Evidence forN-methyl-d-aspartic acid receptor-mediated modulation of the commissural input to central vestibular neurons of the frog

We have investigated the role ofN-methyl-d-asparte (NMDA) receptors in the excitatory synaptic transmission to central vestibular neurons in the isolated superfused brainstem of the frog. In superfusate containing 1 mM Mg²⁺ field potentials in the vestibular nuclei evoked by electrical stimulation of either the ipsi- or the contralateral VIIIth nerve were not affected by bath-appliedd-2-amino-5-phosphonovaleric acid (D-APV, 25–50 μM), a selective NMDA antagonist. In a low Mg²⁺ solution postsynaptic field potential components were larger than control but still unaffected by D-APV. Ipsi- and contralaterally evoked excitatory postsynaptic potentials (EPSPs) differed in their shape parameters as well as their pharmacological sensitivity. Ipsilaterally evoked EPSPs were not affected by D-APV and had a rise time that was faster than that of contralaterally evoked EPSPs. The peak amplitude of the latter was reduced by D-APV (25–50 μM) to about 65% of the control value in the presence of 1 mM Mg²⁺. During bath application of NMDA (100 μM) an increased input resistance and repetitive de- and hyperpolarizing membrane potential shifts were observed. Similar events were observed during a reduction of the Mg²⁺ concentration. Bath application of NMDA (0.1–1 μM) resulted in an enhanced size of the recorded EPSPs. Dendritic and somatic EPSPs were stimulated on a computer with the assumption of a constant NMDA receptor activation and a pulse-like non-NMDA receptor activation. The results of these stimulations are consistent with the hypothesis that the efficacy of non-NMDA-mediated vestibular commissural synaptic transmission is modulated through tonically activated NMDA receptors.     

GABAergic inhibition and epileptiform discharges in the turtle hippocampus in vitro

The effects of excitatory amino acid (EAA) receptor antagonists were examined on intracellularly recorded epileptiform discharges in turtle hippocampal (ventromedial cortical) pyramidal neurons in vitro. Afferent synaptic activation of turtle hippocampal neurons evoked monophasic or biphasic GABAergic inhibitory postsynaptic potentials (IPSPs). In the presence of bicuculline (5 microM) or picrotoxin (100 microM) IPSPs were reduced, and long-lasting ictal-like discharges were transiently observed prior to the establishment of a regular rhythm of discharge of spontaneous paroxysmal depolarization shifts (PDSs). Bicuculline-induced PDSs were reversibly reduced in amplitude and duration, but not abolished by the EAA receptor antagonists kynurenic acid (1 mM), cis-2,3-piperidine dicarboxylic acid (cis-2,3-PDA) (1 mM), or DL-2-amino-5-phosphonovalerate (DL-AP-5) (100 microM), revealing a long-lasting hyperpolarizing afterpotential. These results indicate that the blockade of GABAergic inhibition leads to the genesis of epileptiform discharges, and EAA receptor antagonists (particularly those of the N-methyl-D-aspartate (NMDA) receptor subtype) block the maintained depolarization underlying PDSs, but do not prevent their spontaneous discharge in turtle hippocampus.     

Streptomycin blocks the postsynaptic effects of excitatory amino acids on the vestibular system primary afferents

It has been suggested that streptomycin might be an antagonist of the glutamate receptors, and that it selectively blocks quisqualic acid receptors. We studied whether streptomycin blocks the responses to excitatory amino acid agonists on the vestibular system primary afferents, and if it allows us to differentiate between kainate (KA) and quisqualate (QA) receptor mediated responses. The experiments were performed in the axolotl (Ambystoma tigrinum). Intra- and extracellular records of the electrical activity of semicircular canal afferent fibers were obtained. Drugs were applied by pressure ejection in volumes of 20 μl in a 10 ml bath. Streptomycin (0.01–10 mM), induced a dose dependent reversible inhibition of the basal spike discharge of the afferent fibers. This coincided with a reduction in the amplitude of excitatory postsynaptic potentials (EPSP) recorded recorded intracellularly in the afferent fibers. Streptomycin also blocked the excitatory action produced by KA and QA; increasing concentrations of streptomycin produced a rightward shift in the concentration-response curves for both KA and QA. This action persisted even in a high Mg²⁺ (10 mM), low Ca²⁺ (0.09 mM) Ringer solution, indicating its postsynapsic nature. These results show that streptomycin might be a non-selective excitatory amino acid (EAA) receptor antagonist.     

