Brown adipose tissue sympathetic nerve activity is potentiated by activation of 5-hydroxytryptamine (5-HT)1A/5-HT7 receptors in the rat spinal cord

In urethane-chloralose anesthetized, neuromuscularly blocked, ventilated rats, microinjection of NMDA (12 pmol) into the right fourth thoracic segment (T4) spinal intermediolateral nucleus (IML) immediately increased ipsilateral brown adipose tissue (BAT) sympathetic nerve activity (SNA; peak +492% of control), expired CO2 (+0.1%) heart rate (+48 beats min(-1)) and arterial pressure (+8 mmHg). The increase in BAT SNA evoked by T4 IML microinjection of NMDA was potentiated when it was administered immediately following a T4 IML microinjection of 5-hydroxytryptamine (5-HT, 100 pmol) or the 5-HT1A/5-HT7 receptor agonist, 8-OH-DPAT (600 pmol), (area under the curve: 184%, and 259% of the NMDA-only response, respectively). In contrast, T4 IML microinjection of the 5-HT2 receptor agonist, DOI (28 pmol) did not potentiate the NMDA-evoked increase in BAT SNA (101% of NMDA-only response). Microinjection into the T4 IML of the selective 5-HT1A antagonist, WAY-100635 (500 pmol), plus the 5-HT7 antagonist, SB-269970 (500 pmol), prevented the 5-HT-induced potentiation of the NMDA-evoked increase in BAT SNA. When administered separately, WAY-100635 (800 pmol) and SB-269970 (800 pmol) attenuated the 8-OH-DPAT-induced potentiation of the NMDA-evoked increase in BAT SNA through effects on the amplitude and duration of the response, respectively. The selective 5-HT2 receptor antagonist, ketanserin (100 pmol), did not attenuate the potentiations of the NMDA-evoked increase in BAT SNA induced by either 5-HT or 8-OH-DPAT. These results demonstrate that activation of 5-HT1A/5-HT7 receptors can act synergistically with NMDA receptor activation within the IML to markedly increase BAT SNA.     

Effects of arginine-vasopressin on neuronal interaction from the area postrema to the nucleus tractus solitarii in rat brain slices

The effects of vasopressin (AVP) on area postrema (AP) neurons and the neuronal connection between the AP and nucleus tractus solitarii (NTS) were investigated electrophysiologically in slices preparation of the medulla oblongata of rats. In the AP, 27.9% of 129 neurons were excited by AVP and 20.5% were inhibited. The excitation was blocked by a V1 receptor antagonist. Synaptic transmission of the AP to the NTS was mainly mediated by non-NMDA receptors. Local application of AVP to the AP activated the NTS neurons. This activation was blocked by an NMDA antagonist. These results suggest that the excitation originating in the AP is conveyed to the NTS via non-NMDA receptors and modified by NMDA receptor activation secondly. These processes may be important in regulation of the arterial baroreceptor reflex.     

Differential regulation of brown adipose and splanchnic sympathetic outflows in rat: roles of raphe and rostral ventrolateral medulla neurons

1. The medullary premotor neurons determining the sympathetic outflow regulating cardiac function and vasoconstriction are located in the rostral ventrolateral medulla (RVLM). The present study sought evidence for an alternative location for the sympathetic premotor neurons determining the sympathetic nerve activity (SNA) controlling brown adipose tissue (BAT) metabolism and thermogenesis. 2. The tonic discharge on sympathetic nerves is determined by the inputs to functionally specific sympathetic preganglionic neurons from supraspinal populations of premotor neurons. Under normothermic conditions, BAT SNA was nearly silent, while splanchnic (SPL) SNA, controlling mesenteric vasoconstriction, exhibited sustained large-amplitude bursts. 3. The rostral raphe pallidus (RPa) contains potential sympathetic premotor neurons that project to the region of sympathetic preganglionic neurons in the thoracic spinal cord. Disinhibition of neurons in RPa elicited a dramatic increase in BAT SNA, with only a small rise in SPL SNA. 4. Splanchnic SNA was strongly influenced by the baroreceptor reflex, as indicated by a high coherence with the arterial pressure wave, a significant amplitude modulation over the time-course of the cardiac cycle and a marked inhibition of SPL SNA during a sustained increase in arterial pressure. When activated, the bursts in BAT SNA exhibited no correlation with arterial pressure and were not affected by increases in arterial pressure. 5. Because these characteristics and reflex responses in sympathetic outflow have been shown to arise from the on-going or altered discharge of sympathetic premotor neurons, the marked differences between SPL and BAT SNA provide strong evidence supporting the hypothesis that vasoconstriction and thermogenesis (metabolism) are controlled by distinct populations of sympathetic premotor neurons, the former in the RVLM and the latter, potentially, in the RPa.     

