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The tryptophan metabolite kynurenic acid (KYNA) may play an important role in normal and abnormal cognitive processes, most likely by interfering with α7 nicotinic and NMDA receptor function. KYNA is formed from its immediate precursor kynurenine either by non-enzymatic oxidation or through irreversible transamination by kynurenine aminotransferases. In the mammalian brain, kynurenine aminotransferase II (KAT II) is the principal enzyme responsible for the neosynthesis of rapidly mobilizable KYNA, and therefore constitutes an attractive target for pro-cognitive interventions. N-acetylcysteine (NAC), a brain-penetrant drug with pro-cognitive efficacy in humans, has been proposed to exert its actions by increasing the levels of the anti-oxidant glutathione (GSH) in the brain. We report here that NAC, but not GSH, inhibits KAT II activity in brain tissue homogenates from rats and humans with IC50 values in the high micromolar to low millimolar range. With similar potency, the drug interfered with the de novo formation of KYNA in rat brain slices, and NAC was a competitive inhibitor of recombinant human KAT II (Ki: 450 μM). Furthermore, GSH failed to S-glutathionylate recombinant human KAT II treated with the dithiocarbamate drug disulfiram. Shown by microdialysis in the prefrontal cortex of rats treated with kynurenine (50 mg/kg, i.p.), peripheral administration of NAC (500 mg/kg, i.p., 120 and 60 min before the application of kynurenine) reduced KYNA neosynthesis by ∼50%. Together, these results suggest that NAC exerts its neurobiological effects at least in part by reducing cerebral KYNA formation via KAT II inhibition.

Kynurenine pathway metabolites (KPM) are thought to be synthesized mainly by non-neuronal cells in the mammalian brain. KPM are of particular interest because several studies demonstrated their implication in various disorders of the nervous system. Among KPM is xanthurenic acid (XA) deriving from the catabolism of 3-hydroxykynurenine. Based on its chemical structure, XA appears as a close analog of kynurenic acid which has been extensively investigated and is considered as a potent neuroprotective compound. Contrary to kynurenic acid (KYNA), XA has received little attention and its role in the brain remains not elucidated. We have previously described several characteristics of XA, suggesting its possible involvement in neurotransmission. XA is also proposed as a potential modulator at glutamatergic synapses. Here, we used a selective antibody against XA and various neuronal, glial and synaptic markers to show that XA is essentially localized in the soma and dendrites of brain neurons, but is absent from axonal compartments and terminal endings. Our results also reveal that XA-like immunoreactivity is not expressed by glial cells. To double-check our findings, we have also used another XA antibody obtained from a commercial source to confirm the neuronal expression of XA. Together, our results suggest that, differently to several other KPM produced by glial cells, XA exhibits a neuronal distribution in the mouse brain.

The brain of children affected by organic acidemias develop acute neurodegeneration linked to accumulation of endogenous toxic metabolites like glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids. Excitotoxic and oxidative events are involved in the toxic patterns elicited by these organic acids, although their single actions cannot explain the extent of brain damage observed in organic acidemias. The characterization of co-adjuvant factors involved in the magnification of early toxic processes evoked by these metabolites is essential to infer their actions in the human brain. Alterations in the kynurenine pathway (KP) - a metabolic route devoted to degrade tryptophan to form NAD(+) - produce increased levels of the excitotoxic metabolite quinolinic acid (QUIN), which has been involved in neurodegenerative disorders. Herein we investigated the effects of subtoxic concentrations of GA, 3-OHGA, MMA and PA, either alone or in combination with QUIN, on early toxic endpoints in rat brain synaptosomes. To establish specific mechanisms, we pre-incubated synaptosomes with different protective agents, including the endogenous N-methyl-d-aspartate (NMDA) receptor antagonist kynurenic acid (KA), the antioxidant S-allylcysteine (SAC) and the nitric oxide synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME). While the incubation of synaptosomes with toxic metabolites at subtoxic concentrations produced no effects, their co-incubation (QUIN+GA, +3-OHGA, +MMA or +PA) decreased the mitochondrial function and increased reactive oxygen species (ROS) formation and lipid peroxidation. For all cases, this effect was partially prevented by KA and l-NAME, and completely avoided by SAC. These findings suggest that early damaging events elicited by organic acids involved in metabolic acidemias can be magnified by toxic synergism with QUIN, and this process is mostly mediated by oxidative stress, and in a lesser extent by excitotoxicity and nitrosative stress. Therefore, QUIN can be hypothesized to contribute to the pathophysiology of brain degeneration in children with metabolic acidemias.

Xanthurenic acid (XA), formed from 3-hydroxykynurenine (3-HK) in the kynurenine pathway of tryptophan degradation, may modulate glutamatergic neurotransmission by inhibiting the vesicular glutamate transporter and/or activating Group II metabotropic glutamate receptors. Here we examined the molecular and cellular mechanisms by which 3-HK controls the neosynthesis of XA in rat, mouse and human brain, and compared the physiological actions of 3-HK and XA in the rat brain. In tissue homogenates, XA formation from 3-HK was observed in all three species and traced to a major role of kynurenine aminotransferase II (KAT II). Transamination of 3-HK to XA was also demonstrated using human recombinant KAT II. Neosynthesis of XA was significantly increased in the quinolinate-lesioned rat striatum, indicating a non-neuronal localization of the process. Studies using rat cortical slices revealed that newly produced XA is rapidly released into the extracellular compartment, and that XA biosynthesis can be manipulated experimentally in the same way as the production of kynurenic acid from kynurenine (omission of Na+ or glucose, depolarizing conditions, or addition of 2-oxoacids). The synthesis of XA from 3-HK was confirmed in vivo by striatal microdialysis. In slices from the rat hippocampus, both 3-HK and XA reduced the slopes of dentate gyrus field EPSPs. The effect of 3-HK was reduced in the presence of the KAT inhibitor aminooxyacetic acid. Finally, both 3-HK and XA reduced the power of gamma-oscillatory activity recorded from the hippocampal CA3 region. Endogenous XA, newly formed from 3-HK, may therefore play a physiological role in attentional and cognitive processes.

Since brain ischemia is one of the leading causes of adult disability and death, neuroprotection of the ischemic brain is of particular importance. Acute neuroprotective strategies usually have the aim of suppressing glutamate excitotoxicity and an excessive N-methyl-d-aspartate (NMDA) receptor function. Clinically tolerated antagonists should antagonize an excessive NMDA receptor function without compromising the normal synaptic function. Kynurenic acid (KYNA) an endogenous metabolite of the tryptophan metabolism, may be an attractive neuroprotectant in this regard. The manipulation of brain KYNA levels was earlier found to effectively enhance the histopathological outcome of experimental ischemic/hypoxic states. The present investigation of the neuroprotective capacity of L-kynurenine sulfate (L-KYNs) administered systemically after reperfusion in a novel distal middle cerebral artery occlusion (dMCAO) model of focal ischemia/reperfusion revealed that in contrast with earlier results, treatment with L-KYNs worsened the histopathological outcome of dMCAO. This contradictory result indicates that post-ischemic treatment with L-KYNs may be harmful.

