the rat brain. Using the rat cerebellum, a region of low KAT activity and high DAAO activity, the present experiments were designed to examine KYNA production from L-KYN or D-KYN by KAT and DAAO, respectively, and to investigate the effect of ROS on KYNA synthesis. In chemical combinatorial systems, both L-KYN and D-KYN interacted directly with peroxynitrite (ONOO−) and hydroxyl radicals (OH•), resulting in the formation of KYNA. In tissue homogenates, the non-specific KAT inhibitor aminooxyacetic acid (AOAA; 1 mM) reduced KYNA production from L-KYN and D-KYN by 85.1 ± 1.7% and 27.1 ± 4.5%, respectively. Addition of DAAO inhibitors (benzoic acid, kojic acid or 3-methylpyrazole-5-carboxylic acid; 5 μM each) attenuated KYNA formation from L-KYN and D-KYN by ~35% and ~66%, respectively. ONOO− (25 μM) potentiated KYNA production from both L-KYN and D-KYN, and these effects were reduced by DAAO inhibition. AOAA attenuated KYNA production from L-KYN + ONOO− but not from D-KYN + ONOO−. In vivo, extracellular KYNA levels increased rapidly after perfusion of ONOO− and, more prominently, after subsequent perfusion with L-KYN or D-KYN (100 μM). Taken together, these results suggest that different mechanisms are involved in KYNA production in the rat cerebellum, and that, specifically, DAAO and ROS can function as alternative routes for KYNA production.

Targeting Kynurenine Aminotransferase II in Psychiatric Diseases: Promising Effects of an Orally Active Enzyme Inhibitor

Increased brain levels of the tryptophan metabolite kynurenic acid (KYNA) have been linked to cognitive dysfunctions in schizophrenia and other psychiatric diseases. In the rat, local inhibition of kynurenine aminotransferase II (KAT II), the enzyme responsible for the neosynthesis of readily mobilizable KYNA in the brain, leads to a prompt reduction in extracellular KYNA levels, and secondarily induces an increase in extracellular glutamate, dopamine, and acetylcholine levels in several brain areas. Using microdialysis in unanesthetized, adult rats, we now show that the novel, systemically active KAT II inhibitor BFF-816, applied orally at 30 mg/kg in all experiments, mimics the effects of local enzyme inhibition. No tolerance was seen when animals were treated daily for 5 consecutive days. Behaviorally, daily injections of BFF-816 significantly decreased escape latency in the Morris water maze, indicating improved performance in spatial and contextual memory. Thus, systemically applied BFF-816 constitutes an excellent tool for studying the neurobiology of KYNA and, in particular, for investigating the mechanisms linking KAT II inhibition to changes in glutamatergic, dopaminergic, and cholinergic function in brain physiology and pathology.

Targeted Deletion of Kynurenine 3-Monooxygenase in Mice: A NEW TOOL FOR STUDYING KYNURENINE PATHWAY METABOLISM IN PERIPHERY AND BRAIN

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo(-/-) mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo(-/-) mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo(-/-) mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD(+), did not differ between Kmo(-/-) and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo(-/-) mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo(-/-) mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.

Early developmental elevations of brain kynurenic acid impair cognitive flexibility in adults: Reversal with galantamine

Levels of kynurenic acid (KYNA), an endogenous α7 nicotinic acetylcholine receptor (α7nAChR) antagonist, are elevated in the brain of patients with schizophrenia (SZ) and might contribute to the pathophysiology and cognitive deficits seen in the disorder. As developmental vulnerabilities contribute to the etiology of SZ, we determined, in rats, the effects of perinatal increases in KYNA on brain chemistry and cognitive flexibility. KYNA’s bioprecursor l-kynurenine (100mg/day) was fed to dams from gestational day 15 to postnatal day 21 (PD21). Offspring were then given regular chow until adulthood. Control rats received unadulterated mash. Brain tissue levels of KYNA were measured at PD2 and PD21, and extracellular levels of KYNA and glutamate were determined by microdialysis in the prefrontal cortex in adulthood (PD56–80). In other adult rats, the effects of perinatal l-kynurenine administration on cognitive flexibility were assessed using an attentional set-shifting task. l-Kynurenine treatment raised forebrain KYNA levels ∼3-fold at PD2 and ∼2.5-fold at PD21. At PD56–80, extracellular prefrontal KYNA levels were moderately but significantly elevated (+12%), whereas extracellular glutamate levels were not different from controls. Set-shifting was selectively impaired by perinatal exposure to l-kynurenine, as treated rats acquired the discrimination and intra-dimensional shift at the same rate as controls, yet exhibited marked deficits in the initial reversal and extra-dimensional shift. Acute administration of the α7nAChR-positive modulator galantamine (3.0mg/kg, i.p.) restored performance to control levels. These results validate early developmental exposure to l-kynurenine as a novel, naturalistic animal model for studying cognitive deficits in SZ.

