Search

Anti-glutamatergic agents may have neuroprotective effects against excitotoxicity that is known to be involved in the pathogenesis of Parkinson's disease (PD). One of these agents is kynurenic acid (KYNA), a tryptophan metabolite, which is an endogenous N-methyl-D-aspartic acid (NMDA) receptor antagonist. However, its pharmacological properties of poor water solubility and limited blood-brain barrier (BBB) permeability rules out its systemic administration in disorders affecting the central nervous system. Our aim in the present study was to investigate the neuroprotective effects of KYNA-loaded micelles (KYNA-MICs) against PD in vitro and in vivo . Lipid-based micelles (MICs) in conjunction with KYNA drug delivery have the potential to enhance the penetration of therapeutic drugs into a diseased brain without BBB obstacles. KYNA-MICs were characterized by particle size (105.8 ± 12.1 nm), loading efficiency (78.3 ± 4.23%), and in vitro drug release (approximately 30% at 24 h). The in vitro experiments showed that KYNA-MICs effectively reduced 2-fold protein aggregation. The in vivo studies revealed that KYNA was successfully delivered by 5-fold increase in neurotoxin-induced PD brains. The results showed significant enhancement of KYNA delivery into brain. We also found that the KYNA-MICs exhibited several therapeutic effects. The KYNA-MICs reduced protein aggregation of an in vitro PD model, ameliorated motor functions, and prevented loss of the striatal neurons in a PD animal model. The beneficial effects of KYNA-MICs are probably explained by the anti-excitotoxic activity of the treatment's complex. As the KYNA-MICs did not induce any appreciable side-effects at the protective dose applied to a chronic PD mouse model, our results demonstrate that KYNA provides neuroprotection and attenuates PD pathology.

Objectives: 6-Hydroxykynurenic acid (6-HKA) is an organic acid component in extracts of Ginkgo biloba leaves and acts as a major contributor to neurorestorative effects, while its oral bioavailability was low. Therefore, using prodrug method to improve the bioavailability and brain content of 6-HKA is significant., Methods: Three structural modified compounds of 6-HKA were synthesized, and ultra performance liquid chromatography-tandem mass spectrometry methods for quantification of these structural modified compounds in rat plasma and rat brain homogenate were established and comprehensively validated. The methods were effectively applied to investigate the effects of structural modification on apparent permeability coefficients in cells, the pharmacokinetics and the brain distribution in rats., Key Findings: The results illustrated that esterification can greatly improve the apparent permeability coefficient and bioavailability of 6-HKA. Comparing with direct oral administration of 6-HKA, the bioavailability of isopropyl ester was greatly improved (from 3.96 ± 1.45% to 41.8 ± 15.3%), and the contents of 6-HKA in rat brains (49.7 ± 9.2 ng/g brain) were significantly higher after oral administration., Conclusions: The bioavailability and the brain content of 6-HKA can be improved by the prodrug method. Among three structural modified compounds, isopropyl-esterified 6-HKA was the most promising treatment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Hamilton Search Task (HST) is a test of nonnavigational spatial memory that is dependent on the hippocampus. The parahippocampal cortex (PHC) is a major route for spatial information to reach the hippocampus, but the extent to which the PHC and hippocampus function independently of one another in the context of nonnavigational spatial memory is unclear. Here, we tested the hypotheses that (1) bilateral pharmacological inactivation of the PHC would impair HST performance, and (2) that functional disconnection of the PHC and hippocampus by contralateral (crossed) inactivation would likewise impair performance. Transient inactivation of the PHC impaired HST performance most robustly with 30 s intertrial delays, but not when color cues were introduced. Functional disconnection of the PHC and hippocampus, but not separate unilateral inactivation of either region, also selectively impaired long-term spatial memory. These findings indicate a critical role for the PHC and its interactions with the hippocampus in nonnavigational spatial memory., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Kynurenic acid (KA), an endogenous product of L-tryptophan metabolism in the kynurenine pathway, regulates adipose tissue energy homeostasis and inflammation. However, its role in palmitate-induced insulin resistance and detailed underlying mechanisms in skeletal muscles and adipose tissues are unclear. Herein, we report that KA ameliorated palmitate-induced inflammation and insulin resistance in differentiated C2C12 and 3T3-L1 cell lines as well as soleus skeletal muscle and subcutaneous adipose tissues in mice. Palmitate-induced inflammatory markers, such as nuclear factor κB translocation, inhibitory κBα phosphorylation, pro-inflammatory cytokine expression, and impaired insulin signaling, were markedly attenuated by KA both in vitro and in vivo. KA significantly increased AMP-activated protein kinase (AMPK) phosphorylation and sirtuin 6 (SIRT6) expressions in C2C12 myocytes and 3T3-L1 adipocytes and skeletal muscle and adipose tissues of mice. siRNA-mediated AMPK or SIRT6 inhibition significantly mitigated the suppressive effects of KA on palmitate-induced inflammation and insulin resistance. KA significantly stimulated expression of genes involved in fatty acid oxidation in C2C12 myocytes and skeletal muscle of mice. Moreover, KA inhibits lipogenesis in 3T3-L1 adipocytes. AMPK or SIRT6 siRNA markedly reversed these changes. The siRNA targeting Gpr35 abrogated the effects of KA on AMPK phosphorylation in C2C12 myocytes and 3T3-L1 adipocytes, except SIRT6 expression. It has therefore been shown that KA could potentially alleviate inflammation and insulin resistance in skeletal muscle and adipose tissues through Gpr35/AMPK and SIRT6-mediated pathways., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Kynurenic acid (KYNA) is one of the most significant metabolite of the kynurenine pathway both in terms of functional and potential therapeutic value. It is an N-methyl-D-aspartate (NMDA) receptor antagonist, but it can also activate the G-protein coupled receptor 35 (GPR35), which shares several structural and functional properties with cannabinoid receptors. Previously our group demonstrated that systemic chronic KYNA treatment altered opioid receptor G-protein activity. Opioid receptors also overlap in many features with cannabinoid receptors. Thus, our aim was to examine the direct in vitro and systemic, chronic in vivo effect of KYNA on type 1 cannabinoid receptor (CB 1 R) binding and G-protein activity. Based on competition and [ 35 S]GTPγS G-protein binding assays in rat brain, KYNA alone did not show significant binding towards the CB 1 R, nor did it alter CB 1 R ligand binding and agonist activity in vitro. When rats were chronically treated with KYNA (single daily, i.p., 128 mg/kg for 9 days), the KYNA plasma and cerebrospinal fluid levels significantly increased compared to vehicle treated group. Furthermore, in G-protein binding assays, in the whole brain the amount of G-proteins in basal and in maximum activity coupled to the CB 1 R also increased due to the treatment. At the same time, the overall stimulatory properties of the receptor remained unaltered in vehicle and KYNA treated samples. Similar observations were made in rat hippocampus, but not in the cortex and brainstem. In saturation binding assays the density of CB 1 Rs in rat whole brain and hippocampus were also significantly enhanced after the same treatment, without significantly affecting ligand binding affinity. Thus, KYNA indirectly and brain region specifically increases the abundance of functional CB 1 Rs, without modifying the overall binding and activity of the receptor. Supposedly, this can be a compensatory mechanism on the part of the endocannabinoid system induced by the long-term KYNA exposure., Competing Interests: Declaration of competing interest There are none., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Kynurenic acid (KYNA) and quinolinic acid (QUIN) are metabolites formed in the degradation of tryptophan (Trp). QUIN is a selective NMDA receptor antagonist and may exert neurotoxic effects, whereas KYNA is an agonist of glutamatergic and cholinergic receptors and presents antioxidant properties. KYNA/QUIN ratio is decreased in several central nervous system disorders, but the mechanisms involved are not well elucidated. In the present study, we try to determine the neuroprotective capacity of KYNA on the QUIN effects in redox homeostasis changes (H 2 DCF oxidation, superoxide dismutase/catalase (SOD/CAT) ratio, glutathione peroxidase (GPx) activity, sulfhydryl content, and nitrite levels), as well as on inflammatory parameters (levels of TNF-α, IL-1β, and IL-6). KYNA and QUIN effects on the activities of Na + ,K + -ATPase and acetylcholinesterase (AChE) were also evaluated. Thirty-day-old male Wistar rats underwent stereotactic surgery and received intrastriatal injections as follows: group 1-control (PBS-injected), group 2-KYNA (100 μM), group 3-QUIN (150 nM), and group 4-KYNA + QUIN (KYNA-injected followed QUIN-injected). Results demonstrated that the KYNA administration was able to prevent the increase in reactive oxygen species, SOD/CAT ratio, and pro-inflammatory cytokines (IL-1β and IL-6) and the decrease in GPx activity, sulfhydryl content, and nitrite levels caused by QUIN. KYNA was also able to partially prevent the decrease in Na + ,K + -ATPase activity and the increase in AChE activity caused by QUIN. This study may help in the elucidation of neuroprotective effects of KYNA against oxidative and inflammatory insults caused by QUIN in the striatum of young male Wistar rats.