Endogenous activation of NMDA and non-NMDA glutamate receptors on respiratory neurones in cat medulla.

The aim of this study was to evaluate the involvement of dicarboxylic amino acid neurotransmission in the periodic discharges of respiratory neurones. Respiratory neurones of the ventral and dorsal respiratory groups in the medulla of the cat were subjected to iontophoretic applications of (1) N-methyl-D-aspartate (NMDA) and a blocker of the NMDA subtype of glutamate receptor, D-2-amino-7-phosphonoheptanoic acid (AP7) and (2) an agonist and an antagonist of the non-NMDA subtypes of receptor: quisqualate and 6,7-dinitroquinoxaline-2,3-dione (DNQX), respectively. All five main types of respiratory neurones (all-, early- and late-inspiratory, transitional "off-switch", late expiratory) were excited by NMDA and quisqualate. Both agonists increased the peak firing rate but exerted different effects on the discharge pattern of respiratory neurones, within the respiratory cycle. Quisqualate induced discharges in the "silent" period of the neurone more readily than did NMDA which, in turn had a more pronounced effect during the burst period of the neurone. The effects of quisqualate and NMDA were suppressed by prior application of their selective antagonists, AP7 and DNQX. These antagonists decreased the spontaneous neuronal discharge of all cell types, throughout the entire firing phase, by a maximum of 24-63% with AP7 and by 30-50% with DNQX. The non-selective antagonist, gamma-D-glutamyl-glycine and the selective NMDA antagonists, CPP and MK-801, were also effective. It is concluded that respiratory neurones, of all types, within the medullary respiratory network are subjected to endogenous glutamate-like excitations, which may possibly shape the respiratory train of action potentials through the sequential activation of non-NMDA and NMDA subtypes of receptor.     

Participation of NMDA and kainate receptors of paraventricular nucleus in cardiovascular responses to glutamate receptor agonist

The nuclei of the hypothalamus have been shown to be involved in central cardiovascular homeostasis. Recent studies suggest that glutamate-containing neurons have an important role in the regulation of central cardiovascular function. We report first on the effects of intracerebrally injected NMDA and non-NMDA receptor ligands on blood pressure and heart rate in conscious Sprague-Dawley rats. In the second part, we describe the effect of blockade of NMDA or kainate receptors in the paraventricular nucleus on glutamate receptor agonist-induced blood pressure responses. Intracerebroventricular injections of L-glutamic acid, NMDA and kainic acid produced increases in mean arterial pressure. Kainic acid produced significant decreases in heart rate. Microinjection of DL-2-amino-5-phosphonopentanoic acid (APV; 25 and 50 nmol), a competitive NMDA receptor antagonist, into the paraventricular nucleus blunted the increases in the mean arterial pressure evoked by intracerebroventricular injections of NMDA (1 nmol), whereas microinjection of dinitroquinoxaline (DNQX; 20, 40 and 80 pmol), which acts as an antagonist at kainate receptors, failed to antagonize the cardiovascular effects of intracerebroventricular kainic acid (10 pmol). Microinjections of NMDA (100 pmol) into the paraventricular nucleus produced pressor responses, but kainic acid (5 and 10 pmol) failed to affect either mean arterial pressure or heart rate. These results suggest participation of the glutamergic system in cardiovascular regulation via NMDA receptors located within the paraventricular nucleus of the hypothalamus in rats.     

Stimulation of noradrenaline release in human cerebral cortex mediated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors.