Using two in vivo methods, microdialysis and rapid in situ electrochemistry, this study examined the modulation of extracellular glutamate levels by endogenously produced kynurenic acid (KYNA) in the prefrontal cortex (PFC) of awake rats. Measured by microdialysis, intraperitoneal (i.p.) administration of KYNA's bioprecursor L-kynurenine dose-dependently elevated extracellular KYNA and reduced extracellular glutamate (nadir after 50 mg/kg kynurenine: 60% decrease from baseline values). This dose-dependent decrease in glutamate levels was also seen using a glutamate-sensitive microelectrode array (MEA) (31% decrease following 50 mg/kg kynurenine). The kynurenine-induced reduction in glutamate was blocked (microdialysis) or attenuated (MEA) by co-administration of galantamine (3 mg/kg, i.p.), a drug that competes with KYNA at an allosteric potentiating site of the α7 nicotinic acetylcholine receptor. In separate experiments, extracellular glutamate levels were measured by MEA following the local perfusion (45 min) of the PFC with kynurenine (2.5 μM) or the selective KYNA biosynthesis inhibitor S-ethylsulfonylbenzoylalanine (S-ESBA; 5 mM). In agreement with previous microdialysis studies, systemic kynurenine application produced a reversible reduction in glutamate (nadir: −29%), whereas perfusion with S-ESBA increased glutamate levels reversibly (maximum: +38%). Collectively, these results demonstrate that fluctuations in the biosynthesis of KYNA in the PFC bi-directionally modulate extracellular glutamate levels, and that qualitatively very similar data are obtained by microdialysis and MEA. Since KYNA levels are elevated in the PFC of individuals with schizophrenia, and since prefrontal glutamatergic and nicotinic transmission mediate cognitive flexibility, normalization of KYNA levels in the PFC may constitute an effective treatment strategy for alleviating cognitive deficits in schizophrenia.

The N-methyl-d-aspartate (NMDA) subtype of glutamate receptors plays an important role in brain physiology, but excessive receptor stimulation results in seizures and excitotoxic nerve cell death. NMDA receptor-mediated neuronal excitation and injury can be prevented by high, non-physiological concentrations of the neuroinhibitory tryptophan metabolite kynurenic acid (KYNA). Here we report that endogenous KYNA, which is formed in and released from astrocytes, controls NMDA receptors in vivo. This was revealed with the aid of the dopaminergic drugs d-amphetamine and apomorphine, which cause rapid, transient decreases in striatal KYNA levels in rats. Intrastriatal injections of the excitotoxins NMDA or quinolinate (but not the non-NMDA receptor agonist kainate) at the time of maximal KYNA reduction resulted in two- to threefold increases in excitotoxic lesion size. Pre-treatment with a kynurenine 3-hydroxylase inhibitor or with dopamine receptor antagonists, i.e., two classes of pharmacological agents that prevented the reduction in brain KYNA caused by dopaminergic stimulation, abolished the potentiation of neurotoxicity. Thus, the present study identifies a previously unappreciated role of KYNA as a functional link between dopamine receptor stimulation and NMDA neurotoxicity in the striatum.

The effects of a ketogenic diet in controlling seizure activity have been proven in many studies, although its mechanism of action remains elusive in many regards. We hypothesize that the ketogenic diet may exert its antiepileptic effects by influencing tryptophan (TRP) metabolism. The aim of this study was to investigate the influence of octanoic and decanoic fatty acids (FAs), the main components in the MCT diet (medium-chain triglyceride diet, a subtype of the ketogenic diet), on the metabolism of TRP, the activity of the kynurenic pathway and the concentrations of monoamines and amino acids, including branched-chain amino acids (BCAA) and aromatic amino acids (AAA) in rats. The acute effects of FA on the sedation index and hippocampal electrical after-discharge threshold were also assessed. We observed that intragastric administration of FA increased the brain levels of TRP and the central and peripheral concentrations of kynurenic acid (KYNA), as well as caused significant changes in the brain and plasma concentrations of BCAA and AAA. We found that the administration of FA clearly increased the seizure threshold and induced sedation. Furthermore, we have demonstrated that blocking TRP passage into the brain abolished these effects of FA but had no similar effect on the formation of ketone bodies. Given that FAs are major components of a ketogenic diet, it is suggested that the anticonvulsant effects of a ketogenic diet may be at least partly dependent on changes in TRP metabolism. We also propose a more general hypothesis concerning the intracellular mechanism of the ketogenic diet.

The olfactory bulb, first relay of olfactory pathways, is densely innervated by serotoninergic centrifugal fibers originating from the raphe nuclei. Although serotonin innervation was reported to be involved in olfactory learning in mammals, the action of this neurotransmitter on its putative cellular targets has been never described through unitary recordings. This lack of data initiated the present study where the effects of 5HT on juxtaglomerular and mitral cells are analyzed using whole-cell recordings on olfactory bulb slices. Serotonin depolarizes 34% of 525 JG cells. A multivariate statistical analysis of juxtaglomerular cells characteristics shows that the serotonin responsive cell group can be individualized regarding their tonic discharge-mode in response to a direct current injection, their lower expression of hyperpolarization-activated cation current and their low membrane capacities. The use of ion channel blockers and ramp voltage protocol indicate that serotoninergic depolarization of juxtaglomerular cells may be due to a nonselective cation current with a reversal potential of −44 mV. Pharmacological tests with serotonin receptor antagonists and agonists reveal that 5HT action on juxtaglomerular cells would be mainly mediated by 5HT2C receptors. In mitral cells, serotonin acts on 49.1% of the 242 tested cells, inducing two types of responses. A first subset of mitral cells (26.8%, n=65) were hyperpolarized by serotonin. This response would be indirect and mediated by action of GABA on GABAA receptors since it was antagonized by bicuculline. The involved GABAergic neurons are hypothesized to be juxtaglomerular and granular cells, on which serotonin would act mainly via 5HT2C and via 5HT2A receptors respectively. The second subset of mitral cells (22.3%, n=54) were directly depolarized by serotonin acting through 5HT2A receptors. Our data on serotonin action on juxtaglomerular cells and mitral cells reveal a part of functional mechanisms whereby serotonin can act on olfactory bulb network. This is expected to enrich the understanding of its determining role in olfactory learning.

Long-term potentiation (LTP) is a use-dependent and persistent enhancement of synaptic strength. In the CA1 region of the hippocampus, LTP has Hebbian characteristics and requires precisely timed interaction between presynaptic firing and postsynaptic depolarization. Although depolarization is an absolute requirement for plasticity, it is still not clear whether the postsynaptic response during LTP induction should be subthreshold or suprathreshold for the generation of somatic action potential. Here, we use the whole-cell patch-clamp technique and different pairing protocols to examine systematically the postsynaptic induction requirements for LTP. We induce LTP by changes only in membrane potential while keeping the afferent stimulation constant and at minimal levels. This approach permits differentiation of two types of LTP: LTP induced with suprathreshold synaptic responses (LTP(AP)) and LTP induced with subthreshold excitatory postsynaptic current (EPSCs; LTP(EPSC)). We found that LTP(AP) (>40%) required pairing of depolarization (V(m)>or=-40 mV, for 40-60 s) with four to six (0.1 Hz) single synaptically initiated action potentials. LTP(EPSC) was of smaller magnitude (

The startle reflex is elicited by acoustic, trigeminal or vestibular stimulation, or by combinations of these stimuli. Acoustic startle is mediated largely by ibotenate-sensitive neurons in the ventrocaudal pontine reticular formation (PnC). In these studies we tested whether startle elicited by stimulation of different modalities is affected by infusion of the non-selective glutamate antagonist, kynurenate, into the PnC. In awake rats, startle responses evoked by either acoustic or spinal trigeminal nucleus stimulation were inhibited by kynurenate, but not saline, infusions, with the most effective placements nearest PnC. In chloral hydrate-anesthetized rats, kynurenate in the PnC reduced trigeminal nucleus-evoked hindlimb EMG responses, but not vestibular nucleus-evoked startle. Kynurenate in the vestibular nucleus had no effect on trigeminal nucleus-evoked startle. These results indicate that trigeminal nucleus stimulation evokes startle largely through glutamate receptors in the PnC, similarly to acoustic startle, but vestibular nucleus-evoked startle is mediated through other pathways, such as the vestibulospinal tract.

