Antagonist Kynurenic Acid Research Articles

Osmotic modulation in glutamatergic excitatory synaptic inputs to neurons in the supraoptic nucleus of rat hypothalamus in vitro.

To clarify influence of osmotic stimulation on the excitatory synaptic inputs to the neurosecretory cells of the supraoptic nucleus (SON), the blind patch technique was used in rat hypothalamic slice preparations. Stable whole-cell recordings were made from 22 neurons in the SON. To observe spontaneous excitatory postsynaptic currents (sEPSCs) in the SON neurons, membrane potentials were clamped between -50 and -90mV. The effects of hypertonic stimulation on the frequency of the sEPSCs were tested in 18 SON neurons. Bath application of mannitol 30 or 60 mM increased the frequency of the sEPSCs. During the application of mannitol (60 mM), the frequency of the sEPSCs increased in 12 of 15 neurons without a change in amplitude. Hypertonic stimulation with NaCl (30 mM) had similar effects to that of mannitol. The increased frequency of miniature EPSCs (mEPSCs) during mannitol application persisted in the presence of TTX in all 8 SON neurons tested with no change in amplitude. Both the non-NMDA antagonist CNQX at 10-30 microM (n = 6) and the non-selective glutamate antagonist kynurenic acid at 1 mM (n = 3) almost completely blocked the EPSCs while the NMDA antagonist AP-5 at 10 microM had no effect on the frequency of the EPSCs in the 4 neurons tested. During application of CNQX, mannitol (60 mM) was added to the perfusion medium in 3 SON neurons. Under these conditions, mannitol had no effect on the frequency of EPSCs. We conclude that hypertonic stimulation directly influences glutamatergic inputs to the neurosecretory cells of the SON by an action on the presynaptic terminals and enhances the excitatory synaptic events.

Central integration of the Bezold-Jarish reflex in the cat

The medullary structures involved in the central Bezold-Jarisch reflex pathway were studied by recording unit activity of sympatho-excitatory (SE) or inhibitory (SI) cardiovascular neurons in pentobarbital-anesthetized cats. The neurons were selected based upon their spontaneous activity and upon their sensitivity to baroreceptor reflex activation by l-phenylephrine. The Bezold-Jarisch reflex was induced by i.v. injection of chlorophenylbiguanide 10 µg/kg which produced a short-lasting decrease in blood pressure, heart rate and renal nerve activity. 76 neurons were studied. In 10 out of 12 SE neurons of the rostral ventrolateral medulla the activity was inhibited by chlorophenylbiguanide whereas in 10 out of 11 SI neurons and 6 out of 6 baroreflex-insensitive cells of the caudal ventrolateral medulla it was activated. The others cells were insensitive. Three types of neurons: excitatory, inhibitory or non-barosensitive, were recorded in the lateral tegmental field (27 cells) and the medullary raphe (20 cells). These neurons were either activated, inhibited or insensitive to Bezold-Jarisch reflex activation. Microinjection of the glutamate receptor antagonist kynurenic acid (2.5 nmol/site) or the GABAergic agonist muscimol (1 nmol/site) into the nucleus tractus solitarii abolished the effects of both l-phenylephrine and chlorophenyl-biguanide on heart rate and renal nerve activity. These results indicate that the cardiovascular neurons (sympatho-excitatory and sympatho-inhibitory) located in the medullary areas, involved in cardiovascular and baroreflex mechanisms, are implicated in the central Bezold-Jarisch reflex pathway.

Contribution of the Dorsal Nucleus of the Lateral Lemniscus to Binaural Responses in the Inferior Colliculus of the Rat: Interaural Time Delays