1. Human brain cortical slices from patients undergoing neurosurgery for treatment of epilepsy resistant to antiepileptic drugs were used to identify and characterize N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediating stimulation of noradrenaline release. The slices preincubated with [3H]-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg2+ (1.2 mmol l-1) and were stimulated by 2-min exposure to NMDA, kainic acid or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). 2. In slices superfused without Mg2+, NMDA induced a concentration-dependent tritium overflow. 3. The NMDA-evoked tritium overflow was almost abolished by tetrodotoxin (TTX), Mg2+ or by omission of Ca2+ from the superfusion fluid. 2-Amino-5-phosphonopentanoic acid (AP5; a competitive NMDA receptor antagonist) or dizocilpine (formerly MK-801; an antagonist at the phencyclidine receptor within the NMDA-gated ion channel) inhibited the NMDA-evoked tritium overflow. The stimulatory effect of NMDA was not significantly enhanced by glycine added to the superfusion fluid but was reduced by 7-chlorokynurenic acid (an antagonist at the glycine site coupled to the NMDA receptor). 4. In slices superfused with solution containing Mg2+, kainic acid or AMPA induced a concentration-dependent tritium overflow which was susceptible to blockade by TTX. 5. The kainic acid-evoked tritium overflow was not affected by DL-(E)-2-amino-4-methyl-5-phosphono-3-pentanoic acid (CGP37849; a competitive NMDA receptor antagonist), but was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist at glutamate receptors of the non-NMDA type). 6. The AMPA-evoked tritium overflow was also inhibited by CNQX.2ń     

Glutamatergic-cholinergic synergistic interaction in the pontine reticular formation. Effects on catalepsy

Microinjections of small amounts of the cholinergic receptor agonists carbachol and nicotine into the pontine reticular formation (PRF) of rats were shown to induce catalepsy. Catalepsy was used in this work as an experimental model for studying the interactions between cholinergic mechanisms and excitatory amino acid mechanisms in the PRF. The excitatory amino acid (EAA) receptor agonists glutamate, NMDA, kainate and AMPA were microinjected in subcataleptic doses before carbachol in the same location into the PRF. All the EAA receptor agonists injected induced a significant potentiation of the cataleptogenic effect of carbachol. The NMDA receptor antagonist MK-801 and the non-NMDA receptor antagonist DNQX microinjected in picomol doses before the EAA receptor agonists attenuated their potentiating effect. These results support the suggestion that EAA neuronal mechanisms contribute synergistically with the cholinergic mechanisms to the PRF neuronal interactions involved in the generation of catalepsy. Similar synergistic interactions might be active in the generation of other pontine behavioral manifestations like REM sleep.     

GABAergic and glutamatergic modulation of exploratory behavior in the dorsomedial hypothalamus

Systemic injection of glutamate NMDA receptor antagonists or drugs that facilitate GABA(A)-mediated neurotransmission produces anxiolytic effects. The dorsomedial hypothalamic (DMH) region is proposed to be a possible site of action of these drugs. The objective of the present study was to investigate if facilitation of GABA(A)-mediated neurotransmission or blockade of NMDA receptors in the DMH would produce anxiolytic effects in the elevated plus-maze (EPM). Seven days after surgery, male Wistar rats with unilateral cannulas in the DMH were submitted to the behavioral studies. Results showed that midazolam, a benzodiazepine anxiolytic (30-60 nmol/0.3 microl), produced a dose-dependent increase in open arm exploration without changing the number of enclosed arm entries, indicating an anxiolytic effect. This effect was antagonized by previous treatment with flumazenil, a benzodiazepine receptor antagonist (60 nmol/0.3 microl). Flumazenil alone had an anxiogenic effect, decreasing exploration of the open arms of the EPM. 2-Amino-7-phosphonoheptanoic acid (AP7), an NMDA receptor antagonist (0.2-2 nmol/0.3 microl), did not modify open arm exploration but decreased general exploratory activity. These results indicate that benzodiazepine receptors located in the DMH could modulate anxiety. Interference with NMDA receptor-mediated neurotransmission in this region, however, seems to change general exploratory activity rather than anxiety.     