We have previously shown that conditioned fear to context increases Fos expression in the caudal ventrolateral region of the periaqueductal gray in the rat. To understand the reason for this activation and its role in the expression of the contextual fear response, the ventrolateral periaqueductal gray was temporarily blocked with bilateral microinjections (0.4 μl) of the GABA agonist muscimol (0.2 mM) or the glutamate antagonist kynurenic acid (0.1 M). Cardiovascular changes and activity were recorded by radio-telemetry and the microinjections were made immediately before testing the conditioned response in the aversive context. Muscimol and kynurenic acid had the same effects: when compared to saline controls, freezing immobility and ultrasonic vocalizations were reduced and replaced by marked locomotor activity, and the increase in heart rate was enhanced; however, the increase in arterial blood pressure remained the same. Interesting changes were also observed when animals were returned to the safe context of their home box after fear (recovery). Basically, the recovery response was either prevented or delayed: instead of returning to resting immobility, the rats remained agitated in their home box with a moderately elevated activity, heart rate and blood pressure. However, the effect of ventrolateral periaqueductal gray blockade on heart rate, arterial pressure and activity did not appear to be specific to the fear response or its recovery because they were also observed in animals returned to the safe context of their home box immediately after injection. The later response was also a recovery response from the milder stress of handling and the injection procedure. We discuss the results by arguing that the ventrolateral periaqueductal gray is involved in the immobility component of both the fear response and poststress recovery responses. To explain our interpretation we consider the findings in relation to the classic descending defence-arousal system and the hyporeactive-hypotensive immobility pattern that has been attributed to the ventrolateral periaqueductal gray. We propose that there is a dual activation of the defence-arousal system and of the ventrolateral periaqueductal gray during fear, with the ventrolateral periaqueductal gray acting as a brake on the defence-arousal system. The role of this brake is to impose immobility and hold off active defence responses such as fight and flight. The result of this combination of arousal and immobility is a hyperreactive freezing immobility associated with ultrasonic vocalizations, and a pressor response accompanied with a slow rise in heart rate. Basically, the animal is tense and ready for action but temporarily immobilised. The ventrolateral periaqueductal gray also acts to impose immobility during recovery; however, this is without coactivation of the defence-arousal system. The result is a return to resting immobility, associated with a return to baseline blood pressure and heart rate. This is an active process that insures a faster and complete return to rest. We conclude that the ventrolateral periaqueductal gray is an immobility center involved not only in the fear response but also in poststress recovery responses.

In urethane-anaesthetised artificially ventilated Sprague-Dawley rats, bilateral microinjection of the divalent cation nickel chloride (Ni(2+); 50 mM, 50 nl) into the rostral ventrolateral medulla elicited a dramatic inhibition of splanchnic sympathetic nerve activity (-44+/-6%) and a marked depressor response (-35+/-7 mmHg). Selective blockade of high-voltage activated Ca(2+) channels with omega-agatoxin IVA (P/Q-type), omega-conotoxin GVIA (N-type) and nifedipine (L-type) did not decrease arterial pressure or splanchnic sympathetic nerve activity when injected separately into the rostral ventrolateral medulla, or combined with kynurenate. Injection of caesium chloride or ZD 7288, a blocker of the hyperpolarization-activated cation current, into the rostral ventrolateral medulla had no effect on arterial pressure or splanchnic sympathetic nerve activity. Bilateral microinjection of nickel chloride into the caudal ventrolateral medulla/pre-Bötzinger complex elicited small increases in splanchnic sympathetic nerve activity (+17+/-13%) and arterial pressure (+12+/-4 mmHg). These were substantially smaller than those evoked by blockade of glutamatergic receptors or high-voltage activated Ca(2+) channels in this area. Injection of kynurenate or high-voltage activated Ca(2+) channel blocker, but not Ni(2+), in this area evoked respiratory termination. The results indicate the existence of a distinct mechanism maintaining the tonic activity of rostral ventrolateral medulla presympathetic neurons that is different from that maintaining the tonic activity in the caudal ventrolateral medulla/pre-Bötzinger region. We conclude that ion channels that are sensitive to Ni(2+), but are insensitive to high-voltage activated (L, P/Q, N) Ca(2+) channel blockers, and are located postsynaptically on the presympathetic rostral ventrolateral medulla neurons are responsible for the tonic activity of the presympathetic neurons in rostral ventrolateral medulla. These channels could well be the low-voltage-activated (or T-type) Ca(2+) channels although other conductances cannot be conclusively excluded.

Intracellular recordings were made in a mid-pontine slice preparation of the rat brain containing the nucleus locus coeruleus. Focal electrical stimulation evoked biphasic synaptic potentials consisting of early depolarizing (d.p.s.p.) and late hyperpolarizing (i.p.s.p.) components. The alpha(2)-adrenoceptor antagonist idazoxan inhibited the i.p.s.p. without altering the d.p.s.p. All of the following experiments were carried out in the presence of kynurenic acid and picrotoxin to block the glutamatergic and GABAergic fractions of the d.p.s.p., respectively. Guanethidine, which is known to inhibit noradrenaline and ATP release from nerve terminals of postganglionic sympathetic nerves, depressed both the d.p.s.p. and the i.p.s.p. in a concentration-dependent manner. Damage of catecholaminergic nerve terminals by 6-hydroxydopamine also decreased both the d.p.s.p. and the i.p.s.p. The P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) depressed the d.p.s.p., whereas the i.p.s.p. remained unaffected. The further application of PPADS did not increase the depression of the d.p.s.p. by guanethidine. Superfusion with the mixed alpha-adrenoceptor agonist noradrenaline or the selective P2 receptor agonist adenosine 5'-O-(2-thiodiphosphate) inhibited both the d.p.s.p. and the i.p.s.p. The inhibitory effects of these agonists were prevented by the respective antagonists idazoxan or suramin. In the presence of suramin noradrenaline failed to inhibit the residual d.p.s.p. Superfused noradrenaline potentiated rather than inhibited responses to pressure-applied alpha,beta-methylene-ATP; superfused adenosine 5'-O-(2-thiodiphosphate) did not interact with pressure-applied noradrenaline. In conclusion, we present electrophysiological evidence for the co-release of ATP and catecholamines in the CNS. At the cell somata of neurons in the locus coeruleus, noradrenaline and ATP activate inhibitory alpha(2)-adrenoceptors and excitatory P2 receptors, respectively. In addition, inhibitory presynaptic autoreceptors of the alpha(2) and P2 types appear to regulate release of the two co-transmitters.