The contribution of the dorsal nucleus of the lateral lemniscus (DNLL) to binaural responses in the inferior colliculus of the rat was determined for a wide range of interaural time differences (ITDs). Single-unit action potentials were recorded from the inferior colliculus before and after local injection of the excitatory amino acid antagonist kynurenic acid into the DNLL. Binaural properties were determined by manipulating the time difference between paired clicks delivered to the ears ipsilateral and contralateral to the recording site. The probability of an action potential decreased as contralateral stimulation was delayed, relative to ipsilateral stimulation. These data generated a sigmoidal ITD curve for delays between -1.0 and + 1.0 msec. By extending the time intervals beyond 1 msec, it was possible to determine the trailing edge of the inhibition produced by ipsilateral stimulation. The duration of the inhibitory effect varied from cell to cell but lasted as long as 20 msec in some cases. Injection of kynurenic acid into the DNLL contralateral to the recording site reduced the extent of both short (0-1 msec) and long-lasting (1-20 msec) inhibition in the inferior colliculus. No effect was seen after injections ipsilateral to the recording site. The data demonstrate that the DNLL plays an important role in shaping ITD responses in the inferior colliculus and contributes to both the short and long-lasting inhibition produced by stimulation of the ipsilateral ear.

Non-NMDA receptors transmit cardiopulmonary C fibre input in nucleus tractus solitarii in rats.

1. We sought first to determine whether neurones in caudomedial aspects of commissural nucleus tractus solitarii (NTS) received input from cardiopulmonary C fibre endings supplied by the pulmonary versus systemic circulation. We then examined the role of N-methyl-D-aspartate (NMDA) and non-NMDA receptors in transmitting cardiopulmonary C fibre input to such NTS neurones. 2. Extracellular NTS unit activity, phrenic nerve activity and arterial blood pressure were recorded in urethane-anaesthetized rats. Unit responses to right atrial and left ventricular phenylbiguanide injections were compared in rats with arterial baroreceptors, carotid chemo-receptors and subdiaphragmatic vagal inputs eliminated. Right atrial phenylbiguanide injections produced greater peak responses (27 +/- 11 impulses s-1) than did left ventricular injections (11 +/- 3 impulses s-1) (n = 9). 3. The non-NMDA receptor agonist quisqualic acid (QUIS) and NMDA were ionophoresed onto NTS neurones that were synaptically activated by right atrial phenylbiguanide injection. Responses were compared before and during ionophoresis of the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulphamoylbenzo(f)quinoxaline (NBQX), the NMDA receptor antagonist DL-2-amino-5-phosphovaleric acid (AP5), or the broad spectrum antagonist kynurenic acid (KYN). 4. NBQX, which blocked QUIS-but spared NMDA-evoked responses, significantly attenuated synaptic activation by 65% (n = 9). AP5, which blocked NMDA- but spared QUIS-evoked responses, did not significantly diminish synaptic activation (11%; n = 7). KYN, which blocked QUIS- and NMDA-evoked responses, decreased synaptic activation by 70% (n = 9). 5. The results suggest that input from cardiopulmonary C fibre endings, primarily supplied by the pulmonary circulation, is transmitted to this commissural NTS region largely via non-NMDA receptors.

Glutamate receptor agonists decrease extracellular dopamine in the rat nucleus accumbens in vivo.

Intracerebral microdialysis was used to investigate the effects of local application of L-glutamate, N-methyl-D-aspartate, and the glutamate uptake inhibitor 1-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) in the nucleus accumbens (NAc) on extracellular dopamine (DA) concentrations. The effects of locally applied PDC on extracellular glutamate concentrations were also examined. Glutamate produced a concentration-dependent decrease in extracellular DA that could be blocked by concurrent, local application of the broad spectrum ionotropic glutamate receptor antagonist kynurenic acid (KYN:1 mM). N-Methyl-D-aspartate had a concentration-dependent effect on DA release, with a low concentration (0.1 mM) producing a decrease and a higher concentration (1.0 mM) resulting in an increase. Both effects were blocked by KYN. PDC (1 mM) increased extracellular glutamate concentrations to 102% above baseline. The same concentration of PDC decreased extracellular DA concentrations, and coapplication of KYN attenuated this effect. These results indicate that glutamate receptor agonists can have both facilitatory and inhibitory effects on extracellular DA concentrations. However, the effects of PDC indicate that inhibition of DA release is the more physiologically relevant effect. Furthermore, the results of these and other experiments suggest that glutamate's inhibitory effects on DA release in the NAc are not due to direct actions of this excitatory amino acid on DA terminals. A multisynaptic model that accounts for glutamate's actions on DA release is proposed.