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan. KYNA naturally occurs in breast milk and its content increases with lactation, indicating the role of neonatal nutrition in general growth with long-term health effects. KYNA is also an antagonist of ionotropic glutamate receptors expressed in bone cells. The aim of this study was to establish the effects of chronic KYNA supplementation on bone homeostasis in young rats, using mandible as a model bone. Female and male newborn Wistar rats were divided into control and KYNA-administered groups until 60 days of age (25x101 mg/L or 25x102 mg/L in drinking water). Hemimandibles were subjected to densitometry, computed tomography analysis and mechanical testing. Rats supplemented with KYNA at both doses showed a decrease in body weight. There were no effects of KYNA administration and mandible histomorphometry. In males, a significant quadratic effect (P < 0.001) was observed in the densitometry of the hemimandible, where BMD increased in the group supplemented with 2.5x101 mg/L of KYNA. Analysis of mechanical tests data showed that when fracture forces were corrected for bone geometry and rats body weight the improvement of bone material properties was observed in male and female rats supplemented with lower dose of KYNA. This study showed that chronic supplementation with KYNA may limit weight gain in the young, without adversely affecting the development of the skeleton., Competing Interests: The authors have declared that no competing interests exist.

The global increase in resorting to artificial nutritional formulas replacing breastfeeding has been identified among the complex causes of the obesity epidemic in infants and children. One of the factors recently recognized to influence metabolism and weight gain is kynurenic acid (KYNA), an agonist of G protein-coupled receptor (GPR35). Therefore the aim of the study was to determine the concentration of KYNA in artificial nutritional formulas in comparison with its level in human breast milk and to evaluate developmental changes in rats exposed to KYNA enriched diet during the time of breastfeeding. KYNA levels were measured in milk samples from 25 heathy breast-feeding women during the first six months after labor and were compared with 21 time-adjusted nutritional formulas. Animal experiments were performed on male Wistar rats. KYNA was administered in drinking water. The content of KYNA in human milk increases more than 13 times during the time of breastfeeding while its level is significantly lower in artificial formulas. KYNA was detected in breast milk of rats and it was found that the supplementation of rat maternal diet with KYNA in drinking water results in its increase in maternal milk. By means of the immunoblotting technique, GPR35 was evidenced in the mucosa of the jejunum of 1-day-old rats and distinct morphological changes in the jejunum of 21-day-old rats fed by mothers exposed to water supplemented with KYNA were found. A significant reduction of body weight gain of rats postnatally exposed to KYNA supplementation without changes in total body surface and bone mineral density was observed. The rat offspring fed with breast milk with artificially enhanced KYNA content demonstrated a lower mass gain during the first 21 days of life, which indicates that KYNA may act as an anti-obesogen. Further studies are, therefore, warranted to investigate the mechanisms regulating KYNA secretion via breast milk, as well as the influence of breast milk KYNA on mass gain. In the context of lifelong obesity observed worldwide in children fed artificially, our results imply that insufficient amount of KYNA in baby formulas could be considered as one of the factors associated with increased mass gain.

Background: The G protein-coupled receptor 35 (GPR35), is considered important for nociceptive transmission, as suggested by accumulating evidence. This receptor was discovered in 1998; however, a lack of pharmacological tools prevented a complete understanding of its function and how to exploit it therapeutically. We studied the influence of CXCL17, kynurenic acid and zaprinast on nociceptive transmission in naïve and neuropathic mice. Additionally, we investigated the influence of kynurenic acid and zaprinast on morphine effectiveness in neuropathic pain., Methods: The chronic constriction injury (CCI) of the sciatic nerve in Swiss mice was performed. The CXCL17, kynurenic acid, zaprinast and morphine were injected intrathecally into naive and CCI-exposed mice at day 14. To evaluate tactile and thermal hypersensitivity, the von Frey and cold plate tests were used, respectively., Results: Our results have shown, for the first time, that administration of CXCL17 in naïve mice induced strong pain-related behaviours, as measured by von Frey and cold plate tests. Moreover, we demonstrated that kynurenic acid and zaprinast diminished CXCL17-evoked pain-related behaviours in both tests. Kynurenic acid and zaprinast reduced thermal and tactile hypersensitivity developed by sciatic nerve injury and strongly enhanced the effectiveness of morphine in neuropathy., Conclusions: Our study highlights the importance of GPR35 as a receptor involved in neuropathic pain development. Therefore, these results suggest that the modulation of GPR35 could become a potential strategy for the treatment of neuropathic pain., (Copyright © 2018 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Background: Kynurenic acid (KYNA) is a biologically active metabolite of tryptophan exerting action on several receptors located in the brain and periphery. KYNA can be synthesized endogenously or supplied in the diet. It was documented that KYNA is present in various types of food. However, its presence in beverages was not yet investigated. Here, we measured content of KYNA in tea and coffee as well as analyzed distribution and fate of intragastrically administered labelled KYNA in mice., Methods: 16 and 13 studied samples of tea and coffee, respectively were of commercial origin. Tea and coffee infusions were prepared according to the producers' guidelines. KYNA content in beverages was measured by means of HPLC detection. Adult male mice were used for analysis of fate of intragastrically administered labelled KYNA and collected samples were analyzed using liquid scintillation counter., Results: KYNA was identified in all studied beverages. Amounts of KYNA found in various types of beverages differed significantly. The highest content of KYNA in tea and coffee was 8.7 μg/100 ml and 0.63 μg/100 ml, respectively. It was found that KYNA administered intragastrically as a liquid is absorbed from the digestive system and readily excreted in urine. The atypical kinetics of KYNA distribution were found in intestinal content of cecum, where it appeared later and persisted longer than in other tissues., Conclusions: Our data show that tea and coffee intake may contribute to KYNA content in the human organism. The distribution pattern of KYNA delivered as a liquid suggests that it either directly affects digestive system's functioning and intestinal microbiome composition, or participates in the whole body pool of KYNA., (Copyright © 2018 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Scarring is a consequence of biological tissue repair following trauma. Currently, there are no generally agreed ways to prevent scarring. Recently, kynurenic acid has shown to be a potent modulator of extracellular matrix deposition and remodeling. Kynurenic acid can reduce matrix deposition and other fundamental characteristics of fibrosis in vitro and in vivo. Specifically, kynurenic acid has shown to increase matrix metalloproteinase-1 activity and subsequently reduce collagen deposition in a rabbit ear scar model. In the present study kynurenic acid cream in different concentrations was topically applied on healthy skin on volunteers to assess skin reactions and skin sensitivity in both acute and chronic application settings. Skin reactions were assessed, and concentrations for kynurenic acid were assessed both form serum and urine. Results showed to acute or delayed skin reactions. Kynurenic acid was not detectable in blood at any time point, and only trace elements of kynurenic acid were found in urine. This study supports safety and tolerability of topically administered FS2 when using a liposomal, compounding base carrier., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

To assess the possible function of glutamate in the interaction between the dorsomedial hypothalamic nucleus-perifornical area (DMH-PeF) and the A5 pontine region (A5), cardiovascular and respiratory changes were studied in response to electrical stimulation of the DMH-PeF (1 ms pulses, 30-50 μA given at 100 Hz for 5 s) before and after the microinjection of kynurenic acid (non-specific glutamate receptor antagonist; 50 nl, 5 nmol), MK-801 (NMDA receptor antagonist; 50 nl, 50 nmol), CNQX (non-NMDA receptor antagonist; 50 nl, 50 nmol) or MCPG (metabotropic glutamate receptor antagonist; 50 nl, 5 nmol) within the A5 region. DMH-PeF electrical stimulation elicited a pressor (p < 0.001) and tachycardic response (p < 0.001) which was accompanied by an inspiratory facilitation characterised by an increase in respiratory rate (p < 0.001) due to a decrease in expiratory time (p < 0.01). Kynurenic acid within the A5 region decreased the tachycardia (p < 0.001) and the intensity of the blood pressure response (p < 0.001) to DMH-PeF stimulation. After the microinjection of MK-801 and CNQX into the A5 region, the magnitude of the tachycardia and the pressor response were decreased (p < 0.05 and p < 0.01; p < 0.001 and p < 0.05, respectively). After MCPG microinjection into the A5 region, a decrease in the tachycardia (p < 0.001) with no changes in the pressor response was observed during DMH-PeF stimulation. The respiratory response elicited by DMH-PeF stimulation was not changed after the microinjection of kynurenic acid, MK-801, CNQX or MCPG within the A5 region. These results suggest that A5 region glutamate receptors play a role in the cardiovascular response elicited from the DMH-PeF. The possible mechanisms involved in these interactions are discussed.