Kynurenine 3-mono-oxygenase, one of the key enzymes of the "kynurenine pathway", catalyses the formation of 3-hydroxykynurenine and may direct the neo-synthesis of quinolinic and kynurenic acids. While 3-hydroxykynurenine and quinolinic acid have neurotoxic properties, kynurenic acid antagonizes excitotoxic neuronal death. Here we report that the expression and activity of kynurenine 3-mono-oxygenase significantly increased in the spinal cord of rats with experimental allergic encephalopathy, an experimental model of multiple sclerosis. As a consequence of this increase, the spinal cord content of 3-hydroxykynurenine and quinolinic acid reached neurotoxic levels. We also report that systemic administration of Ro 61-8048, a selective kynurenine 3-mono-oxygenase inhibitor, reduced the increase of both 3-hydroxykynurenine and quinolinic acid, and caused accumulation of kynurenic acid. In the brain and spinal cord of the controls, kynurenine 3-mono-oxygenase immunoreactivity was located in granules (probably mitochondria) present in the cytoplasm of both neurons and astroglial cells. In the spinal cord of rats with experimental allergic encephalopathy, however, cells with a very intense kynurenine 3-mono-oxygenase immunoreactivity, also able to express class II major histocompatibility complex and inducible nitric oxide synthase, were found in perivascular, subependymal and subpial locations. These cells (most probably macrophages) were responsible for the large increase in 3-hydroxykynurenine and quinolinic acid found in the spinal cords of affected animals. The results show that cells of the immune system are responsible for the increased formation of 3-hydroxykynurenine and quinolinic acid, two neurotoxic metabolites that accumulate in the central nervous system of rats with experimental allergic encephalomyelitis. They also demonstrate that selective kynurenine 3-mono-oxygenase inhibitors reduce the neo-synthesis of these toxins.

Resting and evoked extracellular dopamine levels in the striatum of the anesthetized rat were measured by fast-scan cyclic voltammetry in conjunction with carbon fiber microelectrodes. Identification of the substance detected in vivo was achieved by inspection of background-subtracted voltammograms. Intrastriatal microinfusion of kynurenate, a broad-spectrum antagonist of ionotropic glutamate receptors, caused a decrease in the resting extracellular level of dopamine. The kynurenate-induced decrease was unaffected by systemic pretreatment with pargyline, an inhibitor of monoamine oxidase, but was significantly attenuated by systemic pretreatment with α-methyl- p -tyrosine, an inhibitor of tyrosine hydroxylase. Although glutamate by itself did not affect resting extracellular dopamine levels, glutamate did attenuate the kynurenate-induced decrease. Kynurenate decreased dopamine release in response to electrical stimulation of the medial forebrain bundle, an effect that was also attenuated by glutamate. These results suggest that both spontaneous and evoked dopamine release in the rat striatum are under the local tonic excitatory influence of glutamate. Interactions between central dopamine and glutamate systems that have been implicated in the etiologies of Parkinson's disease, schizophrenia, stress, and substance abuse. The precise nature of those interactions, however, remains a matter of some controversy.

Glutathione (γ-glutamylcysteinylglycine) is a neuromodulator at glutamate receptors, but may also act as a neurotransmitter at sites of its own. The Na + -independent binding of [ 3 H]glutathione to pig cortical synaptic membranes was characterized here using glycine, cysteine analogs, dipeptides and glutathione derivatives, and ligands selective for known glutamate receptors. l -Glutamate, pyroglutamate, quinolinate, ( S )-5-fluorowillardiine and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione were weak inhibitors at concentrations of 0.5 or 1 mM. d -Glutamate, l - and d -aspartate, glutamine, quisqualate, kynurenate, other N -methyl- d -aspartate receptor ligands and non- N -methyl- d -aspartate receptor ligands failed to displace [ 3 H]glutathione. Except for weak inhibition by d -serine (0.5 mM), glycine and other ligands of the glycine co-activatory site in the N -methyl- d -aspartate receptors had no displacing effect. Similarly, metabotropic glutamate group I, II and III receptor agonists and antagonists and compounds acting at the glutamate uptake sites were generally inactive. Glutathione, oxidized glutathione, S -nitrosoglutathione, γ- l -glutamylcysteine, cysteinylglycine, cysteine, cysteamine and cystamine were the most potent displacers ( ic 50 values in the micromolar range), followed by dithiothreitol, glutathione sulfonate and the S -alkyl derivatives of glutathione ( S -methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione). l -Homocysteinate and aminomethanesulfonate exhibited a moderate efficacy. Thiokynurenate, a cysteine analog and an antagonist at the N -methyl- d -aspartate receptor glycine co-activatory site, was a potent activator of glutathione binding. At 1 mM, some dipeptides also slightly activated the binding, γ- l -glutamylleucine and γ- l -glutamyl-GABA being the most effective. The specific binding sites for glutathione in brain synaptic membranes are not identical to any known excitatory amino acid receptor. The cysteinyl moiety is crucial in the binding of glutathione. The oxidation or alkylation of the cysteine thiol group reduces the binding affinity. The strong activation by thiokynurenate may indicate that the glutathione receptor protein contains a modulatory site to which co-agonists may bind and allosterically activate glutathione binding. The novel population of specific binding sites of glutathione gives rise to the possibility that they may have profound effects on synaptic functions in the mammalian central nervous system. The glutathione binding sites may be an important, and for the most part unrecognized, component in signal transduction in the brain.

Excitatory amino acids are known to activate the spinal neural network that organize locomotor activity in various species. In this study, the role of various compounds which alter the functioning of the N -methyl- d -aspartate receptor (glycine, Mg 2+ ions and spermine) was investigated during fictive locomotion, using an in vitro isolated spinal cord preparation from neonatal rats. Locomotor-like activity induced by excitatory amino acids was recorded both extra- and intracellularly. 7-chlorokynurenic acid, an antagonist of the glycine site at the N -methyl- d -aspartate receptor, depressed the N -methyl- d -aspartate component of the synaptic inputs received by the motoneurons. Glycine at low concentrations had no effect on locomotor activity, while 7-chlorokynurenic acid increased the locomotor period and decreased the burst amplitude in a dose-dependent manner. Removal of Mg 2+ ions from the saline facilitated the N -methyl- d -aspartate-mediated response, and triggered spontaneous bursting activity, abolished by 2-amino-5-phosphonovaleric acid, an antagonist of the N -methyl- d -aspartate receptor. The polyamine, spermine, did not change the locomotor parameters. On the contrary, arcaine, a putative antagonist of the polyamine site on the N -methyl- d -aspartate receptor, increased locomotor activity. The effects of arcaine were counteracted by spermine. These results suggest that glycine and spermine are present at saturating concentrations on the N -methyl- d -aspartate receptor during ongoing locomotion. Together with Mg 2+ ions, these endogenous regulators contribute to control the level of activity of the N -methyl- d -aspartate receptor in the spinal cord of the neonatal rat.

The effect of transitory blockage of substantia nigra pars compacta glutamatergic inputs by intracranial injections of kynurenic acid were evaluated in two monkey treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The MPTP protocol was designed to mimic the gradual evolution of human Parkinson's disease. No effects were observed before MPTP treatment or in the first stage of treatment. Once clinical signs appeared, however, motor abnormalities were significantly aggravated by blockage of these inputs (P < 0.001). Conversely, after full Parkinsonism was established, blockage no longer had any behavioural effect. These results confirm the postulated compensatory role of the glutamatergic pathways feeding the substantia nigra pars compacta. This added insight into the physiopathology of the basal ganglia, when compared with previous data on the presymptomatic revelation of experimental Parkinsonism, should help elucidation of the time pattern of evolution of Parkinson's disease.