Hippocampal AstrocytesIn SituRespond to Glutamate Released from Synaptic Terminals

A long-standing question in neurobiology is whether astrocytes respond to the neuronal release of neurotransmitters in vivo. To address this question, acutely isolated hippocampal slices were loaded with the calcium-sensitive dye Calcium Green-1 and the responses of the astrocytes to electrical stimulation of the Schaffer collaterals were monitored by confocal microscopy. To confirm that the responsive cells were astrocytes, the slices were immunostained for the astrocytic marker glial fibrillary acidic protein. Stimulation of the Schaffer collaterals (50 Hz, 2 sec) resulted in increases in the concentration of intracellular calcium ([Ca2+]i) in the astrocytes located in the stratum radiatum of CA1. The astrocytic responses were blocked by the sodium channel blocker tetrodotoxin, the voltage-dependent calcium channel blocker omega-conotoxin-MVIIC, and the selective metabotropic glutamate receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG). These results suggest that the astrocytic responses were induced by stimulation of metabotropic glutamate receptors on the astrocytes by neuronally released glutamate. The astrocytic responses to neuronal stimulation were enhanced in the presence of the K+ channel antagonist 4-aminopyridine (4-AP). Inhibition of the astrocytic responses in the presence of 4-AP required the presence of both MCPG and the ionotropic glutamate receptor antagonist kynurenic acid. These results suggest that higher levels of neuronal activity result in stimulation of both metabotropic and ionotropic glutamate receptors on the astrocytes. Overall, the results indicate that hippocampal astrocytes in situ are able to respond to the neuronal release of the neurotransmitter glutamate with increases in [Ca2+]i.

In vitro brain slice studies of the rat's dorsal nucleus of the lateral lemniscus. III. synaptic pharmacology

1. The synaptic pharmacology of the dorsal nucleus of the lateral lemniscus (DNLL) of the rat was investigated in a brain slice preparation of the auditory midbrain. The brain slice was cut in the coronal plane and placed in a small recording chamber where warm, oxygenated saline was continuously perfused over and underneath the tissue. Intracellular recordings were made with glass microelectrodes filled with 4 M potassium acetate. Synaptic potentials were elicited by electrical stimulation of the lateral lemniscus or commissure of Probst and pharmacological effects were tested by bath application of amino acid agonists and antagonists. 2. The cells in DNLL were challenged with the excitatory amino acid (EAA) agonists, alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), N-methyl-D-aspartic acid (NMDA) in 0 Mg2+, and L-glutamate. Each of these caused a depolarization of the cell membrane, a reduction in cell membrane resistance, and the onset of spontaneous firing. 3. Short-latency excitatory postsynaptic potentials (EPSPs) were evoked by stimulation of the lateral lemniscus in 77% of the neurons tested. The mean latency to initial depolarization was 0.9 ms. A single spike with relatively constant latency (mean 1.5 ms) was typically elicited when the strength of lemniscal stimulation was increased. A longer-latency EPSP (mean 2.9 ms) was seen in 34% of the neurons tested either with the slice in normal saline or after pharmacological block of the earlier, short-latency EPSP. The long-latency EPSP was followed by a single spike of multiple spikes with highly variable latencies (range 3.2-24 ms). In 28% of the neurons tested, both early and late EPSPs were observed in response to stimulation of a single location on the lateral lemniscus. 4. Stimulation of the commissure of Probst elicited short-latency EPSPs (mean 0.9 ms) in 37% of the neurons tested. Longer-latency EPSPs (mean 3.0 ms) were found in only 3% of the neurons in response to commissural stimulation. 5. The nonspecific EAA antagonist kynurenic acid blocked both short-and long-latency EPSPs evoked by either lemniscal or commissural stimulation. The non-NMDA antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), at very low concentrations, blocked the short-latency EPSPs but had no effect on the longer-latency EPSPs. The short-latency EPSPs were unaffected by the NMDA antagonist D,L-2-amino-5-phosphonovaleric acid (APV). In contrast, the longer-latency EPSPs were blocked by APV, but never by CNQX. 6. DNLL neurons were affected by the inhibitory amino acid agonists gamma-aminobutyric acid (GABA) and glycine. The membrane resistance of the neurons was decreased by GABA and glycine in a solution of either normal or calcium-free saline in a concentration-dependent manner. 7. Inhibitory postsynaptic potentials (IPSPs) were elicited by stimulation of the lateral lemniscus in 53% of the neurons and the commissure of the Probst in 18% of the neurons tested. The mean latencies were 1.0 and 0.9 ms, respectively. The reversal potentials of the IPSPs were around -70 mV. 8. The IPSPs evoked by stimulation of the lateral lemniscus were blocked by the glycine receptor antagonist strychnine, but not by the GABA receptor antagonist bicuculline, whereas the IPSPs elicited by stimulation of the commissure of Probst were blocked by bicuculline but not strychnine.