Modulation of the locomotor responses induced by D1-like and D2-like dopamine receptor agonists and D-amphetamine by NMDA and non-NMDA glutamate receptor agonists and antagonists in the core of the rat nucleus accumbens

Dopamine and glutamate interactions in the nucleus accumbens (NAcc) play a crucial role in both the development of a motor response suitable for the environment and in the mechanisms underlying the motor-activating properties of psychostimulant drugs such as amphetamine. We investigated the effects of the infusion in the NAcc of NMDA and non-NMDA receptor agonists and antagonists on the locomotor responses induced by the selective D(1)-like receptor agonist SKF 38393, the selective D(2)-like receptor agonist quinpirole, alone or in combination, and D-amphetamine. Infusion of either the NMDA receptor agonist NMDA, the NMDA receptor antagonist D-AP5, the non-NMDA receptor antagonist CNQX, or the non-NMDA receptor agonist AMPA resulted in an increase in basal motor activity. Conversely, all of these ionotropic glutamate (iGlu) receptor ligands reduced the increase in locomotor activity induced by focal infusion of D-amphetamine. Interactions with dopamine receptor activation were not so clear: (i). infusion of NMDA and D-AP5 respectively enhanced and reduced the increase in locomotor activity induced by the infusion of the D(1)-like receptor agonist of SKF 38393, while AMPA or CNQX decreased it; (ii). infusion of NMDA, D-AP5, and CNQX reduced the increase in locomotor activity induced by co-injection of SKF 38393+quinpirole--a pharmacological condition thought to activate both D(1)-like and D(2)-like presynaptic and postsynaptic receptors, while infusion of AMPA potentiated it; (iii). infusion of either NMDA, D-AP5 or CNQX, but not of AMPA, potentiated the decrease in motor activity induced by the D(2)-like receptor agonist quinpirole, a compound believed to act only at presynaptic D(2)-like receptors when injected by itself. Our results show that NMDA receptors have an agonist action with D(1)-like receptors and an antagonist action with D(2)-like receptors, while non-NMDA receptors have the opposite action. This is discussed from a anatamo-functional point of view.     

Enhanced NMDA receptor NR1 phosphorylation and neuronal activity in the arcuate nucleus of hypothalamus following peripheral inflammation

AbstractAim:To investigate the role of glutamate and N-methyl-D-aspartate (NMDA) receptors in central sensitization following peripheral inflammation in the arcuate nucleus (ARC) of the mediobasal hypothalamus.Methods:Mediobasal hypothalamic slices were prepared from rats undergoing peripheral inflammation, which was induced by a unilateral injection of complete Freund's adjuvant (CFA) into hind paw. Neuronal activation levels in the ARC were monitored by recording extracellular unit discharges. The NMDA receptor NR1 subunit (NR1) was measured using Western blot analysis.Results:Enhanced NR1 phosphorylation was observed in the ARC of CFA-inflamed rats. Compared with the control rats, the firing rate of spontaneous discharges in ARC neurons of inflamed rats was significantly higher, and it was significantly reduced both by an NMDA receptor antagonist (MK-801, 300 μmol/L) and by a non-NMDA receptor antagonist (CNQX, 30 μmol/L). Application of exogenous glutamate (200 μmol/L) or NMDA (25 μmol/L) resulted in increased neuronal discharges for ARC neurons, which was enhanced to a greater extent in inflamed rats than in control rats.Conclusion:Glutamate receptor activation in the hypothalamic ARC plays a crucial role in central sensitization associated with peripheral inflammation.     

NMDA和非NMDA受体参与介导猫脊髓内脏作害性信息传递

AIM: To study the role of N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors in processing nociceptive visceral information in the spinal cord. METHODS: The firing of spinal dorsal horn neurons to colorectal distension (3-15 kPa, 20 s) by inflation with air of latex balloon was recorded in 25 anesthetized cats. RESULTS: 1) According to the patterns of responses to colorectal distension, the neurons with increase and decrease in firing were classified as excitatory and inhibitory, respectively. The former consisted of 17 short-latency abrupt (SLA) neurons, 11 short-latency sustained (SLS) neurons, 9 long-latency (LL) neurons. The 15 inhibited (Inh) neurons were recorded. 2) Microelectrophoretic administration of NMDA, quisqualic acid (QA), and kainic acid (KA) activated 67.6%, 78.4%, and 59.5% of the colorectal distension-excited neurons tested. Also, 60%, 86.7%, and 53.3% of Inh neurons were activated by these 3 amino acids. 3) Colorectal distension-induced excitatory responses were reduced by 35% +/- 10% and 65% +/- 14% by a selective NMDA receptor antagonist d,l-2-amino-5-phosphonovalerate (APV) and a selective non-NMDA receptor antagonist 6,7-dinitro-quinoxaline-2,3-dione (DNQX), respectively. Such DNQX-induced inhibition was significantly more potent than that by APV (P < 0.05). Colorectal distension-induced inhibitory responses were partially relieved by 30%-50% in 3/7 Inh neurons by DNQX, but not APV. CONCLUSION: Both NMDA and non-NMDA receptors are involved in transmission and/or modulation of spinal visceral nociceptive information and non-NMDA receptors may play more important role than NMDA receptors.     