Neuroinflammatory disturbances have been closely associated with depression and many other neuropsychiatric diseases. Although targeting neuroinflammatory mediators with centrally acting drugs has shown certain promise, its translation is faced with several challenges especially drug delivery and safety concerns. Here, we report that neuroinflammation-induced behavioral abnormality could be effectively attenuated with immunomodulatory agents that need not to gain brain penetration. In a rat model with intracerebral lipopolysaccharide (LPS) challenge, we validated that ginsenoside Rg1 (Rg1), a well-established anti-inflammatory agent, was unable to produce a direct action in the brain. Interestingly, peripherally restricted Rg1 could effectively attenuate the weight loss, anorexic- and depressive-like behavior as well as neurochemical disturbances associated with central LPS challenge. Biochemical assay of neuroimmune mediators in the periphery revealed that Rg1 could mitigate the deregulation of the hypothalamic-pituitary-adrenal axis and selectively blunt the increase in circulating interleukin-6 levels. Furthermore, these peripheral regulatory effects were accompanied by dampened microglial activation, mitigated expression of pro-inflammatory mediators and neurotoxic species in the central compartment. Taken together, our work suggested that targeting the peripheral immune system may serve as a novel therapeutic approach to neuroinflammation-induced neuropsychiatric disorders. Moreover, our findings provided the rationale for employing peripherally active agents like Rg1 to combat mental disturbances.

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.

The mechanisms by which activation of excitatory amino acid receptors is coupled to the regulation of gene transcription were studied using cultured hippocampal neurons from neonatal rats. Voltage recording, calcium imaging, specific RNA analysis and immunocytochemistry were carried out on sister cultures. This allowed analysis of the expression of functional glutamate receptor subtypes, examination of their role in controlling intracellular free calcium ([Ca2+]i), and determination of their relative contributions to the transcriptional regulation of six immediate early genes c-fos, fosB, c-jun, junB, zif/268 (also termed Egr-1; NGFI-A; Krox-24) and nur/77 (also termed NGFI-B). Expression of all six immediate early genes was induced in hippocampal neurons by glutamate treatment. Nuclear run-on assays demonstrated that this induction occurred at the transcriptional level. Activation of the N-methyl-D-aspartate subtype of glutamate receptor was necessary and sufficient for the transcriptional response. Non-N-methyl-D-aspartate receptors, while present in cultured hippocampal neurons, contributed relatively little to the regulation of transcription. Calcium imaging showed that glutamate-induced changes in [Ca2+]i were almost entirely mediated by N-methyl-D-aspartate receptors, rather than by L-type voltage-sensitive calcium channels. Previous studies have shown that stimulation with selective agonists of either N-methyl-D-aspartate receptors, non-N-methyl-D-aspartate receptors, or L-type calcium channels can lead to an increase in [Ca2+]i and c-fos expression. Here we demonstrate that in our hippocampal culture system glutamate controls [Ca2+]i and induces immediate early gene transcription primarily by activating N-methyl-D-aspartate receptors.

Hypoxia evokes a regulated decrease in the body core temperature, which response is mediated, at least in part, by noradrenaline (NA) and nitric oxide (NO) in the rostromedial preoptic area (POA) of the hypothalamus. In the accompanying paper, it was shown that glutamatergic activation of the lateral POA also evokes hypothermic responses. Here, I tested the hypothesis that the glutamatergic transmission in the lateral POA is critically involved in the neural mechanism of hypoxia-induced hypothermia. Hypoxic ventilation (10% O(2)-90% N(2), 5 min) as well as a single microinjection of NA (50 pmol) or the NO donor sodium nitroprusside (8.4 nmol) into the rostromedial POA evoked an increase in the tail skin temperature and a decrease in the colonic temperature in urethane-chloralose-anesthetized, neuromuscularly blocked, artificially ventilated rats. All of these responses were greatly attenuated by pretreatment with multiple microinjections of kynurenic acid (10 nmol, four locations), a nonselective glutamate receptor antagonist, but not by those with saline solution, in the bilateral rostral and central parts of the lateral POA. These results suggest that the NA- and NO-sensitive structure in the rostromedial POA activated the glutamatergic transmission in the lateral POA to mediate hypoxia-induced hypothermia.

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 (GD15) 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.

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.

Kynurenate is an endogenous antagonist of the ionotropic glutamate receptors. It is synthesized from kynurenine, a tryptophan metabolite, and a significant increase in its brain concentration could be useful in pathological situations. We attempted to increase its neosynthesis by modifying kynurenine catabolism. Several kynurenine analogues were synthesized and tested as inhibitors of kynurenine hydroxylase (E.C.1.14.13.9) and of kynureninase (E.C.3.7.1.3), the two enzymes which catalyse the conversion of kynurenine to excitotoxin quinolinate. Among these analogues we observed that nicotinylalanine, a compound whose pharmacological properties have previously been reported, had an IC50 of 900 +/- 180 microM as inhibitor of kynurenine hydroxylase and of 800 +/- 120 microM as inhibitor of kynureninase. In the search for more potent molecules we noticed that meta-nitrobenzoylalanine had an IC50 of 0.9 +/- 0.1 microM as inhibitor of kynurenine hydroxylase and of 100 +/- 12 microM as inhibitor of kynureninase. When administered to rats meta-nitrobenzoylalanine (400 mg/kg) significantly increased the concentration of kynurenine (up to 10 times) and kynurenate (up to five times) in the brain. Similar results were obtained in the blood and in the liver. Furthermore meta-nitrobenzoylalanine increased in a dose dependent, long lasting (up to 13 times and up to 4 h) manner the concentration of kynurenate in the hippocampal extracellular fluid, as evaluated with a microdialysis technique. This increase was associated with a decrease in the locomotor activity and with protection from maximal electroshock-induced seizures in rats or from audiogenic seizures in DBA/2 mice. The conclusions drawn from the present study are: (i) meta-nitrobenzoylalanine is a potent inhibitor of kynurenine hydroxylase also affecting kynureninase; (ii) the inhibition of these enzymes causes a significant increase in the brain extracellular concentration of kynurenate; (iii) this increase is associated with sedative and anticonvulsant actions, suggesting a functional antagonism of the excitatory amino acid receptors.

Acetylcholine released from the cerebral cortex was collected using microdialysis while stimulating the region of the pedunculopontine tegmentum in urethane-anesthetized rats. Electrical stimulation in the form of short trains of pulses delivered once per minute produced a 350% increase in acetylcholine release and a desynchronization of the electroencephalogram, as measured by relative power in the 20-45 Hz range (low-voltage fast activity). Perfusion of the region of cholinergic neurons believed to be responsible for the cortical release of acetylcholine, the nucleus basalis magnocellularis, was carried out using a second microdialysis probe. Exposure of the nucleus basalis magnocellularis to blockers of neural activity (tetrodotoxin or procaine) or to blockers of synaptic transmission (calcium-free solution plus magnesium or cobalt) produced a substantial decrease in the release of acetylcholine and desynchronization evoked by brainstem stimulation. Exposure of the nucleus basalis magnocellularis to the glutamate antagonist, kynurenate, resulted in a decrease in evoked acetylcholine release and electroencephalogram desynchronization similar in magnitude to that produced by nonspecific blockers, whereas application of muscarinic or nicotinic cholinergic blockers to the nucleus basalis magnocellularis did not reduce acetylcholine release or electroencephalogram desynchronization. Application of tetrodotoxin to the collection site in the cortex abolished the stimulation-evoked acetylcholine release, but not the low baseline release indicating that cholinergic nucleus basalis magnocellularis neurons have a low spontaneous firing rate in urethane-anesthetized animals. The results of this study suggest that the major excitatory input to the cholinergic neurons of the nucleus basalis magnocellularis from the pedunculopontine tegmentum is via glutamatergic and not cholinergic synapses.