807 Cloning of rat S182 cDNA

Caffeine (100 mg/kg, i.p.) induces a rapid increase in the expression of mRNA for the immediate early genes (IEGs) c-fos and NGFI-A in rat striatum. We have examined how this response is affected by pretreatment with either the noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist MK-801 (1 and 3 mg/kg, i.p.), the competitive NMDA receptor antagonist D-CPP (6 mg/kg, i.p.), or the non-selective excitatory amino acid receptor antagonist kynurenic acid (300 mg/kg, i.p.). The two NMDA receptor antagonists significantly reduced the caffeine-induced expression of both c-fos mRNA and NGFI-A mRNA in the medial part of the caudate putamen. The effect was less pronounced in the lateral part of the caudate putamen. MK-801 caused an enancement of c-fos and NGFI-A mRNA expression in nucleus accumbens. Pretreatment with kynurenic acid caused no marked alterations in the caffeine-induced expression of c-fos mRNA and NGFI-A mRNA in any brain region. These findings suggest that glutamatergic transmission via NMDA receptors contributes to the induction of c-fos mRNA and NGFI-A mRNA by caffeine in striatum. In addition we show that MK-801 can either increase or decrease the caffeine effect of IEGs depending on the region studied.

Cyclic AMP-dependent modulation of giant depolarizing potentials by metabotropic glutamate receptors in the rat hippocampus.

1. Intracellular recordings were used to study the role of metabotropic glutamate receptors (mGluRs) in modulating GABA-mediated giant depolarizing potentials (GDPs) in immature rat hippocampal CA3 neurones. 2. The mGluR antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) reduced the frequency of GDPs. The broad-spectrum ionotropic glutamate receptor antagonist kynurenic acid (1 mM) blocked GDPs. 3. In the presence of kynurenic acid, both tetanic stimulation of the hilus or bath application of quisqualic acid (1 microM) and trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD, 20 microM) induced the appearance of GDPs. These effects were antagonized by MCPG (1 mM) or L(+)-2-amino-3-phosphonopropionic acid (L-AP3) and blocked by bicuculline (10 microM). 4. 8-Bromo-cAMP (8-Br-cAMP, 0.3 mM), 3-isobutyl-1-methylxanthine (IBMX, 200 microM) or forskolin (30 microM) mimicked the effects of mGluR agonists on GDPs. The forskolin analogue 1,9-dideoxyforskolin (30 microM), which does not activate adenylate cyclase, was ineffective. 5. Incubation of slices in the presence of the protein kinase A inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS) (500 microM) or superfusion of Rp-cAMPS (20 microM) prevented the effects of forskolin or t-ACPD on GDPs. In the presence of kynurenic acid, the protein kinase C activator, phorbol 12,13-diacetate (2 microM) induced the appearance of GDPs. This effect was prevented by staurosporine (1 microM). However, staurosporine (1-3 microM) did not modify the effects of t-ACPD on GDPs. 6. It is suggested that, during development, mGluRs enhance the synchronous release of GABA, responsible for GDPs, through cAMP-dependent protein kinase.