Regulation of spontaneous inhibitory synaptic transmission by endogenous glutamate via non-NMDA receptors in cultured rat hippocampal neurons

The regulation of gamma-aminobutyric acid (GABA)-mediated spontaneous inhibitory synaptic transmission by endogenously released glutamate was studied in cultured rat hippocampal neurons. After 7 days in vitro (DIV), both spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) could be detected. After 15 DIV, most postsynaptic spontaneous currents occurred as sEPSC/sIPSC sequences when recorded at a holding voltage of -30 mV. In the presence of the glutamate alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtype antagonist LY303070, both the frequency and amplitude of sIPSC were strongly and reversibly reduced. The N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoic acid (AP5), had no effect on sIPSC while cyclothiazide strongly increased sIPSC frequency. Under blockade of AMPA receptors, the kainate- and GluR5-selective kainate receptor agonists, (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid) (ATPA) and (S)-5-iodowillardiine (5IWill), induced a large enhancement of the frequency of small-amplitude sIPSC which was blocked by the non-NMDA receptor antagonist, 2,3-dihydro-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX). All of these effects were sensitive to tetrodotoxin (TTX). In the presence of LY303070 and TTX, kainate could induce a small inward current while GluR5 agonists had no effect. In the presence of NMDA and AMPA receptor antagonists, the glutamate uptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) could restore sIPSC. When NBQX was used as an AMPA antagonist, the stimulatory effect of t-PDC was blocked while the group I metabotropic glutamate agonist, 3,5-dihydroxyphenylglycine (DHPG), induced a strong enhancement of sIPSC. Therefore, both AMPA and kainate receptors can regulate inhibitory synaptic transmission in cultured hippocampal neurons, the former by tonic activation, the latter when the glutamate concentration is increased by impairing glutonate uptake.     

The non-NMDA glutamate receptor antagonist GYKI 52466 counteracts locomotor stimulation and anticataleptic activity induced by the NMDA antagonist dizocilpine

Summary The effects of the non-NMDA glutamate receptor antagonist GYKI 52466 (2.4 and 4.8 mg/kg, i.p.) on spontaneous locomotor activity and haloperidol-induced catalepsy (0.5 mg/kg, i.p.) were assessed in naive rats and in rats pretreated with the NMDA antagonist dizocilpine (0.08 mg/kg, i.p.). GYKI 52466 given alone did not alter locomotor activity and haloperidol-induced catalepsy, but significantly antagonized the dizocilpine-induced locomotor stimulation and counteracted the anti-cataleptic effects of dizocilpine on haloperidol-induced catalepsy. Thus blockade of non-NMDA glutamate receptors antagonized the behavioural stimulant effects of a NMDA receptor blockade. Correspondence to: W. Hauber at the above address     

Synthesis and biological evaluation of cyclopropyl analogues of 2-amino-5-phosphonopentanoic acid.

A series of cyclopropyl analogues related to 2-amino-5-phosphonopentanoic acid (AP5) were synthesized and their biological activity was assessed as competitive antagonists for the N-methyl-D-aspartate (NMDA) receptor. In vitro receptor binding using [3H]-L-glutamate as the radioligand provided affinity data, while modulation of [3H]MK-801 binding was used as a functional assay. The analogues were also evaluated in [3H]kainate binding to assess selectivity over non-NMDA glutamate receptors. Of the compounds tested, 4,5-methano-AP5 analogue 26 was the most potent selective NMDA antagonist; however, potency was lower than that for [[(+/-)-2-carboxypiperidin-4-yl]methyl]phosphonic acid (CGS 19755, 5).     