Intermittent hypoxia causes a persistent increase in sympathetic activity that progresses to hypertension in chronic conditions such as obstructive sleep apnea. Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neurotransmitter that causes long-lasting sympathetic excitation. We aimed to determine if intermittent activation of the rostral ventrolateral medulla (RVLM) causes PACAP-mediated elevation of sympathetic nerve activity, termed sympathetic long-term facilitation (sLTF). The role of PACAP in mediating sLTF in response to intermittent activation of the RVLM was investigated in urethane-anaesthetized and artificially ventilated rats ( n = 65, Sprague-Dawley). Bilateral RVLM microinjections of the PACAP type 1 receptor/vasoactive intestinal polypeptide receptor type 2 receptor antagonist PACAP-(6-38) [ n = 6, change (Δ): -16.4 ± 6.5%) or an ionotropic glutamate antagonist, kynurenate ( n = 6, Δ:-7.2 ± 2.3%), blocked the development of acute intermittent hypoxia-induced sLTF ( n = 6, Δ: 49.2 ± 14.2%). Intermittent RVLM microinjections of glutamate caused sLTF ( n = 5, Δ: 56.9 ± 14.7%) that was abolished by PACAP-(6-38) pretreatment ( n = 5, Δ:-1.2 ± 4.7%). Conversely, intermittent microinjections of PACAP in the RVLM did not elicit sLTF. Intermittent bilateral disinhibition of the RVLM by microinjection of γ-aminobutyric acid in the caudal ventrolateral medulla did not elicit sLTF. Direct activation of RVLM neurons is crucial for the development of sLTF. PACAP and glutamate act synergistically in the RVLM, with both being necessary for the sLTF response. We found that activation of glutamate but not PACAP receptors is necessary and sufficient to generate sLTF, even in the absence of intermittent hypoxia. Our results demonstrate that PACAP within the RVLM may contribute to the development of obstructive sleep apnea -induced hypertension. NEW & NOTEWORTHY Pharmacological blockade of either pituitary adenylate cyclase-activating polypeptide (PACAP) or ionotropic glutamate receptors in the rostral ventrolateral medulla prevents development of sympathetic long-term facilitation. PACAP receptor inhibition prevents the occurrence of hypoxia-induced peripheral chemoreflex sensitization. Thus, PACAP receptors may be a potential therapeutic target serving to reduce heightened sympathetic tone and hypersensitized cardiovascular reflexes.

Background: Migraine is a debilitating neurological disorder where trigeminovascular activation plays a key role. We have previously reported that local application of Complete Freund's Adjuvant (CFA) onto the dura mater caused activation in rat trigeminal ganglion (TG) which was abolished by a systemic administration of kynurenic acid (KYNA) derivate (SZR72). Here, we hypothesize that this activation may extend to the trigeminal complex in the brainstem and is attenuated by treatment with SZR72., Methods: Activation in the trigeminal nucleus caudalis (TNC) and the trigeminal tract (Sp5) was achieved by application of CFA onto the dural parietal surface. SZR72 was given intraperitoneally (i.p.), one dose prior CFA deposition and repeatedly daily for 7 days. Immunohistochemical studies were performed for mapping glutamate, c-fos, PACAP, substance P, IL-6, IL-1β and TNFα in the TNC/Sp5 and other regions of the brainstem and at the C 1 -C 2 regions of the spinal cord., Results: We found that CFA increased c-fos and glutamate immunoreactivity in TNC and C 1 -C 2 neurons. This effect was mitigated by SZR72. PACAP positive fibers were detected in the fasciculus cuneatus and gracilis. Substance P, TNFα, IL-6 and IL-1β immunopositivity were detected in fibers of Sp5 and neither of these molecules showed any change in immunoreactivity following CFA administration., Conclusion: This is the first study demonstrating that dural application of CFA increases the expression of c-fos and glutamate in TNC neurons. Treatment with the KYNA analogue prevented this expression.

Background and Aims: Neuropathic pain is a significant medical problem needing more effective treatments with fewer side effects. Overactive glutamatergic transmission via N-methyl-d-aspartate receptors (NMDARs) are known to play a role in central sensitization and neuropathic pain. Although ketamine, a NMDAR channel-blocking antagonist, is often used for neuropathic pain, its side-effect profile and abusive potential has prompted the search for a safer effective oral analgesic. A novel oral prodrug, AV-101 (l-4 chlorokynurenine), which, in the brain, is converted into one of the most potent and selective GlyB site antagonists of the NMDAR, has been demonstrated to be active in animal models of neuropathic pain. The two Phase 1 studies reported herein were designed to assess the safety and pharmacokinetics of AV-101, over a wide dose range, after daily dosing for 14-days. As secondary endpoints, AV-101 was evaluated in the capsaicin-induced pain model., Methods: The Phase 1A study was a single-site, randomized, double-blind, placebo-controlled, single oral ascending dose (30-1800mg) study involving 36 normal healthy volunteers. The Phase 1B study was a single-site randomized, double-blind, placebo-controlled, study of multiple ascending doses (360, 1080, and 1440mg/day) of AV-101 involving 50 normal healthy volunteers, to whom AV-101 or placebo were administered orally daily for 14 consecutive days. Subjects underwent PK blood analyses, laboratory assessments, physical examination, 12-lead ECG, ophthalmological examination, and various neurocognitive assessments. The effect of AV-101 was evaluated in the intradermally capsaicin-induced pain model (ClinicalTrials.gov Identifier: NCT01483846)., Results: Two Phase 1, with an aggregate of 86 subjects, demonstrated that up to 14 days of oral AV-101, up to 1440mg per day, was safe and very well tolerated with AEs quantitively and qualitatively like those observed with placebo. Mean half-life values of AV-101 were consistent across doses, ranging with an average of 1.73h, with the highest C max (64.4μg/mL) and AUC 0-t (196μgh/mL) values for AV-101 occurring in the 1440-mg dose group. In the capsaicin induce-pain model, there was no significant change in the area under the pain time curve (AUPC) for the spontaneous pain assessment between the treatment and the placebo groups on Day 1 or 14 (the primary endpoint). In contrast, there were consistent reductions at 60-180min on Day 1 after dosing for allodynia, mechanical hyperalgesia, heat hyperalgesia, and spontaneous pain, and on Day 14 after dosing for heat hyperalgesia., Conclusions: Although, AV-101 did not reach statistical significance in reducing pain, there were consistent reductions, for allodynia pain and mechanical and heat hyperalgesia. Given the excellent safety profile and PK characteristics demonstrated by this study, future clinical trials of AV-101 in neuropathic pain are justified., Implications: This article presents the safety and PK of AV-101, a novel oral prodrug producing a potent and selective GlyB site antagonist of the NMDA receptor. These data indicate that AV-101 has excellent safety and PK characteristics providing support for advancing AV-101 into Phase 2 studies in neuropathic pain, and even provides data suggesting that AV-101 may have a role in treating depression., (Copyright © 2017 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Increased concentrations of kynurenic acid (KYNA) in the prefrontal cortex (PFC) are thought to contribute to the development of cognitive deficits observed in schizophrenia. Although this view is consistent with preclinical studies showing a negative impact of prefrontal KYNA elevation on executive function, the mechanism underlying such a disruption remains unclear. Here, we measured changes in local field potential (LFP) responses to ventral hippocampal stimulation in vivo and conducted whole-cell patch-clamp recordings in brain slices to reveal how nanomolar concentrations of KYNA alter synaptic transmission in the PFC of male adult rats. Our data show that prefrontal infusions of KYNA attenuated the inhibitory component of PFC LFP responses, a disruption that resulted from local blockade of α7-nicotinic acetylcholine receptors (α7nAChR). At the cellular level, we found that the inhibitory action exerted by KYNA in the PFC occurred primarily at local GABAergic synapses through an α7nAChR-dependent presynaptic mechanism. As a result, the excitatory-inhibitory ratio of synaptic transmission becomes imbalanced in a manner that correlates highly with the level of GABAergic suppression by KYNA. Finally, prefrontal infusion of a GABA A R positive allosteric modulator was sufficient to overcome the disrupting effect of KYNA and normalized the pattern of LFP inhibition in the PFC. Thus, the preferential inhibitory effect of KYNA on prefrontal GABAergic transmission could contribute to the onset of cognitive deficits observed in schizophrenia because proper GABAergic control of PFC output is one key mechanism for supporting such cortical functions. SIGNIFICANCE STATEMENT Brain kynurenic acid (KYNA) is an astrocyte-derived metabolite and its abnormal elevation in the prefrontal cortex (PFC) is thought to impair cognitive functions in individuals with schizophrenia. However, the mechanism underlying the disrupting effect of KYNA remains unclear. Here we found that KYNA biases the excitatory-inhibitory balance of prefrontal synaptic activity toward a state of disinhibition. Such disruption emerges as a result of a preferential suppression of local GABAergic transmission by KYNA via presynaptic inhibition of α7-nicotinic acetylcholine receptor signaling. Therefore, the degree of GABAergic dysregulation in the PFC could be a clinically relevant contributing factor for the onset of cognitive deficits resulting from abnormal increases of cortical KYNA., (Copyright © 2017 the authors 0270-6474/17/377921-09$15.00/0.)