Systemic administration of kainate induces marked increases of endogenous kynurenic acid in various brain regions and plasma of rats

The endogenous neuroinhibitory and neuroprotective excitatory amino acid receptor antagonist kynurenic acid has been hypothetically linked to the pathogenesis of epilepsy and several other brain disorders. In the present study, alterations in kynurenic acid levels were examined in the kainate model of temporal lobe epilepsy. Kainate was systemically injected in rats at a dose (10 mg/kg s.c.) which induces a characteristic behavioural syndrome with stereotypies and focal (limbic) and generalized seizures, eventually progressing into severe status epilepticus. Kynurenic acid was determined 3 h after kainate injection in various brain regions (olfactory bulb, frontal cortex, piriform cortex, amygdala, hippocampus, nucleus accumbens, caudate/putamen, thalamus, superior and inferior colliculus, pons and medulla, and cerebellar cortex) and in plasma, using a sensitive high-performance liquid chromatographic method. When data were analysed irrespective of individual seizure severity, significant increases in kynurenic acid were determined in all brain regions examined except the hippocampus, nucleus accumbens and pons/medulla. The most marked (200–500%) increases above controls were seen in the piriform cortex, amygdala, and cerebellar cortex. Furthermore, a significant kynurenic acid increase of about 200% above control was determined in plasma. When kynurenic acid levels were determined in subgroups of rats with different behavioural alterations in response to kainate, the most marked kynurenic acid increases were seen in subgroups with status epilepticus. Rats which only developed mild (focal) seizures or stereotyped behaviours (wet dog shakes) also exhibited significantly increased kynurenic acid levels, thus indicating that the increase in kynurenic acid in response to kainate was not solely due to sustained convulsive seizure activity. Whereas it was previously proposed that kynurenic acid is involved only in later stages of seizure disorders, the present data demonstrate that marked increases in central and peripheral kynurenic acid levels occur early after the onset of neuroexcitation, at least in the kainate model.

The stimulatory effect of clonidine through imidazoline receptors on locus coeruleus noradrenergic neurones is mediated by excitatory amino acids and modulated by serotonin.

Clonidine and other imidazoline/oxazoline drugs, such as cirazoline and rilmenidine, have been shown to stimulate the activity of noradrenergic neurones in the locus coeruleus (NA-LC) by an alpha 2-adrenoceptor-independent mechanism through the activation of I-imidazoline receptors. The endogenous modulation of the stimulatory effect of clonidine on NA-LC neurones was further investigated after inactivation of alpha 2-adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). In EEDQ-pretreated rats (6 mg/kg, i.p., 6 h), clonidine caused a rapid and dose-dependent (320-5120 micrograms/kg, i.v.) increase in the firing rate of NA-LC neurones (ED50 = 809 micrograms/kg, Emax = 90%). The stimulatory effect of clonidine on NA-LC neurones was completely blocked by pretreatment of rats with the excitatory amino acid receptor antagonist kynurenic acid (1-3 mumol in 10-30 microliters, i.c.v., 2-5 min before clonidine). In contrast, the stimulatory effect of clonidine on NA-LC neurones was potentiated by pretreatment with reserpine (5 mg/kg, s.c., 18 h) (Emax increased by 63%). Pretreatment with alpha-methyl-p-tyrosine (250 mg/kg, i.p., 24 h) did not alter the stimulatory effect of clonidine, but pretreatment with p-chloro-phenylalanine (400 mg/kg, i.p., 24 h) markedly enhanced the stimulatory effect of clonidine on NA-LC neurones (Emax increased by 139%). The present results indicate that the imidazoline receptor-mediated stimulatory effect of clonidine on NA-LC neurones is an indirect effect dependent on an excitatory amino acid pathway and modulated by an inhibitory serotonin mechanism.

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

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

Dysfunction of brain kynurenic acid metabolism in Huntington's disease: focus on kynurenine aminotransferases