Ethanol inhibition of NMDA mediated depolarizations is increased in the presence of Mg

The inhibitory potency of ethanol upon excitatory amino acid induced depolarizations of rat hippocampal CA1 pyramidal cells was assessed in the presence and absence of magnesium (Mg²⁺) using the grease-gap technique. Ethanol shifted theN-methyl-d-aspartate (NMDA) dose-response curves to the right in a non-parallel manner. In the presence of Mg²⁺, ethanol appeared to be a more effective NMDA antagonist (IC₅₀ 47 mM) than in the absence of Mg²⁺ (IC₅₀ 107 mM). The IC₅₀ for ethanol upon non-NMDA mediated CA1 pyramidal cell depolarizations was in excess of 170 mM. These results strongly suggest a preferential inhibitory action of ethanol against NMDA, rather than non-NMDA, mediated responses. Experiments in which ethanol and Mg²⁺ were covaried indicated that these substances act by two distinct mechanisms to antagonize the action of NMDA. These effects of ethanol, at concentrations which elicit intoxication(< 50mM) but not anesthesia, suggest that the NMDA receptor complex may play an important role in the acute effects of ethanol.     

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg²⁺, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 μM ACPD and in 7 of 12 cells with 0.5 μM ACPD. The EPSP amplitudes with 5 and 0.5 μM ACPD were 142±10% (mean±S.E.M., n=19) and 130±13% (n=12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 μM ACPD were not significantly different in cells perfused with the low Ca²⁺-high Mg²⁺ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence ofd-(−)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg²⁺. Five μM ACPD potentiated monosynaptic EPSPs in 4 of 6 cells with 1 mM Mg²⁺ in the solution and in 2 of 17 cells with 4 mM Mg²⁺, and the EPSP amplitude was 123±9% (n=6) and 98±3% (n=17) of those before application of ACPD, respectively. These results suggest that activation of metabotropic glutamate receptors potentiates LC-EPSPs via mechanisms sensitive to Mg²⁺ and may work as a positive feedback mechanism at the excitatory amino acid-mediated synapses between the descending fibers and lumbar spinal motoneurons.     

Quisqualate, kainate and NMDA can initiate spreading depression in the turtle cerebellum

This study evaluated the role of excitatory amino acid (EAA) receptor activation in spreading depression (SD), using the in vitro turtle cerebellum as a model system. SD was triggered by electrical stimulation or by elevated K+ after the cerebellum had been conditioned for at least 30 min with physiological saline in which most of the chloride had been replaced by propionate. SD was recognized as a transient (1-3 min) negative shift of extracellular potential accompanied by depression of evoked potentials (15-30 min) and an increase of extracellular K+ up to 60 mM, which spread across the cerebellum at rates of 1-7 mm/min. SD usually commenced in the granular layer, which apparently contains the 3 major EAA receptor subtypes, quisqualate, kainate and N-methyl-D-aspartate (NMDA), then subsequently spread to the molecular layer, which is largely free of NMDA receptors. Glutamate, aspartate, NMDA, kainate and quisqualate all triggered SD. Kynurenic acid and 2-aminophosphonovaleric acid (APV) inhibited SD under certain conditions further suggesting involvement of EAA receptors. The initiation of SD was blocked by high Mg2+ and facilitated in low extracellular Mg2+, which also eliminated the delay in molecular layer SD onset. Our data suggest that no one EAA receptor subtype is singly responsible for SD.     