The present experiments examined the role of excitatory amino acid receptors in the orofacial stereotypy induced by direct amphetamine microinjection into the ventrolateral striatum. In these experiments, the influence of prior intra-ventrolateral striatum treatment with various excitatory amino acid antagonists on the expression of amphetamine-stimulated oral stereotypy was observed. In all experiments, behavioral observations were conducted in the home cage using a time-sampling procedure. In the first experiment, different groups of rats received bilateral microinfusions of either kynurenic acid, 2-amino-5-phosphonopentanoic acid, 6,7-dinitroquinoxaline or dizocilpine maleate. The excitatory amino acid antagonists were administered immediately prior to bilateral microinfusions of d-amphetamine. Both N-methyl-D-aspartate and non-N-methyl-D-aspartate antagonists dose-dependently attenuated or blocked the expression of dopamine-mediated stereotypy. 2-Amino-5-phosphonopentanoic acid was the most potent of these compounds, totally suppressing stereotypy at a dose of 0.3 micrograms (equivalent to 1.5 nmol). In the second experiment, the same compounds were tested for their ability to suppress physostigmine-induced mouth movements. Cholinergic stimulation of the ventrolateral striatum has previously been shown to elicit non-directed mouth movements, quite distinguishable from stimulus-directed, amphetamine-induced biting. Excitatory amino acid antagonists were administered in the same doses prior to bilateral infusion of physostigmine (2.5 micrograms/0.5 microliters). The expression of physostigmine-induced mouth movements was for the most part not affected by excitatory amino acid antagonists, although dizocilpine maleate slightly reduced this oral behavior. In a third experiment, behavior was observed following infusion of the antagonists alone, using the same doses as in the previous experiments. No behavioral alterations were observed with the exception of a small increase in nonspecific mouth movements induced by kynurenic acid and 2-amino-5-phosphonopentanoic acid. These findings indicate that the expression of dopamine-mediated oral stereotypy, induced by amphetamine stimulation of the ventrolateral striatal region, is highly dependent on activation of striatal excitatory amino acid receptors. In contrast, oral behavior induced by cholinergic stimulation of the ventrolateral region is not mediated by glutamate input. These results are discussed in relation to the synaptic organization of neuronal elements within the striatum. Moreover, the relevance to further understanding of orofacial dyskinesias is noted.

The accessory olfactory system constitutes a sensory system specifically involved in regulating neuroendocrine function and reproductive behavior. The chemosensitive structure of this system, the vomeronasal organ, exclusively innervates the accessory olfactory bulb, which in turn projects via monoor disynaptic pathways to a limited number of regions implicated in endocrine and sexual function, including the amygdala and hypothalamus. The present study investigated synaptic processing between the different levels of this sensory system, with particular focus on the input from the accessory olfactory bulb to the medial amygdala and the reciprocal connections between the medial amygdala and the ventromedial hypothalamus. Extracellular single-unit recordings were obtained from medial amygdala neurons in anesthetized female rats to study the synaptic responses elicited by stimulation of the olfactory bulb and hypothalamus. Locally applied agonists and antagonists of amino acid neurotransmitters were tested for their ability to mimic and block these synaptic responses in an attempt to identify the neurotransmitters involved in the stimulated pathways. Electrical stimulation of the accessory olfactory bulb induced orthodromic responses in 88% of 288 medial amygdala neurons, with 67% showing orthodromic inhibition and 21% showing excitation as the shortest-latency response. Many cells displayed multiphasic responses with both orthodromic excitation and inhibition. In separate experiments, main olfactory bulb stimulation also produced excitatory (39%) or inhibitory (50%) orthodromic responses in medial amygdala neurons (n = 105), but the latency and distribution of responses were significantly different than with accessory olfactory bulb stimulation. Stimulation of the ventromedial hypothalamus also had prominent excitatory (29%) or inhibitory (59%) orthodromic actions on the majority of amygdala neurons. Out of 288 cells tested with both accessory olfactory bulb and ventromedial hypothalamus stimulation, 79% were orthodromically responsive to both areas, whereas only 3% responded to neither area. Fourteen (6%) medial amygdala neurons were antidromically activated from the ventromedial hypothalamus, and 11 of these 14 cells were simultaneously orthodromically responsive to accessory olfactory bulb stimulation, indicating that the accessory olfactory bulb can influence medial amygdala neurons that project directly to the hypothalamus. Iontophoretic application of drugs to the medial amygdala revealed that glutamate and GABA consistently mimicked the orthodromic excitatory and inhibitory responses, respectively, from both the accessory olfactory bulb and ventromedial hypothalamus. In some cases, ejection of bicuculline could block or reverse excitation of the orthodromic inhibitory responses, whereas kynurenic acid could eliminate the orthodromic excitation, indicating that excitatory and inhibitory amino acid transmitters are at least partially involved in these pathways. These findings form a framework for determining the synaptic mechanisms underlying information processing in the accessory olfactory system and olfactory modulation of reproductive function.

Recent anatomical studies in this laboratory have demonstrated that the proisocortex cortex adjacent and dorsal to the rhinal sulcus is one of the major forebrain afferent inputs to the midbrain periaqueductal gray matter in the rat. The physiological influence(s) of this projection has not been examined. The present studies investigated the responses of periaqueductal gray neurons to chemical and electrical stimulation of proisocortex in chloral hydrate-anesthetized rats. In addition, the role of glutamate as a possible transmitter in excitatory proisocortex-periaqueductal gray synaptic responses was tested. Microinjection of D,L-homocysteate into proisocortex excited 44% (19/43), inhibited 37% (16/43) and had no effect on 19% of periaqueductal gray cells. The onset of D,L-homocystic acid-evoked responses ranged from 2 to 60 s; the duration of responses ranged from 1 to 18 min. Low-frequency, single-pulse electrical stimulation of proisocortex robustly altered neuronal discharge in 25% of periaqueductal gray neurons sampled; 10% (74/724) of neurons were excited and 15% (107/724) were inhibited. Insular cortex-evoked excitatory responses had a mean onset latency of 19.5 +/- 4.2 ms and a mean duration of 38.5 +/- 26.9 ms. Inhibitory responses had a mean onset latency of 26.2 +/- 15.6 ms and mean duration of 108.0 +/- 84.9 ms. Trains of high-frequency electrical stimulation of proisocortex excited 22% (13/59) and inhibited 25% (15/59) of periaqueductal gray cells tested. In separate experiments, stimulation electrodes were placed in periaqueductal gray to antidromically activate proisocortex neurons that project to periaqueductal gray.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to determine the neurotransmitter(s) involved in activation of noradrenergic locus coeruleus neurons by urinary bladder distention. The spontaneous discharge rate of single locus coeruleus neurons was recorded from halothane-anesthetized rats during the physiological challenge of bladder distention. Intrabladder saline infusion (0.5 ml) increased bladder pressure by 77 ± 9.7mmHg (n = 19) and this was associated with an increase in locus coeruleus discharge rate of 53 ± 4.8% (n = 29). Simultaneous recordings of cortical electroencephalographic activity demonstrated that electroencephalographic activation, characterized by a decreased amplitude and tendency to shift from low frequency activity to higher frequency activity, was also associated with bladder distention. The role of corticotropin-releasing factor and excitatory amino acid inputs to the locus coeruleus in activation by bladder distention was tested in rats pretreated with a corticotropin-releasing factor antagonist, or excitatory amino acid antagonists. Intracerebroventricular administration of the corticotropin-releasing factor antagonist did not alter locus coeruleus activation by bladder distention. In contrast, both locus coeruleus activation and electroencephalographic activation associated with bladder distention were prevented by intracerebroventricular administration of kynurenic acid. The same dose of kynurenic acid also prevented locus coeruleus activation by repeated sciatic nerve stimulation, as previously reported. Local administration of kynurenic acid into the locus coeruleus greatly attenuated, but did not completely prevent the increase in locus coeruleus discharge elicited by bladder distention. Finally, locus coeruleus activation by bladder distention appeared to be mediated by excitatory amino acid receptors of the non-N-methyl-d-aspartate subtype because this was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione, but not by 2-amino-5-phosphono-pentanoic acid. Taken together, these results implicate excitatory amino acid neurotransmission in locus coeruleus activation by bladder distention, and argue against a role for corticotropin-releasing factor. Additionally, these findings suggest that locus coeruleus activation may be important in arousal associated with bladder distention.