The levels of the neuroprotective excitatory amino acid receptor antagonist kynurenic acid (KYNA) have been previously shown to be reduced in several regions of the brain of Huntington's disease (HD) patients. Thus, KYNA has been speculatively linked to the pathogenesis of HD. We have examined KYNA levels and the activity of its two biosynthetic enzymes (kynurenine aminotransferases (KAT) I and II) in 12 regions of brains from late-stage HD patients and control donors ( n = 17 each). KYNA levels were measured in the original tissue homogenate. Using [ 3H]kynurenine as the substrate, enzyme activities were determined in dialyzed tissue homogenates. KYNA levels in the caudate nucleus decreased from 733 ± 95 in controls to 401 ± 62 fmol/mg tissue in HD ( p < 0.01). The activity of both enzymes was highest in cortical areas (e.g. control frontal cortex: KAT I: 148 ± 18 fmol/mg tissue/h; KAT II: 25 ± 2 fmol/mg tissue/h). The activities of both KAT I and KAT II, when expressed per mg original weight, showed significant decreases (48–55%) in the HD putamen ( p < 0.01). Trends toward lower enzyme activities and KYNA concentrations were detected in other brain areas as well. Kinetic analyses, performed in putamen and cerebellum, showed an approximately 3-fold increase in K m values for both KAT I and KAT II in the putamen only. V max values remained unchanged in the HD brain. These findings indicate a selective impairment in KYNA biosynthesis in the neostriatum of HD patients, possibly due to the loss of (an) endogenous KAT activators). Future studies should examine whether the demonstrated dysfunction in the production of the endogenous neuroprotecive agent KYNA is causally related to the neurodegenerative process in HD.

Interactions of the subthalamic nucleus and the subpallidal area in oro-facial dyskinesia: role of GABA and glutamate.

Previous studies have shown that lowering the GABAergic activity in the sub-pallidal area (SP) in the cat results in the display of oro-facial dyskinesia (OFD). There exists an intense, mutual anatomical connection between the SP and the subthalamic nucleus and the adjoining lateral hypothalamic area (STH). The present study investigated whether the STH is also involved in OFD. Once this turned out to be true (see below), it was investigated whether the SP-specific OFD is funneled via the STH, or vice versa. Bilateral injections of low doses (50-250 ng) of picrotoxin, a non-competitive GABA antagonist, into the STH were found to elicit OFD. This effect which was quantified in terms of numbers of tongue protrusions, was dose-dependent: a bell-shaped dose-response was found (50-500 ng). The OFD elicited by the most effective dose of picrotoxin (250 ng) was significantly antagonized by muscimol, a specific GABAA agonist, in a dose (50 ng) which itself was ineffective, indicating GABA specificity. In addition, it was found that OFD elicited by local injections of picrotoxin (250 ng) into the STH was significantly attenuated by SP injections of the broad spectrum glutamate antagonist kynurenic acid in a dose (1000 ng) which itself was ineffective, but not by muscimol (100 ng), indicating that the STH-elicited OFD needs an intact and functioning glutaminergic, but not GABAergic, transmission process in the SP for its expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical stimulation of the prefrontal cortex increases dopamine release in the nucleus accumbens of the rat: modulation by metabotropic glutamate receptors

In vivo microdialysis was used to assess the effects of electrical stimulation of the prefrontal cortex (PFC) on dopamine (DA) release in the nucleus accumbens (NAC) of awake, unrestrained rats. The PFC was stimulated bilaterally for 20 min at parameters previously shown to support intracranial self-stimulation in this structure. Stimulation at 50 microA evoked a 38% increase in DA release while 100 microA produced a 69% increase. Thus, phasic activation of the PFC increases DA release in the NAC. Additional experiments were performed to establish whether glutamate receptors in the NAC mediated these effects. The noncompetitive NMDA antagonist dizocilpine maleate (MK-801) and the broad spectrum competitive antagonist kynurenic acid were each applied locally to the NAC via reverse dialysis alone or in combination with electrical stimulation of the PFC (100 microA). Both MK-801 (10 microM) and kynurenic acid (5 mM) increased DA release when administered alone. When a "subthreshold" concentration (i.e., the highest concentration employed that did not itself increase DA release) of either compound was administered together with PFC stimulation, neither kynurenic acid (1 mM) nor MK-801 (1 microM) attenuated the effect of stimulation on DA release, thereby indicating that this effect is not mediated by ionotropic glutamate receptors located within the NAC. To examine the possible role of metabotropic glutamate receptors in regulating DA release, the metabotropic glutamate agonist trans(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) was employed. When applied locally to the NAC, ACPD had a dose-dependent effect on DA release with a high concentration (1 mM) causing an increase and a lower concentration (100 microM) causing a small decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitatory amino acid antagonists and pentylenetetrazol-induced seizures during ontogenesis: III. The action of kynurenic acid and glutamic acid diethylester

