Assessment of NMDA receptor activation in vivo by Fos induction after challenge with the direct NMDA agonist (tetrazol-5-yl)glycine: effects of clozapine and haloperidol

Summary Induction of Fos protein by the potent and direct NMDA agonist (tetrazol-5-yl)glycine (TZG) was examined in mice. Effects of antipsychotic drugs were assessed on this in vivo index of NMDA receptor activation. TZG induced the expression of Fos in a neuroanatomically selective manner, with the hippocampal formation showing the most robust response. In mice genetically altered to express low levels of the NR1 subunit of the NMDA receptor, TZG-induced Fos was reduced markedly in comparison to the wild type controls. TZG-induced Fos was also blocked by the selective NMDA antagonist MK-801. Pretreatment of mice with clozapine (3 and 10 mg/kg) reduced TZG-induced Fos in the hippocampal formation but not in other brain regions. Haloperidol at a dose of 0.5 mg/kg did not antagonize TZG induced Fos in any region. Haloperidol at a dose of 1.0 mg/kg did attenuate the induction of Fos by TZG in the hippocampus but not in other brain regions. The relatively high dose (1 mg/kg) of haloperidol required to block effects of TZG suggests that this action may not be related to the D₂ dopamine receptor-blocking properties, since maximal D₂ receptor blockade was probably achieved by the 0.5 mg/kg dose of haloperidol. The antidepressant drug imipramine (10 or 20 mg/kg) did not antagonize TZG induced Fos in any brain region. The data suggest that clozapine can reduce excessive activation of NMDA receptors by TZG administration in vivo at doses relevant to the drugs’ actions in rodent models of antipsychotic activity. Whether or not this action of clozapine contributes to its therapeutic properties will require further study.     

Suramin disrupts insulin-like growth factor-II (IGF-II) mediated autocrine growth in human SH-SY5Y neuroblastoma cells

Suramin, traditionally used in the treatment of trypanosomiasis, is under investigation in the treatment of cancer. One side effect that limits its use is the onset of a sensorimotor polyneuropathy. In order to investigate the mechanism by which suramin induces polyneuropathy, we examined its effects on SH-SY5Y human neuroblastoma cells, an in vitro model of neuronal growth and differentiation. Addition of 50–400 μg/ml suramin to SH-SY5Y cells grown in 0.6% CS inhibited [³H]thymidine ([³H]TdR) incorporation and cell growth. Upon removal of suramin, [³H]TdR incorporation increased, demonstrating that levels of suramin used were cytostatic and not cytotoxic. Analysis of suramin-treated SH-SY5Y cells by flow cytometry revealed growth arrest in the G₁/G₀ phase of the cell cycle. IGF-II-induced SH-SY5Y growth is mediated by the type I IGF receptor (IGF-IR). Therefore, we examined its effect on IGF-IR tyrosine phosphorylation. Suramin prevented IGF-II-stimulated IGF-IR tyrosine phosphorylation. These results indicate that in SH-SY5Y cells, suramin acts as a cytostatic agent and can block IGF-II-dependent cell growth by preventing IGF-IR activation. Thus, suramin toxicity in the peripheral nervous system may be due, in part, to preventing IGFs and other growth factors from activating their receptors.     

Analysis of NMDA Receptors in the Human Spinal Cord

NMDA receptors in postmortem human spinal cord were analyzed using [³H]MK-801 ligand binding and immunoblotting with NMDA receptor subunit-specific antibodies. The averageK_Dfor [³H]MK-801 binding was 1.77 nM with aB_maxof 0.103 pmol/mg. The EC₅₀for stimulation of [³H]MK-801 binding withl-glutamate was 0.34 μM. None of these parameters were affected by postmortem intervals up to 72 h. Immunoblotting of native NMDA receptors showed that NR1, NR2A, NR2C, and NR2D subunits could all be found in the human spinal cord of which NR1 was preferentially located to the dorsal half. Immunoprecipitation of solubilized receptors revealed that NR1, NR2C, and NR2D subunits coprecipitated with the NR2A subunit, indicating that native human spinal cord NMDA receptors are heteroligimeric receptors assembled by at least three different receptor subunits. These results provide a basis for the development of drugs selectively aimed at spinal cord NMDA receptors for the future treatment of spinal cord disorders.     

Electrophysiological studies of NMDA receptors

Recent electrophysiological studies of NMDA receptors and of the associated ion channels (NMDA channels) have revealed five properties of this system: (1) the NMDA channels are blocked by Mg²⁺ in a voltage-dependent way; (2) the NMDA channels are permeable to Ca²⁺ as well as to Na⁺ and K⁺; (3) the NMDA channels may adopt multiple conductance states; some of the minor states (small conductances) resemble the major conductance states opened by non-NMDA agonists; (4) continued exposure to NMDA agonists produces short-term and long-term decreases in the sensitivity of the NMDA system; and (5) glycine potentiates the response to NMDA.     