Adrenaline is an important counter-regulatory hormone that helps restore glucose homeostasis during hypoglycaemia. However, the neurocircuitry that connects the brain glucose sensors and the adrenal sympathetic outflow to the chromaffin cells is poorly understood. We used electrical microstimulation of the perifornical hypothalamus (PeH) and the rostral ventrolateral medulla (RVLM) combined with adrenal sympathetic nerve activity (ASNA) recording to examine the relationship between the RVLM, the PeH and ASNA. In urethane-anaesthetised male Sprague-Dawley rats, intermittent single pulse electrical stimulation of the rostroventrolateral medulla (RVLM) elicited an evoked ASNA response that consisted of early (60±3ms) and late peaks (135±4ms) of preganglionic and postganglionic activity. In contrast, RVLM stimulation evoked responses in lumbar sympathetic nerve activity that were almost entirely postganglionic. PeH stimulation also produced an evoked excitatory response consisting of both preganglionic and postganglionic excitatory peaks in ASNA. Both peaks in ASNA following RVLM stimulation were reduced by intrathecal kynurenic acid (KYN) injection. In addition, the ASNA response to systemic neuroglucoprivation induced by 2-deoxy-d-glucose was abolished by bilateral microinjection of KYN into the RVLM. This suggests that a glutamatergic pathway from the perifornical hypothalamus (PeH) relays in the RVLM to activate the adrenal SPN and so modulate ASNA. The main findings of this study are that (i) adrenal premotor neurons in the RVLM may be, at least in part, glutamatergic and (ii) that the input to these neurons that is activated during neuroglucoprivation is also glutamatergic., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The complex pathophysiology of migraine is not yet clearly understood; therefore, experimental models are essential for the investigation of the processes related to migraine headache, which include cortical spreading depression (CSD) and NO donor-induced neurovascular changes. Data on the assessment of drug efficacy in these models are often limited, which prompted us to investigate a novel combined migraine model in which an effective pharmacon could be more easily identified., Materials and Methods: In vivo electrophysiological experiments were performed to investigate the effect of nitroglycerin (NTG) on CSD induced by KCl application. In addition, sumatriptan and newly synthesized neuroactive substances (analogues of the neuromodulator kynurenic acid [KYNA]) were also tested., Results: The basic parameters of CSDs were unchanged following NTG administration; however, propagation failure was decreased compared to the controls. Sumatriptan decreased the number of CSDs, whereas propagation failure was as minimal as in the NTG group. On the other hand, both of the KYNA analogues restored the ratio of propagation to the control level., Discussion: The ratio of propagation appeared to be the indicator of the effect of NTG. This is the first study providing direct evidence that NTG influences CSD; furthermore, we observed different effects of sumatriptan and KYNA analogues. Sumatriptan changed the generation of CSDs, whereas the analogues acted on the propagation of the waves. Our experimental design overlaps with a large spectrum of processes present in migraine pathophysiology, and it can be a useful experimental model for drug screening., Competing Interests: The authors report no conflicts of interest in this work.

Impaired glucose homeostasis and energy balance are integral to the pathophysiology of diabetes and obesity. Here we show that administration of a glycine transporter 1 (GlyT1) inhibitor, or molecular GlyT1 knockdown, in the dorsal vagal complex (DVC) suppresses glucose production, increases glucose tolerance and reduces food intake and body weight gain in healthy, obese and diabetic rats. These findings provide proof of concept that GlyT1 inhibition in the brain improves glucose and energy homeostasis. Considering the clinical safety and efficacy of GlyT1 inhibitors in raising glycine levels in clinical trials for schizophrenia, we propose that GlyT1 inhibitors have the potential to be repurposed as a treatment of both obesity and diabetes.

Core-shell nanoparticles (CSNPs) were developed to get over therapeutic amount of kynurenic acid (KYNA) across the blood-brain barrier (BBB). Bovine serum albumin (BSA) was used as core for encapsulation of KYNA and the BSA/KYNA composite was finally encapsulated by poly(allylamine) hydrochloride (PAH) polymer as shell. In the interest of the optimization of the synthesis the BSA and KYNA interaction was studied by two-dimensional surface plasmon resonance (SPR) technique as well. The average size of d~100 nm was proven by dynamic light scattering (DLS) and transmission electron microscopy (TEM), while the structure of the composites was characterized by fluorescence (FL) and circular dichroism (CD) spectroscopy. The in vitro release properties of KYNA were investigated by a vertical diffusion cell at 25.0 °C and 37.5 °C and the kinetic of the release were discussed. The penetration capacity of the NPs into the central nervous system (CNS) was tested by an in vitro BBB model. The results demonstrated that the encapsulated KYNA had significantly higher permeability compared to free KYNA molecules. In the neurobiological serial of in vivo experiments the effects of peripherally administered KYNA with CSNPs were studied in comparison with untreated KYNA. These results clearly proved that KYNA in the CSNPs, administrated peripherally is suitable to cross the BBB and to induce electrophysiological effects within the CNS. As the neuroprotective properties of KYNA nowadays are proven, the importance of the results is obvious., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The changes in urine flow and renal sympathetic nerve activity (RSNA) due to pulmonary lymphatic obstruction (PLO) were examined in anesthetized, artificially ventilated New Zealand white rabbits. PLO was produced by pressurizing an isolated pouch created in the right external jugular vein at the points of entry of the right lymphatic ducts. During this maneuver, urine flow increased from 8.5 ± 0.3 mL/10 min to 12 ± 0.5 mL/10 min (P < 0.0001) and RSNA increased from 24.0 ± 4 to 40.0 ± 5 μV·s (P < 0.0001). Bilateral lesioning of the paraventricular nucleus (PVN) of the hypothalamus or cervical vagotomy abolished these responses. PLO increased c-fos gene expression in the PVN. The increase in urine flow due to PLO was attenuated by muscimol and abolished by kynurenic acid microinjections into the PVN. The results show that (i) neurons in the PVN are an important relay site in the reflex arc, which is activated by PLO; and (ii) this activation is regulated by glutamatergic and partly by GABAergic input to the PVN.

Glutamatergic neurotransmission, of special importance in the human brain, is implicated in key brain functions such as synaptic plasticity and memory. The excessive activation of N-methyl- D-aspartate (NMDA) receptors may result in excitotoxic neuronal damage; this process has been implicated in the pathomechanism of different neurodegenerative disorders, such as Alzheimer's disease (AD). Memantine is an uncompetitive antagonist of NMDA receptors with a favorable pharmacokinetic profile, and is therefore clinically well tolerated. Memantine is approved for the treatment of AD, but may additionally be beneficial for other dementia forms and pain conditions. Kynurenic acid (KYNA) is an endogenous antagonist of NMDA receptors which has been demonstrated under experimental conditions to be neuroprotective. The development of a well-tolerated NMDA antagonist may offer a novel therapeutic option for the treatment of neurodegenerative disease and pain syndromes. KYNA may be a valuable candidate for future drug development.

Background: Kynurenic acid (KYNA) is the end stage metabolite of tryptophan produced mainly by astrocytes in the central nervous system (CNS). It has neuroprotective activities but can be elevated in the neuropsychiatric disorders. Toxic effects of KYNA in the CNS are unknown. The aim of this study was to assess the effect of the subdural KYNA infusion on the spinal cord in adult rats., Methods: A total of 42 healthy adult rats were randomly assigned into six groups and were infused for 7 days with PBS (control) or 0.0002 pmol/min, 0.01 nmol/min, 0.1 nmol/min, 1 nmol/min, and 10 nmol/min of KYNA per 7 days. The effect of KYNA on spinal cord was determined using histological and electron microscopy examination. Myelin oligodendrocyte glycoprotein (MOG) was measured in the blood serum to assess a degree of myelin damage., Result: In all rats continuous long-lasting subdural KYNA infusion was associated with myelin damage and myelin loss that was increasingly widespread in a dose-depended fashion in peripheral, sub-pial areas. Damage to myelin sheaths was uniquely related to the separation of lamellae at the intraperiod line. The damaged myelin sheaths and areas with complete loss of myelin were associated with limited loss of scattered axons while vast majority of axons in affected areas were morphologically intact. The myelin loss-causing effect of KYNA occurred with no necrosis of oligodendrocytes, with locally severe astrogliosis and no cellular inflammatory response. Additionally, subdural KYNA infusion increased blood MOG concentration. Moreover, the rats infused with the highest doses of KYNA (1 and 10 nmol/min) demonstrated adverse neurological signs including weakness and quadriplegia., Conclusions: We suggest, that subdural infusion of high dose of KYNA can be used as an experimental tool for the study of mechanisms of myelin damage and regeneration. On the other hand, the administration of low, physiologically relevant doses of KYNA may help to discover the role of KYNA in control of physiological myelination process.