N-methyl- d-aspartate (NMDA) receptor antagonists are anticonvulsant drugs with specific activity against tonic-clonic pentylenetetrazol-induced seizures. However, they do not affect clonic seizures with preserved righting reflexes. In these experiments, we tested the anticonvulsant activity of strychnine-insensitive glycine receptor (at the NMDA site) antagonist kynurenic acid and nonspecific excitatory amino acid receptor antagonist glutamic acid diethylester (GDEE) in the pentylenetetrazol-induced seizure model in developing rats 7, 12, 18, 25, and 90 days old. Control rats were injected with pentylenetetrazol (100 mg/kg subcutaneously). Other rats were pretreated either with kynurenic acid (40, 80, or 240 mg/kg IP) or with GDEE (0.48–480 mg/kg IP), followed by pentylenetetrazol (100 mg/kg). In very young rats (7 and 12 days), both kynurenic acid and GDEE increased the incidence of clonic seizures, whereas the occurrence of tonic-clonic seizures was suppressed or delayed compared to controls. This effect is very similar to the anticonvulsant action of the competitive and non-competitive NMDA receptor antagonists. In adult rats, the pretreatment with rather higher doses of kynurenic acid or GDEE suppressed or delayed clonic seizures as well as tonic-clonic seizures. Both drugs also induced behavioral side effects: repetitive orientation, wet dog shakes, and frequent jumping. Our data show that there are only weak and nonconsistent age-specific anticonvulsant effects resulting from the blockade of strychnine-insensitive glycine receptor often associated with serious side effects, thus decreasing chances to develop effective antiepileptic treatment in this drug class.

Protein synthesis and energy metabolism in hippocampal slices during extended (24 hours) recovery following different periods of ischemia

Hippocampal slices were successfully maintained for 24 hours in vitro in a flow-through chamber by using a modified artificial CSF (amino acids included). Measurement of energy metabolism parameters (adenine nucleotides) and the slice response to KCl-induced depolarization (release of GABA and aspartate) indicated that hippocampal slices were metabolically stable for at least 24 hours. The preparation was used to study recovery of protein synthesis after different periods of in vitro ischemia (5, 10, or 15 min). Protein synthesis inhibition was only partly reversed after 15 min of ischemia, but fully reversible after 5- or 10-min ischemia at 24 hours of recovery. Furthermore, the model was used to study a possible role of glutamate in postischemic inhibition of protein synthesis. Glutamate receptor agonists (glutamate or quinolinic acid) or antagonist (kynurenic acid) were applied during ischemia. Neither treatment affected the late (24 hours) outcome of ischemia, arguing against the critical role of glutamate in ischemic cell damage. The present approach allows use of the hippocampal slice preparation in the study of delayed effects of ischemia of different duration.

A role for non-NMDA excitatory amino acid receptors in regulating the basal activity of rat globus pallidus neurons and their activation by the subthalamic nucleus

We have investigated the hypothesis that excitatory amino acid (EAA) receptors in the globus pallidus (GP) play a significant role in maintaining the firing rates of GP neurons under basal conditions and following activation of the subthalamic nucleus (STN). Drugs were infused directly into the GP and/or STN while the extracellular single unit activity of Type II GP neurons was recorded in ketamine-anesthetized rats. Local infusions of the EAA agonists NMDA (30–300 pmol/200 nl) or AMPA (0.1–1 pmol/200 nl) elicited increases in the firing rate of GP neurons in a dose-dependent fashion. Infusion of the GABA A receptor antagonist bicuculline methiodine (1–10 pmol/100 nl) into the STN also elicited dose-related increases in the firing rate of GP neurons. Intrapallidal infusion of the non-NMDA (AMPA/kainate) receptor antagonist NBQX (0.1–1.0 nmol) reduced the basal firing rate of GP neurons by 40%. In contrast, the NMDA antagonist MK-801 (0.01–0.1 nmol) produced no significant effect on basal firing rate. Intrapallidal infusion of the non-selective EAA receptor antagonist kynurenic acid or NBQX reversed or blocked the increase in firing rate of GP neurons following bicuculline-induced activation of the STN. Similar treatment with MK-801, however, had no significant effect on this response. These results indicate that tonic stimulation of non-NMDA receptors plays an important role in maintaining the basal activity of GP neurons and in mediating the effects of increased excitatory input from subthalamic afferent neurons.