Both glutamate receptor antagonists and prefrontal cortex lesions prevent induction of cocaine sensitization and associated neuroadaptations.

Behavioral sensitization to psychomotor stimulants is accompanied by a number of alterations in the mesoaccumbens dopamine (DA) system, including DA autoreceptor subsensitivity in the ventral tegmental area (VTA) and DA D1 receptor supersensitivity in the nucleus accumbens (NAc). We investigated the role of excitatory amino acid (EAA) transmission in the induction of cocaine sensitization and these accompanying DA receptor alterations. To do so, we used three glutamate receptor antagonists, the noncompetitive NMDA receptor antagonist MK-801 (0.1 mg/kg), the competitive NMDA receptor antagonist CGS 19755 (10.0 mg/kg), and the AMPA receptor antagonist NBQX (12.5 mg/kg). Rats received daily double injections of either one of these antagonists or saline with either cocaine (15.0 mg/kg) or saline for 5 days. Cocaine sensitization was defined as an increase in horizontal locomotor activity in response to cocaine challenge (7.5 mg/kg) on the third day of withdrawal. All three antagonists prevented the induction of cocaine sensitization. Extracellular single cell recordings revealed that these antagonists also prevented the induction of DA autoreceptor subsensitivity in the VTA and DA D1 receptor supersensitivity in the NAc. To determine whether the relevant glutamate receptors were under regulation by medial prefrontal cortex (mPFC) EAA efferents, we next lesioned the mPFC bilaterally with ibotenic acid at least 7 days before repeated cocaine treatment began. These lesions also prevented the induction of cocaine sensitization and the associated neuroadaptations. Our findings indicate that glutamate transmission from mPFC to the mesoaccumbens DA system is critical for the induction of cocaine sensitization and its cellular correlates.     

Functional N-Methyl-D-Aspartate and Non-N-Methyl-D-Aspartate Receptors are Expressed by Rat Supraoptic Neurosecretory Cells in vitro

A considerable amount of evidence has accumulated to support a role for excitatory glutamatergic transmission in the regulation of the hypothalamo-neurohypophysial system. Glutamate immunoreactivity has been found in axon terminals forming asymmetric synapses on to magnocellular neurosecretory cells and kynurenic acid, a broad spectrum glutamate receptor antagonist inhibits 1) spontaneous electrical activity in vivo, 2) excitatory postsynaptic potentials in hypothalamic slices, and 3) osmotically-evoked vasopressin release from hypothalamic explants. While this provides strong evidence for glutamatergic regulation of hypothalamic magnocellular neurosecretory cells, the subtypes of glutamate receptors expressed by these cells have not been defined. We have, therefore, obtained current and voltage clamp recordings from supraoptic magnocellular neurosecretory cells in vitro to investigate the functional and pharmacological properties of their glutamate receptors. Application of micromolar concentrations of L-glutamate, or of the agonists kainate, quisqualate and N-methyl-D-aspartate (NMDA), produced reversible and dose-dependent depolarizations in all cells tested. These responses were mediated by postsynaptic receptors since they persisted during chemical synaptic blockade with Ca^{2 +} -free or tetrodotoxin-containing solutions. The inward current induced by NMDA showed a marked Mg²⁺-sensitive voltage dependence, and was blocked by D, L-2-amino-5-phosphonovalerate. In contrast, currents induced by kainate and quisqualate showed linear current-voltage properties and were antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione. We conclude that both NMDA and non-NMDA receptors are expressed by magnocellular neurosecretory cells of the rat supraoptic nucleus.     