Introduction: There are no data available about the effects of spinally administered drugs on thermoregulation in freely moving animals. The first goal of the present study was to throw light on the consequences of intrathecally administered saline as a vehicle on core temperature and motor activity in unrestrained conditions. The second goal was to characterize the effects of morphine on these parameters as a widely used antinociceptive drug in spinal anesthesia, and reveal the potential role of the N-methyl-d-aspartate (NMDA) receptors in these processes., Methods: For these purposes, male Wistar rats were catheterized intrathecally, and E-Mitter battery-free transponders were implanted intraabdominally to continuously monitor core temperature and locomotor activity., Results: Saline induced a short-lasting hyperactivity accompanied by significant and prolonged hyperthermia that was blunted by systemic paracetamol administration. Morphine had no impact on motor activity; however, it caused high but equivalent degree hyperthermia in a wide dose range (1-15 μg), suggesting that it reached its peak effect. In the highest applied dose (25 μg), the NMDA receptor antagonist kynurenic acid blunted the saline-induced hyperthermia, and all doses caused higher hyperactivity compared to vehicle or morphine injections. In combination, kynurenic acid significantly inhibited the morphine-induced hyperthermia., Discussion: These data suggest that this method might be a valuable tool for investigating the thermoregulatory and locomotor effects of different drugs at spinal level; however, the prolonged effects of intrathecal vehicle injections should also be considered. The results point out that morphine is a very potent hyperthermic drug that may act primarily on the efferent limb of thermoregulation, at least partially, via an indirect NMDA-receptor mediated action mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Kynurenic acid (KYNA), a tryptophan metabolite is an antagonist of ionotropic glutamate receptors and alpha-7 nicotinic receptor. Moreover, it is an agonist of G-protein receptor GPR35. Its neuroprotective, anticonvulsant, anti-inflammatory and antioxidant activity was documented. KYNA is present in food and herbal medicines. However, the data on effects induced by a long-lasting treatment with KYNA is lacking. The aim of the study was the assessment of toxicity of a prolonged administration of KYNA in rodents. The cytotoxicity of KYNA in vitro was also examined., Methods: Adult mice and rats were used. KYNA was administered in the drinking water in concentrations of 25 or 250mg/L for 3-21 days. The following cells were cultured in an in vitro study: mouse fibroblast (NIH/3T3), green monkey kidney cells and primary chick embryo cells (CECC). Cell viability was determined with methyl thiazol tetrazolium reduction assay, neutral red uptake assay and lactate dehydrogenase leakage assay., Results: KYNA affected neither body gain nor body composition. Blood counts were also unaffected. The viability of cells in the culture was lowered at high millimolar concentrations of KYNA. An elevated viability of GMK and CECC cells was detected in the presence of KYNA in micromolar concentrations., Conclusions: The obtained results showed that a long-term application of KYNA in the drinking water is well-tolerated by rodents. No evidence of a toxic response was recorded. Achieved results indicate that diets containing a high amount of KYNA or enriched with KYNA should not cause any risk to the human health., (Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Both NMDA receptor and kynurenic acid (KYNA), a glycine-site NMDA receptor antagonist, are present in the kidney yet their functional role remains unclear. Our aim was to examine effects of intravenous KYNA and glycine on arterial blood pressure (MAP) and renal haemodynamics and excretion in anaesthetized normotensive Sprague-Dawley (S-D) and in spontaneously hypertensive (SHR) rats. Renal blood flow (RBF, renal artery probe) and renal cortical (CBF) and outer- and inner medullary perfusion (laser-Doppler) were measured, along with diuresis (V) and sodium excretion (UNaV). KYNA given alone (150mgkg(-1) iv) or during infusion of glycine at 1gkg(-1)h(-1) iv (G+K) increased or decreased RBF, respectively, in both S-D and SHR. Neither treatment altered MAP. In both strains glycine alone increased RBF and CBF 50-60% and was clearly diuretic and natriuretic, less so in SHR. KYNA increased UNaV by 4.1±1.7μmolmin(-1)and V by 11.1±4.3μlmin(-1) in S-D (P<0.05 for both); the respective increases in SHR were by 1.7±0.6μmolmin(-1) and 4.7±1.7μlmin(-1) (P<0.02 for both). G+K treatment increased UNaV by 5.2±1.4μmolmin(-1) (P<0.01) and V by 29.6±4.6μmolmin(-1) (P<0.001) in S-D, and by 2.7±0.7μmolmin(-1) (P<0.05) and 19.3±3.5μlmin(-1) (P<0.0006) in SHR. In conclusion, KYNA increased renal excretion, apparently by inhibiting tubular reabsorption, whereas glycine substantially increased renal haemodynamics by an ill-defined mechanism, with a secondary increase in the excretion. Combined G+K treatment could be utilised to combat body fluid retention and possibly alleviate hypertension, without endangering renal perfusion and function., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The dorsomedial hypothalamus (DMH) and lateral/dorsolateral periaqueductal gray (PAG) are anatomically and functionally connected. Both the DMH and PAG depend on glutamatergic inputs for activation. We recently reported that removal of GABA-ergic tone in the unilateral DMH produces: asymmetry, that is, a right- (R-) sided predominance in cardiac chronotropism, and lateralization, that is, a greater increase in ipsilateral renal sympathetic activity (RSNA). In the current study, we investigated whether excitatory amino acid (EAA) receptors in the DMH-PAG pathway contribute to the functional interhemispheric difference. In urethane (1.2 to 1.4 g/kg, i.p.) anesthetized rats, we observed that: (i) nanoinjections of N-methyl D-aspartate (NMDA 100 pmol/100 nl) into the unilateral DMH produced the same right-sided predominance in the control of cardiac chronotropy, (ii) nanoinjections of NMDA into the ipsilateral DMH or PAG evoked lateralized RSNA responses, and (iii) blockade of EAA receptors in the unilateral DMH attenuated the cardiovascular responses evoked by injection of NMDA into either the R- or left- (L-) PAG. In awake rats, nanoinjection of kynurenic acid (1 nmol/100 nL) into the L-DMH or R- or L-PAG attenuated the tachycardia evoked by air stress. However, the magnitude of stress-evoked tachycardia was smallest when the EAA receptors of the R-DMH were blocked. We conclude that EAA receptors contribute to the right-sided predominance in cardiac chronotropism. This interhemispheric difference that involves EAA receptors was observed in the DMH but not in the PAG.

The present study was designed to investigate the role of glycine in ischemia reperfusion-induced acute kidney injury (AKI) in rats. The AKI was induced in rats by occluding renal pedicles for 40 min followed by reperfusion for 24 h. The AKI was assessed by measuring creatinine clearance, blood urea nitrogen, plasma uric acid, potassium, fractional excretion of sodium, and microproteinuria. The oxidative stress in renal tissues was assessed by quantification of myeloperoxidase activity, thiobarbituric acid-reactive substances, superoxide anion generation, and reduced glutathione level. Glycine (100, 200, and 400 mg/kg, i.p.) was administered to rats 30 min before subjecting to AKI. The glycinergic receptor blocker, strychnine (0.75 mg/kg i.p.), and glycine-binding site blocker at N-methyl-D-aspartate (NMDA) receptor, kynurenic acid (300 and 600 mg/kg i.p.), were used in the present study. The ischemia reperfusion induced AKI as witnessed by significant change in plasma, urinary, and tissue parameters employed in the present study. Glycine treatment increased ischemia reperfusion-induced AKI. The treatment with strychnine did not show any protection, whereas kynurenic acid ameliorated renal ischemia reperfusion-induced AKI. The results obtained in present study suggest that glycine increases ischemia reperfusion-induced renal damage through NMDA receptor agonism rather than strychnine-sensitive glycinergic receptors. Hence, it is concluded that glycine aggravates ischemia reperfusion-induced AKI. In addition, the activation of strychnine-insensitive glycine-binding site of NMDA receptors is responsible for its renal-damaging effect rather than strychnine-sensitive glycinergic receptors.

The rostral ventrolateral medulla (RVLM) plays a key role in cardiovascular regulation. It has been reported that tonically active glutamatergic input to the RVLM is increased in hypertensive rats, whereas angiotensin-converting enzyme 2 (ACE2) in the brain has been suggested to be beneficial to hypertension. This study was designed to determine the effect of ACE2 gene transfer into the RVLM on tonically active glutamatergic input in spontaneously hypertensive rats (SHRs). Lentiviral particles containing enhanced green fluorescent protein (lenti-GFP) or ACE2 (lenti-ACE2) were injected bilaterally into the RVLM. Both protein expression and activity of ACE2 in the RVLM were increased in SHRs after overexpression of ACE2. A significant reduction in blood pressure and heart rate in SHRs was observed 6 wk after lenti-ACE2 injected into the RVLM. The concentration of glutamate in microdialysis fluid from the RVLM was significantly reduced by an average of 61% in SHRs with lenti-ACE2 compared with lenti-GFP. ACE2 overexpression significantly attenuated the decrease in blood pressure and renal sympathetic nerve activity evoked by bilateral injection of the glutamate receptor antagonist kynurenic acid (2.7 nmol in 100 nl) into the RVLM in SHRs. Therefore, we suggest that ACE2 overexpression in the RVLM attenuates the enhanced tonically active glutamatergic input in SHRs, which may be an important mechanism underlying the beneficial effect of central ACE2 to hypertension., (Copyright © 2014 the American Physiological Society.)