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.     

An electrophysiological study of the action of N-methyl-D-aspartate on excitatory synaptic transmission in the optic tectum of the frog in vitro

Excitatory synaptic field potentials, induced by stimulating optic nerve fibers, were recorded from in vitro preparations of the optic tectum of the frog. Bath-applied N-methyl-D-aspartate (NMDA), glutamate or quisqualate elicited transient enhancement in these field potentials, followed by a sustained depression reversible on washout. Responses to glutamate or quisqualate and the amplitude of control synaptic potentials, were not affected by the NMDA receptor antagonists aminophosphonovalerate (APV), 3(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), ketamine, magnesium ions or dizocipiline (MK 801) which, on the other hand, blocked the effects of NMDA. The antagonist dinitroquinoxaline-2,3-dione (DNQX), which is preferential for non-NMDA receptors, blocked the action of glutamate and synaptic transmission. In the presence of strychnine, glycine reversed the block of NMDA-mediated responses caused by magnesium. It is suggested that in the optic tectum of the frog, glutamate is the excitatory transmitter of at least one class of optic nerve fibers and that it acts through non-NMDA receptors. Although this area of the brain contains a well-developed NMDA receptor system, its function in physiological synaptic transmission remains to be elucidated.     

Evidence that activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors within the nucleus tractus solitarii triggers swallowing.

Swallowing is a patterned motor activity generated by neurons located within the nucleus tractus solitarii (NTS). Previous experiments have shown that administration of excitatory amino acids within the NTS induces swallowing. The present study was undertaken to identify the receptor subtypes involved in this effect. Pressure microinjections of L-glutamate (10-100 pmol), quisqualate (0.1-10 pmol) and N-methyl-D-aspartate (NMDA, 0.1-10 pmol) were performed into the NTS of decerebrate rats. Glutamate and quisqualate microinjections elicited short series of swallows while NMDA microinjections induced long-lasting, rhythmic swallowing. Pretreatment with the selective NMDA antagonist, DL-2-amino-5-phosphonovalerate (50 pmol), almost completely suppressed the response elicited by NMDA (10 pmol) but did not induce a significant modification of swallowing triggered by either glutamate (25 pmol) or quisqualate (10 pmol). Pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione (50 pmol), a selective blocker of non-NMDA receptors, suppressed the swallows elicited by glutamate and strongly inhibited the response elicited by quisqualate microinjections. The same pretreatment induced only a slight modification of the swallowing elicited by NMDA. These data demonstrate that deglutition can be triggered by activating either NMDA or non-NMDA receptors localized within the NTS, and therefore suggest that both receptor subtypes may be involved in swallowing elicited under physiological conditions.     

Endogenous adenosine release from hippocampal slices excitatory amino acid agonists stimulate release,antagonists reduce the electrically-evoked release

Summary The effect of excitatory amino acids and their antagonists on adenosine and inosine release has been investigated on unstimulated and electrically stimulated hippocampal slices.On unstimulated slices N-methyl-D-aspartate (NMDA), quisqualate and glutamate concentration-dependently evoked the release of adenosine and inosine. The effect of NMDA and quisqualate was antagonized by the NMDA receptor antagonist D(–)-2-amino-7-phosphonoheptanoic acid (D-AP7; 100 mol/1) and the non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 mol/1) respectively. Glutamate (2 and 10 mmol/1)-evoked adenosine and inosine release was not antagonized by the NMDA and non-NMDA receptor antagonists indicating that the effect of glutamate is due to a metabolic rather than a receptor-mediated effect.Electrical field stimulation at 10 Hz also evoked a release of endogenous adenosine and inosine. Tetrodotoxin (0.5 gmol/1) abolished and absence of Ca²⁺ markedly reduced the electrically evoked release of adenosine and inosine. Adenosine and inosine release evoked by electrical stimulation at 20 Hz was significantly reduced in the presence of the NMDA receptor antagonist D-AP7, while at 10 Hz no consistent decrease was seen. In the presence of D-AP7 plus DNQX the 10 Hz-evoked adenosine and inosine release was reduced to about half. These data suggest that the electrically evoked release of adenosine and inosine is partly mediated by the release of excitatory amino acids which act at both non-NMDA and NMDA receptors. Send offprint requests to F. Pedata at the above address     