Ionotropic glutamate receptors mediate excitatory drive to caudal medullary expiratory neurons in the rabbit

Most of the neurons of the caudal ventral respiratory group (cVRG) are bulbospinal expiratory neurons that receive their main excitatory drive from more rostral, but not yet defined regions. This study was devoted to investigate the functional role of ionotropic excitatory amino acid (EAA) receptors in the excitatory drive transmission to cVRG expiratory neurons during eupnoeic breathing and some respiratory reflexes including cough induced by mechanical stimulation of the tracheobronchial tree. The experiments were performed on spontaneously breathing rabbits under pentobarbitone anesthesia making use of microinjections (30--50 nl) of EAA receptor antagonists into the cVRG. Phrenic nerve and abdominal muscle activities were recorded. Bilateral microinjections of 50 mM kynurenic acid (KYN), a broad-spectrum EAA antagonist, and 10 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA antagonist, or 5 mM 6-nitro-7-sulphamoylbenzo(f)quinoxaline-2,3-dione (NBQX), a more specific non-NMDA antagonist, completely suppressed spontaneous rhythmic abdominal activity and reflex expiratory responses either to tracheal occlusion at end-inspiration (Breuer-Hering inflation reflex) or to expiratory threshold loading (5 cm H(2)O); they also suppressed both the inspiratory and expiratory components of the cough reflex. Spontaneous rhythmic abdominal activity and the reflex respiratory responses were strongly reduced, but not completely abolished by microinjections of 10 mM d(-)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA antagonist. The results provide evidence that the excitatory drive to cVRG bulbospinal expiratory neurons during eupnoeic breathing and the investigated respiratory reflexes is mediated by EAA receptors. They also support the view that neurons located in the cVRG are not merely elements of the expiratory output system.     

Glutamate receptor regulation of rat nucleus accumbens neurons in vivo

Extracellular single cell recording and microiontophoretic techniques were used to characterize the roles of ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs) in glutamate-induced excitation of rat nucleus accumbens (NAc) neurons in vivo. Pulse-ejected glutamate (16–128 nA) induced a current-dependent increase in the firing of quiescent NAc neurons. A stronger excitatory response to α-amino-3-hydroxy-5-methyl-4-iosoxazole-proprionic acid (AMPA) was observed at much lower ejection currents (0.1–6.4 nA). Compared to AMPA and glutamate, N-methyl-D -aspartate (NMDA) induced a much less potent excitation in a narrow current range (1–4 nA) and only when neurons were previously “primed” with other excitatory amino acids (EAAs). Higher ejection currents of all three EAA agonists drove NAc neurons into a state of apparent depolarization block. AMPA-evoked firing was selectively blocked by the AMPA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) whereas NMDA-induced activity was selectively prevented by the NMDA receptor antagonist 2-amino-5-phosphonovalerate (D-AP5). DNQX, but not D-AP5, significantly attenuated glutamate-evoked activity. The mGluR receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-t-ACPD) failed to evoke activity of NAc neurons, but significantly reduced the excitatory effects of other EAAs. This modulatory effect of 1S,3R-t-ACPD was consistently blocked by the selective mGluR antagonist L(+)-2-amino-3-phosphonopropionic acid (L-AP3) whereas another mGluR antagonist (RS)-4-carboxy-3-hydroxy phenylglycine (4C3HPG) was inconsistent in this regard. These results indicate that the excitatory effects of glutamate on rat NAc neurons in vivo are primarily mediated by non-NMDA iGluRs and that mGluRs function to dampen excessive glutamate transmission through iGluRs. © 1996 Wiley-Liss, Inc.     

Participation of excitatory amino acid receptors in the slow excitatory synaptic transmission in rat spinal dorsal horn