Poster #10 SOCIAL ISOLATION REARING IN RATS ALTERS PLASMA TRYPTOPHAN METABOLISM AND IS REVERSED BY SUB-CHRONIC CLOZAPINE TREATMENT

Levels of kynurenic acid (KYNA), an endogenous α7 nicotinic acetylcholine receptor (α7nAChR) antagonist, are elevated in the brain of patients with schizophrenia (SZ) and might contribute to the pathophysiology and cognitive deficits seen in the disorder. As developmental vulnerabilities contribute to the etiology of SZ, we determined, in rats, the effects of perinatal increases in KYNA on brain chemistry and cognitive flexibility. KYNA’s bioprecursor l-kynurenine (100 mg/day) was fed to dams from gestational day 15 to postnatal day 21 (PD21). Offspring were then given regular chow until adulthood. Control rats received unadulterated mash. Brain tissue levels of KYNA were measured at PD2 and PD21, and extracellular levels of KYNA and glutamate were determined by microdialysis in the prefrontal cortex in adulthood (PD56–80). In other adult rats, the effects of perinatal l-kynurenine administration on cognitive flexibility were assessed using an attentional set-shifting task. l-Kynurenine treatment raised forebrain KYNA levels ∼3-fold at PD2 and ∼2.5-fold at PD21. At PD56–80, extracellular prefrontal KYNA levels were moderately but significantly elevated (+12%), whereas extracellular glutamate levels were not different from controls. Set-shifting was selectively impaired by perinatal exposure to l-kynurenine, as treated rats acquired the discrimination and intra-dimensional shift at the same rate as controls, yet exhibited marked deficits in the initial reversal and extra-dimensional shift. Acute administration of the α7nAChR-positive modulator galantamine (3.0 mg/kg, i.p.) restored performance to control levels. These results validate early developmental exposure to l-kynurenine as a novel, naturalistic animal model for studying cognitive deficits in SZ.

Astrocyte‐derived kynurenic acid modulates basal and evoked cortical acetylcholine release

We tested the hypothesis that fluctuations in the levels of kynurenic acid (KYNA), an endogenous antagonist of the alpha7 nicotinic acetylcholine (ACh) receptor, modulate extracellular ACh levels in the medial prefrontal cortex in rats. Decreases in cortical KYNA levels were achieved by local perfusion of S-ESBA, a selective inhibitor of the astrocytic enzyme kynurenine aminotransferase II (KAT II), which catalyses the formation of KYNA from its precursor L-kynurenine. At 5 mm, S-ESBA caused a 30% reduction in extracellular KYNA levels, which was accompanied by a two-threefold increase in basal cortical ACh levels. Co-perfusion of KYNA in the endogenous range (100 nm), which by itself tended to reduce basal ACh levels, blocked the ability of S-ESBA to raise extracellular ACh levels. KYNA perfusion (100 nm) also prevented the evoked ACh release caused by d-amphetamine (2.0 mg/kg). This effect was duplicated by the systemic administration of kynurenine (50 mg/kg), which resulted in a significant increase in cortical KYNA formation. Jointly, these data indicate that astrocytes, by producing and releasing KYNA, have the ability to modulate cortical cholinergic neurotransmission under both basal and stimulated conditions. As cortical KYNA levels are elevated in individuals with schizophrenia, and in light of the established role of cortical ACh in executive functions, our findings suggest that drugs capable of attenuating the production of KYNA may be of benefit in the treatment of cognitive deficits in schizophrenia.

Kynurenic acid leads, dopamine follows: A new case of volume transmission in the brain?