Microinjections of kynurenic acid and kainic acid into the general visceral nucleus (nGV), homologous to the mammalian nucleus tractus solitarius of the medulla, in anesthestized, spontaneously breathing catfish were used to identify central areas and mechanisms controlling resting normoxic heart rate and blood pressure and the cardiovascular responses to hypoxia. Kynurenic acid, an antagonist of ionotropic glutamate receptors, significantly reduced resting normoxic heart rate but did not block the bradycardia associated with aquatic hypoxia. Kainic acid (an excitotoxic glutamatergic receptor agonist) also significantly reduced normoxic heart rate, but blocked the hypoxia-induced bradycardia. Neither kynurenic acid nor kainic acid microinjections affected blood pressure in normoxia or hypoxia. The results of this study indicate that glutamatergic receptors in the nGV are involved in the maintenance of resting heart rate and the destruction of these neurons with kainic acid abolishes the bradycardia associated with aquatic hypoxia., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Recent studies have shown that pancreatic exocrine secretions (PES) are modulated by dorsal motor nucleus of the vagus (DMV) neurones, whose activity is finely tuned by GABAergic and glutamatergic synaptic inputs. Group II metabotropic glutamate receptors (mGluR) decrease synaptic transmission to pancreas-projecting DMV neurones and increase PES. In the present study, we used a combination of in vivo and in vitro approaches aimed at characterising the effects of caerulein-induced acute pancreatitis (AP) on the vagal neurocircuitry modulating pancreatic functions. In control rats, microinjection of bicuculline into the DMV increased PES, whereas microinjections of kynurenic acid had no effect. Conversely, in AP rats, microinjection of bicuculline had no effect, whereas kynurenic acid decreased PES. DMV microinjections of the group II mGluR agonist APDC and whole cell recordings of excitatory currents in identified pancreas-projecting DMV neurones showed a reduced functional response in AP rats compared to controls. Moreover, these changes persisted up to 3 weeks following the induction of AP. These data demonstrate that AP increases the excitatory input to pancreas-projecting DMV neurones by decreasing the response of excitatory synaptic terminals to group II mGluR agonist.

Sunifiram is a novel pyrrolidone nootropic drug structurally related to piracetam, which was developed for neurodegenerative disorder like Alzheimer's disease. Sunifiram is known to enhance cognitive function in some behavioral experiments such as Morris water maze task. To address question whether sunifiram affects N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic function in the hippocampal CA1 region, we assessed the effects of sunifiram on NMDAR-dependent long-term potentiation (LTP) by electrophysiology and on phosphorylation of synaptic proteins by immunoblotting analysis. In mouse hippocampal slices, sunifiram at 10-100 nM significantly enhanced LTP in a bell-shaped dose-response relationship which peaked at 10 nM. The enhancement of LTP by sunifiram treatment was inhibited by 7-chloro-kynurenic acid (7-ClKN), an antagonist for glycine-binding site of NMDAR, but not by ifenprodil, an inhibitor for polyamine site of NMDAR. The enhancement of LTP by sunifilam was associated with an increase in phosphorylation of α-amino-3-hydroxy-5-methylisozazole-4-propionate receptor (AMPAR) through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and an increase in phosphorylation of NMDAR through activation of protein kinase Cα (PKCα). Sunifiram treatments at 1-1000 nM increased the slope of field excitatory postsynaptic potentials (fEPSPs) in a dose-dependent manner. The enhancement was associated with an increase in phosphorylation of AMPAR receptor through activation of CaMKII. Interestingly, under the basal condition, sunifiram treatments increased PKCα (Ser-657) and Src family (Tyr-416) activities with the same bell-shaped dose-response curve as that of LTP peaking at 10 nM. The increase in phosphorylation of PKCα (Ser-657) and Src (Tyr-416) induced by sunifiram was inhibited by 7-ClKN treatment. The LTP enhancement by sunifiram was significantly inhibited by PP2, a Src family inhibitor. Finally, when pretreated with a high concentration of glycine (300 μM), sunifiram treatments failed to potentiate LTP in the CA1 region. Taken together, sunifiram stimulates the glycine-binding site of NMDAR with concomitant PKCα activation through Src kinase. Enhancement of PKCα activity triggers to potentiate hippocampal LTP through CaMKII activation., (Copyright © 2013 Wiley Periodicals, Inc.)

Purpose: Our great interest in this work was study the synergism between l-tryptophan and dipyrone or paracetamol as well as the interaction of kynurenic acid (l-tryptophan metabolite) and these analgesics agents utilizing a robust methodology., Methods: We performed the writhing test induced by acetic acid in mice to evaluate the antinociceptive effect of the treatments isolated and combined (p.o. and i.p.). Dose-response curves were constructed and the values of ED50 for treatment alone and combined were statistically compared. In addition, isobolographic analysis was performed and the experimental values were compared with the theoretical values for simple additive effect., Results: The combined treatment with l-tryptophan and dipyrone or paracetamol reduced significantly the ED50 of these analgesics when compared to the isolated treatments. l-tryptophan alone has no antinociceptive effect. l-Tryptophan increases the central amount of 5-HT and the synergism with dipyrone is antagonized by the 5-HT depletion. The kyna has an antinociceptive dose-related effect and a synergistic interaction with dipyrone and paracetamol verified by isobolographic analyses and confirmed by experimental values of ED50 of combined treatments were statistically lower than theoretical calculated values for simple additive effect. Melatonin antagonist receptor attenuates the antinociceptive synergism between l-tryptophan and dipyrone., Conclusion: Our results demonstrate that the increased 5-HT amount on the central nervous system is not per se capable to induce antinociception. The l-tryptophan interacts synergistically with dipyrone and paracetamol both orally and by i.p. route. This effect is dependent on the biotransformation of l-tryptophan to 5-HT and involves kynurenic acid and melatonin receptors., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Intracerebroventricular (i.c.v.) injection of kynurenic acid (KYNA) had sedative and hypnotic effects during stress in neonatal chicks. However, its mechanism has not been clarified. KYNA is an endogenous antagonist of the α7 nicotinic acetylcholine (α7nACh) receptor and N-methyl-d-aspartate (NMDA) receptor. Therefore, this study clarified the mechanism of sedative and hypnotic effects of KYNA in the brain during an acute stress. In Experiment 1, to investigate the relationship between KYNA and the α7nACh receptor, KYNA was injected i.c.v. with galantamine, an agonist of the allosteric potentiating site of the α7nACh receptor. Galantamine did not attenuate the effect of KYNA, but higher levels of galantamine caused harmful effects. In Experiment 2, the role of the NMDA receptor was investigated using the NMDA receptor antagonist (+)-MK-801, d-serine which has high affinity to a co-agonist glycine site at the NMDA receptors, NMDA as the NMDA receptor agonist, and 2,3-pyridinedicarboxylic acid (QUIN), an agonist of the NMDA receptor subgroup containing the subunits NR2A and NR2B. The behavioral changes following KYNA were partially attenuated by QUIN alone. In conclusion, we suggest that KYNA functioned via the simultaneous inhibition of the α7nACh receptor and NMDA receptor subgroup containing the subunits NR2A and NR2B., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

Cortical spreading depression (CSD) involves a slowly-propagating depolarization wave in the cortex, which can appear in numerous pathophysiological conditions, such as migraine with aura, stroke, and traumatic brain injury. Neurons and glial cells are also depolarized transiently during the phenomena. CSD is followed by a massive increase in glutamate release and by changes in the brain microcirculation. The aim of this study was to investigate the effects of two N-methyl-D-aspartate receptor antagonists, endogenous kynurenic acid (KYNA) and dizocilpine, on CSD and the related blood-brain barrier (BBB) permeability in rats. In intact animals, KYNA hardly crosses the BBB but has some positive features as compared with its precursor L-Kynurenine, which is frequently used in animal studies (KYNA cannot be metabolized to excitotoxic agents such as 3-hydroxy-L-kynurenine and quinolinic acid). We therefore investigated the possible effects of peripherally administered KYNA. Repetitive CSD waves were elicited by the application of 1 M KCl solution to the cortex. Direct current-electrocorticograms were measured for 1 hour. Four parameters of the waves were compared. Evans blue dye and fluorescent microscopy were used to study the possible changes in the permeability of the BBB. The results demonstrated that N-methyl-D-aspartate receptor antagonists can reduce the number of CSD waves and decrease the permeability of the BBB during CSD. These results suggest that KYNA itself or its derivatives may offer a new approach in the therapy of migraines.

Imaging, clinical, and pre-clinical studies have provided ample evidence for a cerebellar involvement in cognitive brain function including cognitive brain disorders, such as autism and schizophrenia. We previously reported that cerebellar activity modulates dopamine release in the mouse medial prefrontal cortex (mPFC) via two distinct pathways: (1) cerebellum to mPFC via dopaminergic projections from the ventral tegmental area (VTA) and (2) cerebellum to mPFC via glutamatergic projections from the mediodorsal and ventrolateral thalamus (ThN md and vl). The present study compared functional adaptations of cerebello-cortical circuitry following developmental cerebellar pathology in a mouse model of developmental loss of Purkinje cells (Lurcher) and a mouse model of fragile X syndrome (Fmr1 KO mice). Fixed potential amperometry was used to measure mPFC dopamine release in response to cerebellar electrical stimulation. Mutant mice of both strains showed an attenuation in cerebellar-evoked mPFC dopamine release compared to respective wildtype mice. This was accompanied by a functional reorganization of the VTA and thalamic pathways mediating cerebellar modulation of mPFC dopamine release. Inactivation of the VTA pathway by intra-VTA lidocaine or kynurenate infusions decreased dopamine release by 50 % in wildtype and 20-30 % in mutant mice of both strains. Intra-ThN vl infusions of either drug decreased dopamine release by 15 % in wildtype and 40 % in mutant mice of both strains, while dopamine release remained relatively unchanged following intra-ThN md drug infusions. These results indicate a shift in strength towards the thalamic vl projection, away from the VTA. Thus, cerebellar neuropathologies associated with autism spectrum disorders may cause a reduction in cerebellar modulation of mPFC dopamine release that is related to a reorganization of the mediating neuronal pathways.