In a rat spinal slice preparation the participation of excitatory amino acid (EAA) receptors in the responses of deep dorsal horn neurons to repetitive stimulation of lumbar dorsal roots was investigated using 3 EAA receptor antagonists, kynurenic acid, D-(-)-2-amino-4-phosphonovaleric acid (D-APV) and 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) and current-clamp and voltage-clamp techniques. We found that the slow excitatory synaptic response evoked by 10-20 Hz electrical stimulation of primary afferent fibers consisted of two depolarizing components: an initial component lasting 1-5 s and a late one of 1-3 min duration. The initial and late components of the slow excitatory synaptic response can also be distinguished on the basis of their voltage-dependence and sensitivity to Mg2+ ions, kynurenate, D-APV and CNQX. In the presence of Mg2+, the initial component of the slow excitatory synaptic response increased with membrane hyperpolarization, whereas the late component decreased in most of the cells examined. In a zero-Mg2+ medium, the initial component was potentiated, but the late component was reduced. In both transverse and longitudinal spinal cord slices perfused with 1.2 mM Mg(2+)-containing medium, bath application of kynurenic acid (0.1-0.5 mM), D-APV (0.05-0.1 mM) and CNQX (5-7 microM) caused a reversible reduction of the peak amplitude of the initial slow depolarizing component that was greater in transverse (kynurenic acid: by 92.6 +/- 5.0%; D-APV: by 69.1 +/- 7.8%; CNQX: by 76.6 +/- 9.8%) than in longitudinal slices (kynurenic acid: by 53.3 +/- 1.3%; D-APV: by 31.5 +/- 9.1%; CNQX: by 35.3 +/- 11.1%). In contrast, all 3 antagonists of EAA receptors produced no consistent change in the peak amplitude or half-duration of the late depolarizing component of the slow excitatory synaptic response. Our results obtained with EAA receptor antagonists, at resting membrane potentials, in the absence and presence of Mg2+ and synaptic inhibition, indicate that the synaptic activation of the NMDA- and non-NMDA-receptor systems of deep spinal dorsal horn neurons by repetitive stimulation of primary afferent fibers may be selectively involved in the mediation of the initial, but not the late depolarizing component of the slow excitatory synaptic response.     

Glutamate, kainate and quisqualate enhance GABA-dependent chloride uptake in cortex

Several lines of evidence indicate a possible interaction between the major inhibitory and excitatory cortical neurotransmitters, GABA and glutamate. To assess the neurochemical basis for such an interaction, we examined the effects of glutamate and several analogs on GABA-dependent chloride uptake in a mouse cortical synaptoneurosome preparation. L-Glutamate and the specific receptor subtype ligands kainate and quisqualate led to a small but significant enhancement in chloride uptake in the presence, but not the absence, of the GABA analog muscimol (5 microM). Enhancement was seen at excitatory amino acid (EAA) concentrations of 2-10 microM, but not at higher concentrations. D-Glutamate, NMDA, the NMDA-related antagonists APV and MK801, and the kainate/quisqualate antagonist CNQX, had no effect on chloride uptake. However, CNQX (50 microM) but not APV (50 microM) blocked the increase in chloride uptake due to kainate or quisqualate (10 microM). In addition, depolarization of synaptoneurosomes using high potassium (40 mM KC1) or ouabain pretreatment (5 microM) blocked the effects of kainate and quisqualate. Glutamate, kainate, and quisqualate had no effect on binding at the benzodiazepine, TBPS, or GABA sites on the GABAA receptor complex.     

Nicotinic acetylcholine receptor-mediated synaptic potentials in rat neocortex

In the neocortex, fast excitatory synaptic transmission can typically be blocked by using excitatory amino acid (EAA) receptor antagonists. In recordings from layer II/III neocortical pyramidal neurons, we observed an evoked excitatory postsynaptic potential (EPSP) or current (EPSC) in the presence of EAA receptor antagonists (40-100 microM D-APV+20 microM CNQX, or 5 mM kynurenic acid) plus the GABA(A)-receptor antagonist bicuculline (BIC, 20 microM). This EAA-antagonist resistant EPSC was observed in about 70% of neurons tested. It had a duration of approximately 20 ms and an amplitude of 61.5+/-6.8 pA at -70 mV (n=35). The EAA-antagonist resistant EPSC current-voltage relation was linear and reversed near 0 mV (n=23). The nonselective nicotinic acetylcholine receptor (nAChR) antagonists dihydro-beta-erythroidine (DH beta E, 100 microM) or mecamylamine (50 microM) reduced EPSC amplitudes by 42 (n=20) and 33% (n=9), respectively. EPSC kinetics were not significantly changed by either antagonist. Bath application of 10 microM neostigmine, a potent acetylcholinesterase inhibitor, prolonged the EPSC decay time. EAA-antagonist resistant EPSCs were observed in the presence of antagonists of metabotropic glutamate, serotonergic (5-HT(3)) and purinergic (P2) receptors. The EAA-antagonist resistant EPSC appears to be due in part to activation of postsynaptic nAChRs. These results suggest the existence of functional synaptic nAChRs on pyramidal neurons in rat neocortex.     