Intrastriatal infusion of nanomolar concentrations of kynurenic acid (KYNA), an astrocyte-derived neuroinhibitory tryptophan metabolite, reduces basal extracellular dopamine (DA) levels in the rat striatum. This effect is initiated by the inhibition of alpha7 nicotinic acetylcholine receptors (alpha7nAChRs) on glutamatergic afferents. The present study was designed to further investigate this functional link between KYNA and DA using striatal microdialysis in awake animals. In rats, increases in KYNA, caused by intrastriatal infusions of KYNA itself (100 nM) or of KYNA's bioprecursor L-kynurenine (2 microM), were associated with substantial reductions in DA. Co-infusion of KYNA with the alpha7nAChR agonist galantamine (5 microM), but not with the NMDA receptor agonist D-serine (100 nM), prevented this effect. Moreover, KYNA also reduced DA levels in the NMDA-lesioned striatum. Conversely, extracellular DA levels were enhanced when KYNA formation was compromised, either by astrocyte poisoning with fluorocitrate or by perfusion with aminooxyacetic acid (AOAA; 5 mM), a non-specific inhibitor of KYNA synthesis. Notably, this effect of AOAA was prevented by co-perfusion with 100 nM KYNA. In the striatum of 21 day-old mice with a targeted deletion of kynurenine aminotransferase II, extracellular KYNA levels were reduced by 67 +/- 6%, while extracellular DA levels were simultaneously increased by 170 +/- 14%. Taken together, a picture emerges where fluctuations in the astrocytic production of KYNA, possibly through volume transmission, inversely regulate dopaminergic tone. This newly uncovered mechanism may profoundly influence DA function under physiological and pathological conditions.

Kynurenate and 7‐Chlorokynurenate Formation in Chronically Epileptic Rats

The tryptophan metabolite kynurenic acid (KYNA) and its synthetic derivative, 7-chlorokynurenic acid (7-Cl-KYNA), are antagonists of the glycine co-agonist ("glycine(B)") site of the N-methyl-D-aspartate (NMDA)-receptor. Both compounds have neuroprotective and anticonvulsive properties but do not readily penetrate the blood-brain barrier. However, KYNA and 7-Cl-KYNA can be formed in, and released from, astrocytes after the peripheral administration of their transportable precursors kynurenine and 4-chlorokynurenine, respectively. The present study was designed to examine these biosynthetic processes, as well as astrogliosis, in animals with spontaneously recurring seizures. The fate and formation of KYNA and 7-Cl-KYNA was studied in vivo (microdialysis) and in vitro (tissue slices) in rats exhibiting chronic seizure activity (pilocarpine model) and in appropriate controls. Neuronal loss and gliosis in these animals were examined immunohistochemically. In vivo microdialysis revealed higher ambient extracellular KYNA levels and enhanced de novo formation of 7-Cl-KYNA in the entorhinal cortex and hippocampus in epileptic rats. Complementary studies in tissue slices showed increased neosynthesis of KYNA and 7-Cl-KYNA in the same two brain areas. Microscopic analysis revealed pronounced astrocytic reactions in entorhinal cortex and hippocampus in epileptic animals. These results demonstrate that the epileptic brain can synthesize glycine(B) receptor antagonists in situ. Astrogliosis probably accounts for their enhanced production in chronically epileptic rats. These results bode well for the use of 4-chlorokynurenine in the treatment of chronic seizure disorders.

Neuronal A1 receptors mediate increase in extracellular kynurenic acid after local intrastriatal adenosine infusion.

The naturally occurring purine nucleoside adenosine has pronounced anticonvulsant and neuroprotective properties and plays a neuromodulatory role in the CNS. Kynurenic acid (KYNA) is an astrocyte-derived, endogenous neuroinhibitory compound, which shares several of adenosine's properties. In a first attempt to examine possible interactions between these two biologically active molecules, adenosine was focally applied into the striatum of freely moving rats by reverse microdialysis, and changes in extracellular KYNA were monitored over time. A 2-h infusion of adenosine increased KYNA levels in a dose-dependent manner, with 10 mm of adenosine causing a twofold elevation within 1 h. This effect was reversible and was effectively blocked by coinfusion of the specific A1 adenosine receptor antagonist 8-cyclopentyltheophylline (100 microm). In contrast, coinfusion of adenosine with MSX-3 (100 microm), an A2A receptor antagonist, did not affect the adenosine-induced increase in KYNA levels. Local striatal perfusion with the A1 receptor agonist N6-cyclopentyladenosine (100 microm) mimicked the effect of adenosine, whereas perfusion with the A2A receptor agonist CGS-21680 (100 microm) was ineffective. Finally, we tested the effect of adenosine (10 mm) on extracellular KYNA in striata that had been injected with quinolinate (60 nmol/1 microL) 7 days earlier. In this neuron-depleted tissue, perfusion with adenosine failed to affect extracellular KYNA levels. These data demonstrate that adenosine is capable of raising extracellular KYNA in the rat striatum by interacting with postsynaptic neuronal A1 receptors. This mechanism may result in a synergism between the neurobiological effects of adenosine and KYNA.