Sustained ethanol inhibition of native AMPA receptors on medial septum/diagonal band (MS/DB) neurons

The direct impact of ethanol on native, non-NMDA glutamate receptors was examined in acutely isolated MS/DB neurons from rat. The impact of ethanol functional tolerance and physical dependence on non-NMDA receptor function was also determined. Non-NMDA receptors were defined pharmacologically as predominantly the AMPA subtype, because both AMPA- or kainate-activated currents were blocked by GYKI 52466, a selective AMPA receptor antagonist. The relative magnitude of potentiation of AMPA-activated currents by 10 or 100 microM cyclothiazide was consistent with recombinant AMPA flop-subtype receptors. Finally, the selective kainate receptor agonist, SYM 8021, induced little current in MS/DB neurons. AMPA receptor currents when activated by kainate were sensitive to ethanol, showing inhibition of approximately 5 - 50% when 10 - 300 mM ethanol and kainate were briefly co-applied (3 s). Ethanol (100 mM) also inhibited both the initial transient peak and sustained currents activated by AMPA. Inhibition was sustained during continuous ethanol superfusions of 5 min, suggesting a lack of acute tolerance to ethanol-induced AMPA receptor blockade. Rapid application of 3 - 3000 microM kainate activated concentration-dependent currents in MS/DB neurons from Control and Ethanol Dependent animals that were not significantly different. Also, direct ethanol inhibition (300 mM) of kainate-activated currents was not reduced by ethanol dependence, suggesting a lack of functional tolerance. These results suggest that native AMPA receptors on MS/DB neurons are inhibited by pharmacologically-relevant concentrations of ethanol. However, these receptors, unlike NMDA receptors, do not undergo adaptation with sustained ethanol exposure sufficient to induce physical dependence. British Journal of Pharmacology (2000) 129, 87 - 94     

Competitive antagonism of glutamate receptor channels by substituted benzazepines in cultured cortical neurons.

Whole-cell recordings from rat cortical neurons in dissociated cell culture were used to study the antagonism of glutamate receptors by several lipophilic benzazepine analogues of 2,5-dihydro-2,5-dioxo-3-hydroxy-1H-benzazepine (DDHB). DDHB and three substituted derivatives, 4-bromo-, 7-methyl-, and 8-methyl-DDHB, inhibited the activation of N-methyl-D-aspartate (NMDA) receptors at both the NMDA recognition site and the glycine allosteric site. In addition, all four compounds blocked the activation of non-NMDA receptors by kainate and L-glutamate. Antagonism by the four benzazepines was equivalent at holding potentials from -80 mV to +50 mV. Both the onset of and recovery from block of the agonist-gated currents were complete within seconds. Antagonist affinity was calculated from the displacement of steady state concentration-response curves for kainate, L-glutamate, glycine, and NMDA, based on the Gaddum-Schild relationship (dose ratio = 1 + [antagonist]/KB). The most potent blocker, 8-Me-DDHB, had an apparent dissociation constant (KB) of 470 nM at the glycine allosteric site and 27 microM at the NMDA recognition site. The apparent dissociation constant of 8-Me-DDHB for non-NMDA receptors was 6.4 microM when kainate was the agonist and 9.6 microM when L-glutamate was the agonist. Unsubstituted DDHB showed slightly higher affinity for the NMDA recognition site (KB = 16 microM) but was less potent than 8-Me-DDHB at the glycine allosteric site and at non-NMDA receptors (KB = 3 and 65 microM, respectively). At all three sites, the inhibitory actions of these benzazepine derivatives were consistent with a simple competitive mechanism of antagonism. In addition, the antagonist potency of the parent compound, DDHB, against kainate, NMDA, and glycine was equal to or greater than that of other bicyclic antagonists, including kynurenic acid, indole-2-carboxylic acid, and quinoxaline-2,3-dione. Substituted benzazepines represent a new class of glutamate receptor antagonists that show competitive action, significant potency at multiple sites, and a high degree of lipophilicity.