Evidence for a role of the N-methyl-d-aspartate (NMDA) receptor in cortical spreading depression in the rat

The neurotransmitter glutamate activates the N-methyl-d-aspartate (NMDA), quisqualate and kainate receptors. It has been proposed, but also disputed, that local release of glutamate would play a pivotal role in cortical spreading depression (SD). We tested this hypothesis by investigating the influence of NMDA antagonists on SD, using the non-competitive NMDA antagonists ketamine, phencyclidine (PCP) and MK-801 and the competitive NMDA antagonist dl-2-amino-7-phosphonoheptanoate (2-APH), injected intraperitoneally in rats anesthetized with alfentanil. SD was elicited by cathodal DC-stimulation of the frontal cortex. SD propagation was followed using two ion-sensitive microelectrodes placed in the parietal and occipital cortex. The NMDA antagonists increased SD threshold, decreased the propagation velocity and decreased the duration of the accompanying extracellular DC, K⁺ and Ca²⁺ changes at the following doses: 40 mg/kg ketamine, 10 mg/kg PCP, 0.63 mg/kg MK-801, 10 and 40 mg/kg 2-APH. With each NMDA antagonist failure of SD propagation between both microelectrodes could be observed. SD elicitation (or propagation) was inhibited completely with 80 mg/kg ketamine, 3.1 mg/kg MK-801 and 160 mg/kg 2-APH. These NMDA antagonists have also anticonvulsant properties. None of these effects on SD were observed with high doses of other anticonvulsants such as 80 mg/kg phenytoin or 40 mg/kg diazepam. These experiments indicate that endogenous release of excitatory amino acids and their action on the NMDA receptor play an important role in the initiation, propagation and duration of SD.     

Action of 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic aci (CPP): a new and highly potent antagonist of N-methyl-d-aspartate receptors in the hippocampus

A new compound, 3-((±)-2-car☐ypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), has been evaluated as an excitatory amino acid receptor antagonist using electrophysiological assays and radioligand binding. In autoradiographic preparations, CPP reduces l-[³H]glutama binding in regions of the hippocampus rich in N-methyl-d-aspartate (NMDA) receptors, but not in regions richin kainate sites. In isolated membrane fraction preparations, CPP displaces l-[³H]glutamate binding to NMDA sites, but does not compete with the binding of selective kainate or quisqualate site ligands. CPP potently reduces depolarizations produced by application of NMDA but not depolarizations produced by quisqualate or kainate. Its order of potency against excitatory amino acid-induced responses in the hippocampus is NMDA > homocysteate > aspartate > glutamate > quisqualate. CPP has no efect on lateral perforant path responses or on inhibition of these responses by 2-amino-4-phosphonobutyrate. Finally, at doses that do not affect Schaffer collateral synpatic transmission, CPP reversibly blocks the induction of long-term potentiation of Schaffer synaptic responses. This new compounds is, therefore, a higly selective brain NMDA receptor blocker, and the most potent such by nearly an order of magnitude.     