In vivo assessment of kynurenate neuroprotective potency and quinolinate excitotoxicity.

Three complementary questions related to the kynurenine pathway and excitotoxicity were addressed in this study: (i) Which extracellular levels of quinolinic acid (QUIN) may be neurotoxic? (ii) Which extracellular levels of kynurenic acid (KYNA) may control excessive NMDA-receptor function? (iii) Can "anti-excitotoxic" levels of KYNA be reached by inhibition of kynurenine-3-hydroxylase (i.e. inhibition of QUIN synthesis and shunts of kynurenine metabolism toward KYNA)? Multifunctional microdialysis probes were used in halothane anaesthetised rats to apply NMDA or QUIN directly to the brain, with or without co-perfusion of KYNA, to record the resulting local depolarisations, and to monitor changes in dialysate KYNA after kynurenine-3-hydroxylase inhibition. QUIN produced concentration-dependent depolarisations with an estimated EC50 (i.e. concentration in the perfusion medium) of 1.22mM. The estimated ED50 for KYNA inhibition of NMDA-responses was 181microM. Kynurenine-3-hydroxylase inhibition (Ro-61-8048, 100mg/kg i.p.) increased dialysate KYNA 11 times (to 33.8nM) but without any reduction of NMDA-responses. These data challenge the notion that extracellular accumulation of endogenous QUIN may contribute to excessive NMDA-receptor activation in some neurological disorders, and the suitability of kynurenine-3-hydroxylase inhibition as an effective anti-excitotoxic strategy.

Neuroprotective potency of kynurenic acid against excitotoxicity.

The aim of this study was to determine in vivo which extracellular levels of kynurenic acid (KYNA) are required to control excessive NMDA receptor activation in the rat cortex. As excitotoxicity is coupled to marked ion movements, local depolarisations induced by perfusion of NMDA or quinolinic acid (QUIN) through microdialysis probes were recorded at the site of excitotoxin application. Perfusion of KYNA through the dialysis fibre inhibited the excitotoxin responses with an IC50 of 32-66 microM (extracellular concentration corrected for microdialysis delivery), but > 10-fold lower levels of endogenous KYNA were reported to be neuroprotective. Accordingly, these results strengthen the notion that KYNA accumulation may protect the brain parenchyma by acting on different molecular target(s), besides the NMDA receptor glycine site.

Systemic d-amphetamine administration causes a reduction of kynurenic acid levels in rat brain

Tissue levels of the endogenous excitatory amino acid receptor antagonist kynurenic acid (KYNA) and of its bioprecursor l-kynurenine were measured in rats of different ages after d-amphetamine administration. In adult animals, extracellular KYNA concentrations were also determined in vivo by hippocampal microdialysis. In the adult brain, d-amphetamine caused a transient, dose-dependent decrease in tissue content and extracellular levels of KYNA, reaching a nadir of approximately 70% of control values after 1 h at 5 mg/kg. Quantitatively similar decrements were observed in four different brain regions. Seven, 14 and 28-day-old pups were particularly sensitive to the drug, showing a reduction in forebrain KYNA levels to 25%, 40% and 35% of control values, respectively, 1 h after the administration of 5 mg/kg d-amphetamine. Notably, no changes in brain l-kynurenine levels and in liver l-kynurenine and KYNA concentrations were found after d-amphetamine administration. Thus, endogenous monoamines released by d-amphetamine may interfere with the transamination of l-kynurenine to KYNA specifically in the brain. These results suggest that d-amphetamine increases excitatory amino acid receptor function temporarily by reducing the levels of endogenous KYNA.

Effect of probenecid on depolarizations evoked by N-methyl-D-aspartate (NMDA) in the rat striatum.