This study bears on the control exerted by neurotransmitters on the expression of a 40-Hz pacemaker activity observed in reticular thalamic cells. Experiments were conducted in urethane-anaesthetized rats using extracellular recordings and local applications of antagonists against the neurotransmitters involved in the modulation of reticular thalamic cells. All drugs were dissolved in a Ringer's solution (pH 7.4) and were applied in small quantities (25–150 nl) by pressure through one barrel of a micropipette assembly. Forty-Hertz firing was abolished by local application of theα 1 antagonist prazosin and by bilateral lesion of the locus coeruleus. Local applications of glutamate antagonists reduced the rate of discharges by 30–50% as did cortical cooling or complete transection of the internal capsule. Conversely, scopolamine exerted a permissive action on the expression of 40-Hz activities; many spontaneously bursting units started firing at 40 Hz under the influence of this muscarinic antagonist. Since reticular thalamic cells are GABAergic and synaptically coupled via axonal collaterals, we investigated how GABAergic drugs affected the regular firing of these cells. Local applications of bicuculline produced a transient increase of the firing rates while the application of GABA induced intermittent pauses on a background of regular discharges. The application of piperidine-4-sulphonic acid, a GABA A receptor agonist, produced a similar effect. The length of pauses generated by piperidine was statistically analysed. It was found that the duration of short pauses was a multiple integer of the mean interspike interval of surrounding discharges. The preservation of the period and phase of the rhythm across the pauses implies that a subthreshold oscillation was presented into the cells during the arrests of discharges. Given the mode of action of noradrenaline and acetylcholine on reticular thalamic neurons, and considering a possible metabotropic action of glutamate, the above results suggest that deactivation of a leaky K conductance is critically involved in the regular firing of these cells in urethane-anaesthetized rats. Alternatively, because reticular cells are coupled via inhibitory synapses, it is proposed that the 40-Hz firing frequency reflects, in the frequency domain, a point of equilibrium in the reticular thalamic network when the leaky K conductance is fully deactivated by the metabotropic effects of monoamines and/or excitatory amino acids.

We attempted to elucidate the cerebrovasomotor function of the caudal ventrolateral medulla. Sixty-one rats were anaesthetized, paralysed and artificially ventilated. The microsphere method was employed for the measurement of blood flow. Microinjection of an antagonist of excitatory amino acids, kynurenate (2 nmol), into functionally identified depressor sites within the caudal ventrolateral medulla produced arterial hypertension of about 140 mmHg. We found that the cerebral blood flow was substantially increased, but was maintained at the same level (17 rats) as that observed under phenylephrine-induced hypertension (26 rats). Bilateral severing of the cervical sympathetic trunks resulted in a further increase in blood flow in all brain regions studied (18 rats). The response was most significant in the cerebral parasagittal cortex (164 ± 31% of baseline without, and 211 ± 43% with sympathectomy; mean ± S.D.; P < 0.001). The contributions of the cerebral metabolic mechanism to this flow increase under denervation was minimal, as evidenced by the observation of disproportionately smaller changes in cerebral metabolic rate for oxygen during any type of hypertension. We conclude that the cerebrovasomotor functions of the caudal ventrolateral medulla may operate to keep an equilibrium between simultaneously working tonic inhibitions against sympathetic vasoconstriction as well as against vasodilatation. This dual effect is mediated by excitatory amino acid receptors located within this particular brain area. The vasodilator mechanism may be of neurogenic origin. When the function of the brain area is suppressed, the subsequently disinhibited vasodilator mechanism dominates the cerebrovascular autoregulatory function. The dilatation is, however, masked by the sympathetically mediated constriction, and an adequate level of cerebral blood flow is thus maintained in the face of concomitantly occurring hypertension.

Following the localization, at the electron microscope level, of the immunoreactivity towards a putative kainate receptor on Bergmann glial cells in the chick cerebellar cortex, cultures of Bergmann glia were used to establish the presence of functional kainate receptor/channels and study their properties. Bergmann glia were identified by their fusiform morphology and their ability to bind an anti-kainate binding protein monoclonal antibody, a kainate receptor high affinity ligand--kainyl-bovine serum albumin--and a glial marker--anti-vimentin monoclonal antibody. Membranes prepared from the culture cells displayed, using 25 nM [3H]kainate, the binding of 4.1 pmol of [3H]kainate/mg protein and showed the presence in Western blots of the two polypeptides of 49 and 93 kDa attributed to the kainate binding protein. Kainate, at concentrations above 0.1 mM, was found to increase the influx into cultured Bergmann glia of 22Na+, 86Rb+, 45Ca2+ and 36Cl- ions. The traffic of 22Na+, induced by kainate and glutamate, observed only in the presence of 1 mM ouabain, was blocked by kainate receptor antagonists and by 0.01 mM quisqualate. Analysis of the kinetics of incorporation of 22Na+ and 45Ca2+ ions showed an initial accumulation of 22Na+ and 45Ca2+ ions followed by their total dissipation. The results indicate that the kainate-induced influx of Na+ ions through the kainate receptor/channel causes the reverse transport of Na+ ions, by activation of the Na+/Ca2+ and Na+/H+ exchangers which remove intracellular Na+ ions. Pre-exposure of the cells to 0.5 mM dibutyryl cAMP was found to greatly enhance the kainate-induced 22Na+ ion influx. We propose that the Bergmann glia kainate receptors modulate the efficacy of the glutamatergic synapses between the parallel fibers and Purkinje cell spines and form part of a glial machinery responsible for plastic changes in synaptic transmission.

Conditioned fear to context in the rat leads to a host of sympathetically mediated physiological changes, including a marked rise in mean arterial pressure, a delayed rise in heart rate and a marked cutaneous vasoconstriction, along with the behavioral responses of freezing and ultrasonic vocalization. In this study we examine the role of the rostral ventromedial medulla (RVM), which includes raphe nuclei pallidus and magnus, in the expression of these changes. RVM is a major premotor sympathetic and somatic center and an important integrating center in the descending emotional motor system. To evaluate its role, conditioned fear was tested after temporary blockade with microinjections (0.4 microl) of the GABA-A receptor agonist muscimol (0.2 mM) or the glutamate receptor antagonist kynurenic acid (0.1 M). Changes in mean arterial pressure, heart rate and activity were recorded by radio-telemetry. Cutaneous vasoconstriction in the tail was recorded indirectly by infrared thermography. Muscimol and kynurenic acid had different, almost complementary effects. Muscimol abolished the skin vasoconstrictor response and significantly reduced the tachycardic response, but did not reduce the pressor response significantly and had little effect on the somatic motor components, freezing and ultrasonic vocalization. In contrast, kynurenic acid abolished ultrasonic vocalization and significantly reduced freezing but had no effect on the cardiovascular components. The results show that neurons in the rostral ventromedial medulla are implicated in the expression of some of the cardiac, vascular and somatic motor components of conditioned fear. Most importantly, these cardiovascular components are not under local glutamatergic control whereas the somatic motor components are.