Nitric oxide and adenosine mediate vasodilation during functional activation in cerebellar cortex

Activation of the cerebellar parallel fibers (PF) releases glutamate and leads to depolarization of Purkinje cells and interneurons. These cells, in turn, release GABA. We have studied the role of glutamate, GABA, nitric oxide (NO) and adenosine in the increases in cerebellar cortex blood flow (BF crb) elicited by PF stimulation. In anesthetized rats (halothane 1%) the cerebellar vermis was exposed and the site was superfused with Ringer (37°C, pH 7.4). The PF were stimulated electrically (50–100 μA; 30 Hz) and the increases in BF crb were recorded using a laser-Doppler flowmeter. Field potentials were recorded using glass microelectrodes. During Ringer superfusion, PF stimulation increased BF crb by 58 ± 5% ( P < 0.001; analysis of variance; n = 6). Superfusion with the broad spectrum glutamate receptor antagonist kynurenic acid (Kyn; 5 mM) abolished the negative component of the field potential ( n = 4), a finding reflecting lack of depolarization of Purkinje cells and interneurons, and blocked the increase in BF crb ( P > 0.05 from Ringer; n = 6). In contrast, Kyn did not influence the increase in BF crb evoked by hypercapnia (pCO 2 55.4 ± 1.1 mmHg) or by superfusion with the NO donor SIN-1 (0.1, 1 mM; P > 0.05; n = 6). Superfusion with the adenosine receptor antagonist 8-sulphophenyltheophylline (8-SPT; 100 μM) reduced the elevation in BF crb by 45 ± 4% ( P < 0.05; n = 6) and co-application of 8-SPT and of the NO synthase inhibitor nitro- l-arginine ( l-NA; 1 mM) attenuated the vasodilation further (−82 ± 4% from Ringer; P < 0.01 from 8-SPT alone). The vasodilation elicited by SIN-1 ( n = 6) and the field potentials evoked by PF stimulation ( n = 4) were not affected by 8-SPT or by 8-SPT plus l-NA. The GABA receptor antagonist bicuculline (300 μM) produced the expected changes in the field potentials ( n = 4) but did not affect the vascular response to PF stimulation ( P . 0.05; n = 5). The data suggest that release of glutamate from PF leads to production of NO and adenosine in Purkinje cells and/or interneurons. NO and adenosine may mediate the vasodilation by diffusing toward resistance arterioles and producing smooth muscle relaxation.

Somato-sympathetic reflex transmission in the ventrolateral medulla oblongata: spatial organization and receptor types

Tonic sympathetic activity in vivo is continuously modulated by inhibitory and excitatory reflex mechanisms. We studied the properties of somato-sympathetic excitatory reflex transmission in the rostral ventrolateral medulla (RVLM) of baroreceptor-denervated and vagotomized chloralose-anesthetized cats. Electrical stimulation of the left intercostal nerve of the 4th thoracic segment (IC-T4) elicited an early spinal and a late supraspinal reflex in the ipsilateral white ramus T3 from which recordings were made. Bilateral cooling of the ventral surface of the RVLM reversibly reduced the supraspinal reflex amplitude to 18.0 ± 3.1% of control (100%). The spinally evoked reflex was enhanced to maximally 154.7 ± 5.3%. Cooling of only the ipsilateral side of the RVLM was nearly equieffective in both, suppressing the supraspinal and enhancing the spinal reflex component. In contrast, cooling of the contralateral side had no significant effects on supraspinal reflex transmission but caused slight increases of the spinal reflex amplitudes. Similar effects were obtained by microinjection (RVLM) of the glutamate antagonist kynurenic acid (5 × 10 −3 M, n = 7) and the specific non-NMDA receptor antagonist CNQX (4 × 10 −3 M, n = 4) which, however, blocked the supraspinal reflex less effectively. These results demonstrate that the RVLM represents an essential relay in the transmission of both somatosympathetic reflex components. The experiments further suggest an almost completely ipsilateral neuronal pathway for the supraspinal reflex component which projects from the RVLM to the intermediolateral cell column (IML). The descending inhibition of the spinal reflex, however, receives neuronal inputs from the contralateral side.