Kynurenic acid is an endogenous, competitive antagonist of the N-methyl-D-aspartate (NMDA) receptor glycine site. Accordingly, increasing the brain extracellular concentration of this metabolite may be a suitable alternative to administration of exogenous NMDA antagonists for the treatment of neurological disorders involving excessive NMDA-receptor activation. As competitive inhibition of organic anion transport by probenecid increased brain extracellular levels of kynurenic acid, the purpose of this study was to examine whether intracerebral application of probenecid reduced depolarizations evoked at the same tissue site by NMDA. Microdialysis probes incorporating an electrode were implanted into the striatum of rats and perfused with artificial cerebrospinal fluid. Local depolarizations were produced by perfusing 200 microM NMDA for 2 min, either alone, or co-applied with 1, 5 or 20 mM probenecid. The lowest concentration of probenecid had no effect. At 5 mM, probenecid abolished the hyperpolarization which consistently followed NMDA-responses, but the slight decrease in depolarization amplitude did not reach significance. Inhibition of post-depolarization hyperpolarization suggests that sustained, high extracellular concentrations of probenecid reduce the capacity of the tissue to recover from a depolarizing stimulus, presumably because intensive transport of probenecid imposes a heavy load on Na+, K(+)-ATPase. At 20 mM, probenecid inhibited NMDA-evoked depolarization by approximately 60% (from 4.7 +/- 0.7 mV to 2.1 +/- 0.2 mV; n = 6, P < 0.005). This effect was more marked 30 min after returning to perfusion with normal artificial cerebrospinal fluid, suggesting that high concentrations of probenecid may be toxic to nerve cells, or initiate long-lasting effects linked to inhibition of the transport of important organic anions. These data suggest that inhibition of organic anion transport is not, by itself, sufficient to protect against neurological disorders involving excessive NMDA-receptor activation. However, results from other studies suggest that it may be a valid strategy for enhancing the neuroprotective actions of treatments which stimulate kynurenic acid synthesis, or those of exogenous glutamate receptor antagonists.

Seizure activity causes elevation of endogenous extracellular kynurenic acid in the rat brain

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 striatum. 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.

Electrical kindling is associated with a lasting increase in the extracellular levels of kynurenic acid in the rat hippocampus

Endogenous kynurenic acid (KYNA), an excitory amino acid receptor antagonist with antineurotoxic and anticonvulsant activity, was assessed by microdialysis in the hippocampus of kindled rats. One week after the completion of amygdala or hippocampal kindling (stage 5), the dialysate concentration of KYNA in the hippocampus of both hemispheres was 1.7 ± 0.1-fold higher than in shams ( P < 0.01). Veratridine (50 μM), applied through the probe, reduced extracellular KYNA by 28% within 1 h in controls ( P < 0.05), but was ineffective in stage 5 kindled rats. At the preconvulsive stage 2, dialysate KYNA concentration and the effect of veratridine were similar to controls. The activity of KYNA's biosynthetic enzyme, kynurenine aminotransferase, did not change in the hippocampus 1 week after stage 5 seizures. These data indicate an enhanced liberation of KYNA in the hippocampus of fully kindled animals due to an impairment of normal regulatory mechanisms. This may be of relevance for the control of hippocampal excitability during epileptogenesis.

L-α-Aminoadipic acid as a regulator of kynurenic acid production in the hippocampus: a microdialysis study in freely moving rats

l-α-Aminoadipic acid is a lysine metabolite with neuroexcitatory properties, and has previously been shown to inhibit the production of the broad spectrum excitatory amino acid receptor antagonist kynurenic acid in brain tissue slices. The effects of l-α-aminoadipic acid on the levels of extracellular kynurenic acid were now studied by microdialysis in the dorsal hippocampus of freely moving rats. Application of l-α-aminoadipic acid through the microdialysis probe dose dependently decreased both the concentration of endogenous kynurenic acid and of kynurenic acid which was produced de novo from its bioprecursor l-kynurenine (500 μM applied through the probe). 500 μM l-α-aminoadipic acid lowered the kynurenic acid concentration in the dialysate by 47% and 28% with and without precursor loading, respectively, whereas d-α-aminoadipic acid was without effect. Co-administration of 500 μM l-α-aminoadipic acid with 50 μM veratridine, which by itself produces a substantial decrease in the levels of extracellular kynurenic acid, did not result in a further reduction in kynurenic acid concentrations. Extensive neuronal degeneration caused by an intrahippocampal injection of quinolinic acid (120 nmol) did not interfere with the effect of l-α-aminoadipic acid. Taken together, these data suggest that the effect of l-α-aminoadipic acid on extracellular kynurenic acid levels is likely due to its direct action on astrocytes, which are known to harbor kynurenic acid's biosynthetic enzyme, kynurenine aminotransferase. l-α-Aminoadipic acid may modulate kynurenic acid function in the brain and thus play a role in the pathogenesis of neurodegenerative and seizure disorders.