A chronic increase in circulating angiotensin II (Ang II) activates an aldosterone-mineralocorticoid receptor-ouabain neuromodulatory pathway in the brain that increases neuronal activation in hypothalamic nuclei, such as the paraventricular nucleus (PVN) and causes progressive hypertension. Several models of chronic sympathetic hyperactivity are associated with an increase in AT1 and glutamate receptor activation in the PVN. The current study evaluated whether increased angiotensin type 1 (AT1) and glutamate receptor-dependent signaling in the PVN contributes to the maintenance of blood pressure (BP) in Ang II-hypertensive Wistar rats, and the role of aldosterone-mineralocorticoid receptor pathway in this enhanced signaling. After subcutaneous infusion of Ang II for 2 weeks, in conscious rats BP and heart rate were recorded after (1) 10-minute bilateral infusions of candesartan and kynurenate in the PVN; (2) 1 hour intracerebroventricular infusion of eplerenone, and (3) candesartan and kynurenate after eplerenone. Candesartan or kynurenate in the PVN fully reversed the increase in BP from circulating Ang II. Kynurenate after candesartan or candesartan after kynurenate did not further lower BP. Intracerebroventricular infusion of eplerenone at 16 hours after its infusion fully reversed the increase in BP from circulating Ang II. After eplerenone, candesartan and kynurenate in the PVN did not further decrease BP. These findings suggest that increased mineralocorticoid receptor activation in the brain activates a slow neuromodulatory pathway that maintains enhanced AT1 and glutamate receptor-dependent signaling in the PVN, and thereby the hypertension from a chronic increase in circulating Ang II.

Increased plasma osmolality elevates mean arterial pressure (MAP) through activation of the sympathetic nervous system, but the neurotransmitters released in the spinal cord to regulate MAP during osmotic stress remain unresolved. Glutamatergic neurons of the rostral ventrolateral medulla project to sympathetic preganglionic neurons in the spinal cord and are likely activated during conditions of osmotic stress; however, this has not been examined in conscious rats. This study investigated whether increased MAP during chronic osmotic stress depends on activation of spinal glutamate receptors. Rats were chronically instrumented with an indwelling intrathecal (i.t.) catheter for antagonist delivery to the spinal cord and a radiotelemetry transmitter for continuous monitoring of MAP and heart rate. Osmotic stress induced by 48 h of water deprivation (WD) increased MAP by ~15 mmHg. Intrathecal kynurenic acid, a nonspecific antagonist of ionotropic glutamate receptors, decreased MAP significantly more after 48 h of WD compared with the water-replete state. Water-deprived rats also showed a greater fall in MAP in response to i.t. 2-amino-5-phosphonovalerate. Finally, i.t. kynurenic acid also decreased MAP more in an osmotically driven model of neurogenic hypertension, the DOCA-salt rat, compared with normotensive controls. Our results suggest that spinally released glutamate mediates increased MAP during 48-h WD and DOCA-salt hypertension.

The paraventricular nucleus of hypothalamus (PVN) is a well known site of integration for autonomic and cardiovascular responses, and the glutamate neurotransmitter plays an important role. The aim of our study was to evaluate the cardiovascular parameters and autonomic modulation by means of spectral analysis after ionotropic glutamate receptor inhibition in the PVN in conscious sedentary (S) or swimming trained (ST) rats. After exercise training protocol, adult male Wistar rats, instrumented with guide cannulae to PVN and artery and vein catheters were submitted to mean arterial pressure (MAP) and heart rate (HR) recording. At baseline, physical training induced a resting bradycardia (S: 379 ± 3, ST: 349 ± 2 bpm, Pb<0.05) and promoted adaptations in HRV characterized by an increase of HF in normalized values and a decrease of LF in absolute and normalized units compared with the sedentary group. Microinjection of kynurenic acid (KYNA) in the PVN of sedentary and trained rats promoted decreases in MAP and HR, but the decrease in HR was smaller in the trained animals (ΔHRS: -48 ± 7, ST: -28 ± 4 bpm, Pb<0.05). Furthermore, the differences in baseline parameters of pulse interval, found between sedentary and trained animals, disappeared after KYNA microinjection in the PVN. Our data suggest that the cardiovascular and autonomic adaptations to the heart induced by exercise training may involve glutamatergic mechanisms in the PVN.

In the brain of neonatal chicks, tryptophan has a sedative effect, and a part of this effect might be dependent upon its metabolite, serotonin. However, the functional mechanisms have not been fully clarified, since l-tryptophan produces kynurenic acid (KYNA) through the kynurenine pathway. The present study aimed to clarify the effect of KYNA on the stress response upon social isolation. Intracerebroventricular injection of KYNA induced a strong sedative effect under stress compared with the effect of l-tryptophan, with or without intracerebroventricular injection of corticotrophin-releasing hormone (CRH). KYNA dose-dependently induced sedative and hypnotic effects under CRH-augmented social isolation stress. Taken together, these results indicate that KYNA is a likely candidate for the sedative and hypnotic effects of tryptophan under acutely stressful conditions., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Rationale and Objectives: Accumulating clinical and preclinical studies have shown that the memories of the rewarding effects of drugs and their paired cues may contribute to relapse and persistent cocaine use. Glutaminergic actions in the ventral tegmental area (VTA) have been shown to regulate the rewarding effect of drugs and conditioned responses to drug-associated cues, but the role of the VTA in the acquisition, retrieval, and reconsolidation of cocaine cues is not yet known., Methods: In the present study, we used 7-chlorothiokynurenic acid (7-CTKA), an N-methyl-D-aspartate (NMDA) receptor glycine modulatory site antagonist with no rewarding effects, to examine the role of the NMDA receptor glycine modulatory site in the acquisition, retrieval, and reconsolidation of cocaine-related reward memory using the conditioned place preference (CPP) paradigm., Results: Separate groups of Sprague-Dawley rats were trained to acquire cocaine-induced CPP. Vehicle or 7-CTKA was microinjected into the VTA or substantia nigra (SN) (5 μg/μl) at different time points: 10 min before each CPP training session (acquisition), 10 min before the reactivation of CPP (retrieval), and immediately after the reactivation of CPP (reconsolidation). Cocaine-induced CPP was retested 24 h and 1 and 2 weeks after 7-CTKA administration. 7-CTKA microinjected into the VTA, but not SN, significantly impaired the acquisition, retrieval, and reconsolidation of cocaine-induced CPP without affecting cocaine-induced locomotion., Conclusions: Our findings suggest that the NMDA receptor glycine modulatory site in the VTA plays a major role in cocaine reward memory, and NMDA receptor glycine site antagonists may be potential pharmacotherapies for the management of relapse.

Several models of chronic sympathetic hyperactivity are associated with an increase in excitatory angiotensinergic and glutamatergic activity, and a decrease in GABAergic activity in the PVN. The present study evaluated whether activation of glutamate and AT1 receptors in the PVN contributes to the maintenance of resting BP in Dahl salt sensitive (S) rats on regular or high salt diet for 4-6 weeks. Candesartan and kynurenate were infused bilaterally into the PVN and BP and heart rate (HR) were recorded. Both candesartan and kynurenate in the PVN did not change MAP and HR in normotensive Dahl salt resistant (R) and S rats on regular salt diet or in R rats on high salt diet. In hypertensive Dahl S rats on high salt diet, candesartan decreased MAP (-14±2 mm Hg), and tended to increase HR (22±5 bpm). Kynurenate decreased both MAP (-22±3 mm Hg) and HR (-42±7 bpm) in these rats. At the peak BP decrease by candesartan, kynurenate in the PVN further decreased BP by ~50% (-14±2 mm Hg), whereas candesartan did not further decrease BP at the peak BP response to kynurenate (-4±2 mm Hg). These results indicate that activation of glutamate and AT1-receptors in the PVN contributes to the maintenance of BP in hypertensive Dahl S rats, but not normotensive Dahl S and R rats. The increased BP response to AT1-receptor activation in the PVN of hypertensive Dahl S appears to be mediated by enhanced local glutamate receptor activation, but another mechanism(s) appears to further enhance glutamate responses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Sympathetic overactivity and altered respiratory control are commonly observed after chronic intermittent hypoxia (CIH) exposure. However, the central mechanisms underlying such neurovegetative dysfunctions remain unclear. Herein, we hypothesized that CIH (6% O(2) every 9 min, 8 h/day, 10 days) in juvenile rats alters glutamatergic transmission in the commissural nucleus tractus solitarius (cNTS), a pivotal site for integration of peripheral chemoreceptor inputs. Using an in situ working heart-brain stem preparation, we found that l-glutamate microinjections (1, 3, and 10 mM) into the cNTS of control rats (n = 8) evoked increases in thoracic sympathetic nerve (tSN) and central vagus nerve (cVN) activities combined with inhibition of phrenic nerve (PN) activity. Besides, the ionotropic glutamatergic receptor antagonism with kynurenic acid (KYN; 250 mM) in the cNTS of control group (n = 7) increased PN burst duration and frequency. In the CIH group (n = 10), the magnitude of l-glutamate-induced cVN excitation was smaller, and the PN inhibitory response was blunted (P < 0.05). In addition, KYN microinjections into the cNTS of CIH rats (n = 9) did not alter PN burst duration and produced smaller increases in its frequency compared with controls. Moreover, KYN microinjections into the cNTS attenuated the sympathoexcitatory response to peripheral chemoreflex activation in control but not in CIH rats (P < 0.05). These functional CIH-induced alterations were accompanied by a significant 10% increase of N-methyl-D-aspartate receptor 1 (NMDAR1) and glutamate receptor 2/3 (GluR2/3) receptor subunit density in the cNTS (n = 3-8, P < 0.05), evaluated by Western blot analysis. These data indicate that glutamatergic transmission is altered in the cNTS of CIH rats and may contribute to the sympathetic and respiratory changes observed in this experimental model.