Excitatory amino acid-induced excitation of dopamine-containing neurons in the rat substantia nigra: Modulation by kynurenic acid

Kynurenic acid (KYNA), an endogenous antagónist of ionotropic excitatory amino acid (EAA) receptors, was tested for its ability to modulate N-methyl-D-aspartate (NMDA)- and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)induced excitation of dopamine (DA)-containing neurons in the zona compacta of the rat substantia nigra (SNc). Experiments were conducted using extracellular recording techniques in conjunction with an in vitro brain slice preparation. Bath application of NMDA (1–20 μ) or AMPA (0.5–10 μ) produced a concentration-dependent increase in the firing rate of SNc DA neurons but had no effect on firing pattern. The highest concentration of both agonists produced a rapid and reversible cessation of activity that was attributed to acute induction of depolarization block. Addition of glycino (GLY) (up to 100 μ) to the bathing solution had no effect on either basal firing rate or the increase in activity produced by NMDA. KYNA (10 μ–1 mM) antagonized the excitatory effects of both NMDA (15 μ) and AMPA (3 μ) in a concentration-dependent fashion (IC50: 102 μ and 64 μ, respectively) without affecting basal firing rate. Perfusion of tissue slices with a modified Ringer's solution containing low Mg²⁺ (0.12 mM) increased NMDA-induced excitation but did not affect the antagonist properties of KYNA. D-serine (100 μ) reversed the ability of KYNA to block the excitatory effects of NMDA, suggesting that KYNA attenuates NMDA-induced excitation of SNc DA neurons via blockade of the GLY allosteric site on the; NMDA receptor. The ability of KYNA to modulate the excitatory effects of both NMDA and non-NMDA agonists implies that endogenous KYNA may play a physiological role it regulating DA cell excitability. © 1994 Wiley-Liss, Inc.     

NBQX is a selective non-NMDA receptor antagonist in rat hippocampal slice°

The effects of NBQX and DNQX on synaptic transmission in rat hippocampal slice were investigated. Both agents produced dosedependent blockade of field potentials evoked by low frequency stimulation of Schaffer collateral-commissural fibers recorded in medium containing 4 mM Mg²⁺ (non-NMDA. mediated transmission), with half-maximal effects at about 0.15μM for NBQX and 1.0μM for DNQX. When the studies were conducted in Mg²⁺-free medium (predominantly NMDA mediated transmission), 100 μM NBQX failed to block transmission; however, the response could be completely blocked by the addition of 10μM of the competitive NMDA antagonist CPP. In contrast, 47 μM DNQX completely blocked secondary field potentials recorded in Mg²⁺-free medium and this effect could be reversed by the addition of 200 μM of the glycine agonist D-serine. Thus, NBQX exhibited selective blockade of non-NMDA mediated synaptic transmission whereas DNQX had effects at both non-NMDA and NMDA receptor sites, the latter effect via an interaction with the glycine site on the NMDA receptor complex.     

Interactions between excitatory amino acids and tachykinins in the rat spinal dorsal horn

Whole-cell patch-clamp technique of freshly isolated rat spinal dorsal horn (DH) neurons, intracellular recording from DH neurons in a slice preparation, and high performance liquid chromatography with fluorimetric detection of release of endogenous glutamate and aspartate from spinal cord slice following activation of primary afferent fibers were employed to investigate interactions between excitatory amino acids (EAA) and tachykinins [substance P (SP) and neurokinin A (NKA)]. Potentiation of N-methyl-D-aspartate (NMDA)-, quisqualate (QA)- and α-amino 3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-, but not kainate-induced currents by SP and NKA was found. Spantide II, a claimed novel nonselective tachykinin antagonist, effectively blocked the SP (2 nM )-induced potentiation of the responses of DH neurons to NMDA. In the presence of glycine (0.1 μM), the SP-evoked increase of the NMDA-induced current was prevented. However, 7-chlorokynurenic acid (2 μM), a competitive antagonist at the glycine allosteric site of the NMDA receptor, led to the reestablishment of the SP effect. Brief high frequency electrical stimulation of primary afferent fibers produced a longlasting potentiation of presumed monosynaptic and polysynaptic excitatory postsynaptic potentials and sustained enhanced release of endogenous glutamate (218.3± 66.1 %) and aspartate (286.3 ± 58.0%). Possible functional implications of the observed phenomena are discussed in relation to transmission and integration of sensory information, including pain.     