The cannabinoid CB1 receptor which is densely located in the basal ganglia is known to participate in the regulation of movement. The present study sought to determine the mechanisms underlying the effect of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) on neurons in the substantia nigra pars compacta (SNpc) using single-unit extracellular recordings in anesthetized rats. Administration of Delta(9)-THC (0.25-2 mg/kg, i.v.) increased the firing rate of SNpc neurons (maximal effect: 33.54+/-6.90%, n=8) without modifying other firing parameters (coefficient of variation and burst firing). This effect was completely blocked by the cannabinoid receptor antagonist rimonabant (0.5 mg/kg, i.v.). In addition, the blockade of excitatory amino acids receptors by kynurenic acid (0.5 microM, i.c.v.) or a chemical lesion of the subthalamic nucleus (STN) with ibotenic acid abolished Delta(9)-THC effect. These results indicate that CB1 receptor activation modulates SNpc neuronal activity by an indirect mechanism involving excitatory amino acids, probably released from STN axon terminals in the SNpc.

The medial prefrontal cortex (mPFC) modulates neurovegetative and behavioral responses, being involved in memory, attention, motivational and executive processes. There is evidence indicating that mPFC modulates cardiovascular correlates of behavioral responses to stressful stimuli. Acute restraint is an unavoidable stress situation that evokes marked and sustained cardiovascular changes, characterized by elevated blood pressure (BP) and intense heart rate (HR) increase. We presently report effects of mPFC pharmacological manipulations on BP and HR responses evoked by acute restraint in rats. Bilateral microinjection of 200 nl of the unspecific synaptic blocker CoCl2 (1 mM) in the mPFC prelimbic area (PL) increased HR response to acute restraint, without significant effect on the BP response. This result indicates that PL synaptic mechanisms have an inhibitory influence on restraint-evoked HR changes. Injections of the non-selective glutamatergic receptor antagonist kynurenic acid (0.02 M) or the selective N-methyl-d-aspartic acid (NMDA) receptor glutamatergic antagonist (LY235959) (0.02 M) caused effects similar to cobalt, suggesting that local glutamatergic neurotransmission and NMDA receptors mediate the PL inhibitory influence on restraint-related HR responses. Pretreatment with the non-non-N-methyl-D-aspartic acid glutamatergic antagonist glutamatergic antagonist glutamatergic receptor antagonist NBQX (0.02 M) did not affect restraint-related cardiovascular responses, reinforcing the idea that NMDA receptors mediate PL-related inhibitory influence. Pretreatment with the glutamatergic-receptor antagonists did not affect baseline BP or HR values. I.v. pretreatment with the quaternary ammonium anticholinergic drug homatropine methyl bromide (0.2 mg/kg) also increased the restraint-related HR response to values similar to those observed after treatment with kynurenic acid or LY235959, thus, suggesting that PL inhibitory influence on restraint-evoked heart rate increase could be related to increased parasympathetic activity. This dose of homatropine had no significant effects on baseline BP or HR values. Results suggest a PL inhibitory influence on restraint-evoked HR increase. They also indicate that local NMDA receptors involved in parasympathetic activation mediate PL inhibitory influence on restraint-evoked HR increase.

Neurons in the region of dorsomedial hypothalamus are involved in the organization of the physiological responses to emotional stress. We have recently shown that the cardiovascular response evoked by activation of dorsomedial hypothalamus neurons is largely dependent on a synaptic relay with the lateral/dorsolateral periaqueductal gray region. In this study, we aimed to investigate whether excitatory amino acid receptors at the lateral/dorsolateral periaqueductal gray region are involved in mediating the response evoked by activation of dorsomedial hypothalamus neurons. In conscious rats, the cardiovascular effects produced by microinjection of GABAA receptor antagonist, bicuculline methiodide into the dorsomedial hypothalamus were evaluated before and after injection of different excitatory amino acid antagonists into lateral/dorsolateral periaqueductal gray region. Pretreatment of lateral/dorsolateral periaqueductal gray region with the non-selective ionotropic excitatory amino acid receptor antagonist kynurenic acid or with the N-methyl- d -aspartate receptor-selective antagonist, MK-801, largely reduced the tachycardic and pressor effects evoked by activation of dorsomedial hypothalamus neurons by bicuculline methiodide microinjection (heart rate 90 and 74%; blood pressure 81 and 84%, respectively). The non-N-methyl- d -aspartate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione, did not alter the cardiovascular response evoked by dorsomedial hypothalamus activation. In an additional series of experiments, microinjection of the N-methyl- d -aspartate receptor agonist, N-methyl- d -aspartate, into the lateral/dorsolateral periaqueductal gray region, evoked an increase in heart rate and a pressor response that was accompanied by an increase in locomotor activity. These effects were not altered by pretreatment of lateral/dorsolateral periaqueductal gray region neurons with 6-cyano-7-nitroquinoxaline-2,3-dione but were completely abolished by MK-801. Altogether, these findings indicate that the cardiovascular response evoked by dorsomedial hypothalamus activation involves a synaptic relay at the lateral/dorsolateral periaqueductal gray region that is mediated at least in large part by excitatory amino acid receptors, possibly N-methyl- d -aspartate receptors.

Excitotoxic mechanisms involving alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)/kainate receptors play an important role in mediating cellular damage in spinal cord injury. However, the precise cellular mechanisms of glutamate release from non-synaptic white matter are not well understood. We examined how the collapse of transmembrane Na(+) and K(+) gradients induces reverse operation of Na(+)-dependent glutamate transporters, leading to glutamate efflux and injury to rat spinal dorsal columns in vitro. Compound action potentials were irreversibly reduced to 43% of control after ouabain/high K(+)/low Na(+) exposure (500 microM ouabain for 30 min to increase [Na(+)](i), followed by 1 h ouabain+high K(+) (129 mM)/low Na(+) (27 mM), to further reverse transmembrane ion gradients) followed by a 2 h wash. Ca(2+)-free perfusate was very protective (compound action potential amplitude recovered to 87% vs. 43%). The broad spectrum glutamate antagonist kynurenic acid (1 mM) or the selective AMPA antagonist GYKI52466 (30 microM) were partially protective (68% recovery). Inhibition of Na(+)-dependent glutamate transport with L-trans-pyrrolidine-2,4-dicarboxylic acid (1 mM) also provided significant protection (71% recovery), similar to that seen with glutamate receptor antagonists. Blocking reverse Na(+)-Ca(2+) exchange with KB-R7943 (10 microM) however, was ineffective in this paradigm (49% recovery). Semiquantitative glutamate immunohistochemistry revealed that levels of this amino acid were significantly depleted in axon cylinders and, to a lesser degree, in oligodendrocytes (but not in astrocytes) by ouabain/high K(+)/low Na(+), which was largely prevented by glutamate transport inhibition. Our data show that dorsal column white matter contains the necessary glutamate pools and release mechanisms to induce significant injury. When Na(+) and K(+) gradients are disrupted, even in the absence of reduced cellular energy reserves, reverse operation of Na(+)-dependent glutamate transport will release enough endogenous glutamate to activate AMPA receptors and cause substantial Ca(2+)-dependent injury. This mechanism likely plays an important role during ischemic and traumatic white matter injury, where collapse of transmembrane Na(+) and K(+) gradients occurs.