It is usually assumed that kynurenic acid (KYNA) modifies neuronal function because it antagonizes the glycine site of the NMDA receptors and/or the neuronal cholinergic α7 nicotine receptors. It is not clear, however, whether the basal levels of KYNA found in brain extracellular spaces are sufficient to interact with these targets. Another reported target for KYNA is GPR35, an orphan receptor negatively coupled to G(i) proteins. GPR35 is expressed both in neurons and other cells (including glia, macrophages and monocytes). KYNA affinity for GPR35 in native systems has not been clarified and the low-affinity data widely reported in the literature for the interaction between KYNA and human or rat GPR35 have been obtained in modified expression systems. Possibly by interacting with GPR35, KYNA may also reduce glutamate release in brain and pro-inflammatory cytokines release in cell lines. The inhibition of inflammatory mediator release from both glia and macrophages may explain why KYNA has analgesic effects in inflammatory models. Furthermore, it may also explain why, KYNA administration (200 mg/kg ip × 3 times) to mice treated with lethal doses of LPS, significantly reduces the number of deaths. Finally, KYNA has been reported as an agonist of aryl hydrocarbon receptor (AHR), a nuclear protein involved in the regulation of gene transcription and able to cause immunosuppression after binding with dioxin. Thus, KYNA has receptors in the nervous and the immune systems and may play interesting regulatory roles in cell function.

The main purpose of this study was to facilitate the delivery of kynurenic acid (KYNA) across the blood-brain barrier (BBB) by applying micelles as nanoscale containers. Non-ionic amphiphilic molecules were used for preparation of spherical micelles for delivery of kynurenic acid in aqueous solution in physiological condition. It was established that Triton X 100 and Lutensol AP 20 non-ionic surfactants are able to produce stable nanocontainers for delivery of kynurenic acid molecules. The incorporation of KYNA molecules was investigated by dynamic light scattering and the size of micelles were calculated between 5 and 10 nm in 150 mM NaCl and pH 7.5-7.6 solutions. Encapsulated kynurenic acid showed a significantly higher blood-brain barrier permeability compared with non-encapsulated kynurenic acid. The in vivo experiments showed that the encapsulated kynurenic acid is able to display effects within the central nervous system, even after its peripheral administration.

Purpose: Serotonin, a neurotransmitter synthesized from tryptophan, has been proposed to play a key role in central fatigue. In this study, we examined whether tryptophan itself and/or its two metabolites, kyneurenic acid (KYNA) and quinolinic acid (QUIN), are involved in central fatigue., Materials and Methods: Experiments were conducted using Sprague-Dawley rats (SDR) and Nagase analbuminemic rats (NAR). Central fatigue was assessed by treadmill running and a Morris water maze test. Microdialysis was used to collect samples for measurement of extracellular concentration of tryptophan, serotonin and 5-hydroxyindoleacetic acid (5-HIAA) and to infuse test agents. To examine the kinetics of release, synaptosomes in the striatum were prepared in vitro to measure intra- and extrasynaptosomal concentration of tryptophan, serotonin and 5-HIAA., Results: The concentration of tryptophan secreted into the extracellular space of the striatum was higher during fatigue only, and quickly returned to basal levels with recovery from fatigue. Running time to exhaustion was reduced by activation of tryptophan receptors. Time to exhaustion was shorter in NAR, which maintain a higher extracellular level of striatum tryptophan than SDR. Impaired memory performance in a water maze task after tryptophan treatment was attributable to high levels of KYNA and QUIN in the hippocampus acting synergistically on N-methyl-D-aspartic acid receptors. When branched-chain amino acids were administered, tryptophan transport to the extracellular space of the striatum was drastically inhibited., Conclusion: Our findings demonstrate that the increase in fatigue which occurs because of excessively elevated brain tryptophan can be further amplified by the use of synthetic KYNA and QUIN.

Background: The rostral ventrolateral medulla (RVLM) in the brainstem is responsible for regulation of the sympathetic nervous system. In the RVLM, nitric oxide (NO)-mediated γ-amino butyric acid (GABA) is a major sympathoinhibitory amino acid neurotransmitter and superoxide is a major sympathoexcitatory factor. In this study, we investigated whether or not NO-mediated GABA release is involved in superoxide-induced sympathoexcitation in the RVLM of hypertensive rats., Methods and Results: For our model hypertensive rats with sympathoexcitation, we used stroke-prone spontaneously hypertensive rats (SHRSP). GABA levels in the RVLM were measured by in vivo microdialysis. Microinjection of tempol, a superoxide scavenger, into the RVLM decreased arterial pressure (AP), heart rate (HR), and renal sympathetic nerve activity (RSNA) with an increase in GABA release in the RVLM. Microinjection of N(G)-monomethyl-L-arginine (L-NMMA), an NO synthase inhibitor, into the RVLM increased AP, HR, and RSNA with a decrease in GABA release in the RVLM. Prior microinjection of L-NMMA into the RVLM attenuated the tempol-induced changes in AP, HR, RSNA, and GABA release in the RVLM. Microinjection of bicuculline, a GABA receptor blocker, into the RVLM attenuated the tempol- and L-NMMA-induced changes in AP, HR, and RSNA., Conclusions: The findings suggest that reduction of NO-mediated GABA release in the RVLM is partly involved in superoxide-induced sympathoexcitation of SHRSP.

Aims: The aims were to provide proofs of mechanism and principle by establishing the ability of kynurenine metabolites to inhibit the liver mitochondrial low K(m) aldehyde dehydrogenase (ALDH) activity after administration and in vivo, and to induce aversion to alcohol., Methods: Kynurenic acid (KA), 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) were administered to normal male Wistar rats and ALDH activity was determined both in vitro in liver homogenates and in vivo (by measuring blood acetaldehyde following ethanol administration). Alcohol consumption was studied in an aversion model in rats and in alcohol-preferring C57 mice., Results: ALDH activity was significantly inhibited by all three metabolites by doses as small as 1 mg/kg body wt. Blood acetaldehyde accumulation after ethanol administration was strongly elevated by KA and 3-HK and to a lesser extent by 3-HAA. All three metabolites induced aversion to alcohol in rats and decreased alcohol preference in mice., Conclusions: The above kynurenine metabolites of tryptophan induce aversion to alcohol by inhibiting ALDH activity. An intellectual property covering the use of 3-HK and 3-HAA and derivatives thereof in the treatment of alcoholism by aversion awaits further development.

Kynurenic acid (KYNA) is an endogenous metabolite of the kynurenine pathway for tryptophan degradation and an antagonist of both N-methyl-D-aspartate (NMDA) and alpha-7 nicotinic acetylcholine (α7nACh) receptors. KYNA has also been shown to scavenge hydroxyl radicals (OH) under controlled conditions of free radical production. In this work we evaluated the ability of KYNA to scavenge superoxide anion (O(2)(-)) and peroxynitrite (ONOO(-)). The scavenging ability of KYNA (expressed as IC(50) values) was as follows: OH=O(2)(-)>ONOO(-). In parallel, the antiperoxidative and scavenging capacities of KYNA (0-150 μM) were tested in cerebellum and forebrain homogenates exposed to 5 μM FeSO(4) and 2.5 mM 3-nitropropionic acid (3-NPA). Both FeSO(4) and 3-NPA increased lipid peroxidation (LP) and ROS formation in a significant manner in these preparations, whereas KYNA significantly reduced these markers. Reactive oxygen species (ROS) formation were determined in the presence of FeSO(4) and/or KYNA (0-100 μM), both at intra and extracellular levels. An increase in ROS formation was induced by FeSO(4) in forebrain and cerebellum in a time-dependent manner, and KYNA reduced this effect in a concentration-dependent manner. To further know whether the effect of KYNA on oxidative stress is independent of NMDA and nicotinic receptors, we also tested KYNA (0-100 μM) in a biological preparation free of these receptors - defolliculated Xenopus laevis oocytes - incubated with FeSO(4) for 1 h. A 3-fold increase in LP and a 2-fold increase in ROS formation were seen after exposure to FeSO(4), whereas KYNA attenuated these effects in a concentration-dependent manner. In addition, the in vivo formation of OH evoked by an acute infusion of FeSO(4) (100 μM) in the rat striatum was estimated by microdialysis and challenged by a topic infusion of KYNA (1 μM). FeSO(4) increased the striatal OH production, while KYNA mitigated this effect. Altogether, these data strongly suggest that KYNA, in addition to be a well-known antagonist acting on nicotinic and NMDA receptors, can be considered as a potential endogenous antioxidant., (Copyright © 2011 Elsevier Inc. All rights reserved.)