Intracellular calcium elevation during plateau potentials mediated by extrasynaptic NMDA receptor activation in rat hippocampal CA1 pyramidal neurons is primarily due to calcium entry through voltage‐gated calcium channels

We reported previously that plateau potentials mediated by extrasynaptic N‐methyl‐d ‐aspartate receptors (NMDARs) can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist or by iontophoresis of NMDA in rat hippocampal CA1 pyramidal neurons. To examine whether the plateau potentials are accompanied by an elevation of intracellular Ca²⁺ and to determine the source of Ca²⁺ elevation, we performed Ca²⁺ imaging during the plateau potential. Neurons were loaded with Ca²⁺ indicator fluo‐4, and the plateau potentials were generated either synaptically in the presence of glutamate transporter antagonist or by iontophoretically applying NMDA. We have found that a transient elevation in intracellular Ca²⁺ accompanies the plateau potential. The synaptically induced plateau potential and the Ca²⁺ elevation were blocked by 5,7‐dichlorokynurenic acid (5,7‐dCK), an antagonist for the glycine‐binding sites of NMDAR. A mixture of Cd²⁺ and tetrodotoxin did not block NMDA‐induced plateau potentials, but completely abolished the accompanying Ca²⁺ elevation in both the presence and absence of Mg²⁺ ions in the bathing solution. The NMDA‐induced plateau potential was blocked by further adding 5,7‐dCK. Our results show that the NMDAR‐mediated plateau potential is accompanied by elevation of intracellular Ca²⁺ that is primarily caused by the influx of Ca²⁺ through voltage‐gated Ca²⁺ channels.     

Comparing long-term depression with pharmacologically induced synaptic attenuations in young rat hippocampi

Field excitatory postsynaptic potentials (EPSPs) were recorded in the CA1 region of hippocampal slices from 12–18-day-old rats. The isolated N-methyl-D-aspartate (NMDA) receptor mediated field EPSP as well as the composite field EPSP of both NMDA and α-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) receptor mediated components were obtained in low Mg²⁺ solutions with 10 μM or 1 μM of the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. The isolated AMPA receptor mediated field EPSP was obtained either in normal Mg²⁺ solution or in a low Mg²⁺ solution in the presence of the NMDA receptor antagonist D-2-amino-5-phosphonopentanoic acid. The waveforms of the field EPSPs were studied and the effect of long-term depression (LTD) on these waveforms was compared with the effects of several pharmacological agents that attenuate the synaptic efficacy. It was shown that LTD occurred without changes in the waveforms of isolated AMPA and NMDA EPSPs. Reducing the number of release sites by lowering the stimulus strength or reducing the probability of transmitter release by an adenosine agonist N⁶-cyclohexyl-adenosine both mimicked the LTD-induced changes. Partial blockade of the AMPA receptors was also without effect on the waveforms of isolated AMPA EPSPs. In contrast, partial blockade of the NMDA receptors in several different ways resulted in waveform changes. A similar result could be inferred from experiments using composite field EPSPs. The synaptic attenuation caused by a partial blockade of NMDA receptors therefore appears to differ mechanistically from that involved in LTD, arguing against a postsynaptic locus of the modification involved in LTD. However, directly testing for alterations in transmitter release using the open channel blocker of NMDA receptors MK-801 failed in revealing such presynaptic changes during LTD. Our results therefore suggest that LTD might be due to a coordinated pre- and postsynaptic change instead of distinct pre- or postsynaptic modifications. Synapse 26:329–340, 1997. © 1997 Wiley-Liss Inc.