Effect of cerebral hypoxia on NMDA receptor binding characteristics after treatment with 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) in newborn piglets

Previous studies have shown that hypoxia modifies the NMDA receptor/ion channel complex in cortical brain cell membranes of newborn piglets. The present study tests the hypothesis that blockade of the glutamate recognition site of the NMDA receptor with the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP) prevents modification of the receptor during hypoxia. Twenty seven anesthetized, ventilated newborn piglets were randomized into four groups: 7 normoxic (Nx), 6 CPP-treated normoxic (CPP-Nx), 8 hypoxic (Hx) and 6 CPP-treated hypoxic (CPP-Hx). Treatment groups received CPP 2 mg/kg i.v. The CPP-Hx group received CPP 30 min: prior to hypoxia, which was induced by lowering the FiO₂, to 5–7% for 1 h. Physiologic data showed no change in heart rate, blood pressure, arterial blood gas values, glucose or lactate following CPP administration. During hypoxia there was a significant decrease in PaO₂, pH and an increase in lactate compared to baseline values. The CPP-Hx group had significantly higher lactate levels than the Hx group during hypoxia. P₂ membrane fractions were prepared and thoroughly washed. Characteristics of the NMDA receptor ion channel were determined by [³H]MK-801 binding assays and characteristics of the glutamate recognition site by specific NMDA-displaceable [³H]glutamate binding assays. Brain tissue ATP and PCr levels confirmed tissue hypoxia, and were not preserved by CPP administration. [³H]MK-801 binding assays revealed that CPP treatment attenuated the hypoxia-induced decrease in the number of receptors (B_max) and receptor binding affinity (K_d) during hypoxia. CPP treatment also decreased receptor affinity (increasedK_d) for [³H]MK-801 binding during normoxia and hypoxia. Assays of [³H]glutamate binding revealed that hypoxia decreased both theB_max and the Kd of the NMDA receptor for [³H]glutamate and both were preserved by CPP treatment prior to hypoxia. CPP had no effect on [³H]glutamate B_max or K_d during normoxia. We conclude that hypoxia decreases theB_max andK_d of the NMDA receptor glutamate recognition site for [³H]glutamate and the ion channel site for [³H]MK-801 in newborn piglets. These changes are prevented by CPP administration prior to hypoxia. The different effects of CPP binding during normoxia and hypoxia suggest a use-dependent mechanism for CPP binding during hypoxia, possibly through an hypoxia-induced alteration of the high-affinity binding site for CPP. During both normoxia and hypoxia CPP binding appeared to induce a conformational change in the receptor causing a decrease in binding affinity for [³H]MK-801. CPP administration did not preserve brain tissue ATP or PCr levels during hypoxia and may alter cellular metabolism in addition to its action at the NMDA receptor. However, even with depletion of the energy precursors ATP and PCr, and with higher lactate levels in the CPP-Hx group, CPP was able to maintain NMDA receptor binding characteristics during hypoxia and may decrease excitotoxic cellular damage from hypoxia.     

Adaptations of NMDA and dopamine D2, but not of muscarinic receptors following 14 days administration of uncompetitive NMDA receptor antagonists

Summary. Behavioral changes have previously been reported following administrations of uncompetitive NMDA receptor antagonists memantine, amantadine and MK-801 for 14 days, at the doses that produce plasma levels comparable to those seen in patients (20, 100 and 0.31 mg/kg/day respectively). Using the same doses, the effect on receptor binding (autoradiography) was studied in rats. [³H]MK-801 binding was increased in the dentate gyrus and CA3 region of the hippocampus (35.2 and 24.3% respectively) following 3 days S.C. infusion of memantine by ALZET minipumps. One daily injection of memantine for 14 days, increased [³H]MK-801 binding in the frontal cortex by 40.3%. The same treatment with amantadine did increase [³H]raclopride binding to dopamine D₂ receptors by 13.5%. None of these treatments changed the expression of muscarinic receptors. It is concluded that subchronic blockade of the NMDA receptor by uncompetitive antagonists at moderate (therapeutically-relevant) doses induced only minor changes in NMDA and dopamine D₂ receptor expression. Received September 18, 1998; accepted November 16, 1998     

Increase of intracellular Ca2+ by P2Y but not P2X receptors in cultured cortical multipolar neurons of the rat

The expression and functionality of P2X/P2Y receptor subtypes in multipolar nonpyramidal neurons of mixed cortical cell cultures were investigated by means of immunocytochemistry and fura‐2 microfluorimetry. The morphological studies revealed that most of the neurons are immunoreactive for GABA and express a range of P2X/P2Y receptors, predominantly of the P2X_2,4,6 and P2Y_1,2 subtypes. P2X₁ and P2X₇ receptor immunoreactivity (IR) was found on thin axon‐like processes and presynaptic structures, respectively. Application of ATP caused a small concentration‐dependent increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in most investigated neurons, whereas only about the half of these cells responded to 2′,3′‐O‐(benzoyl‐4‐benzoyl)‐ATP (BzATP), ADPβS, 2MeSADP, or 2MeSATP and even fewer cells to UTP. In contrast, α,β‐meATP, UDP, and UDP‐glucose failed to produce any [Ca²⁺]_i signaling. The response to ATP itself was inhibited by pyridoxal‐5′‐phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS), Reactive Blue 2, 2′‐deoxy‐N⁶‐methyl adenosine 3′,5′‐diphosphate (MRS2179), and suramin (300 μM) as well as by a cyclopiazonic acid‐induced depletion of intracellular Ca²⁺ stores. A Ca²⁺‐free external medium tended to decrease the ATP‐induced [Ca²⁺]_i transients, although this action did not reach statistical significance. Various blockers of voltage‐sensitive Ca²⁺ channels and the gap junction inhibitor carbenoxolone did not interfere with the effect of ATP, whereas a combination of the ionotropic glutamate receptor antagonists D(–)‐2‐amino‐5‐phosphonopentanoic acid (AP5) and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) decreased it. Cross‐desensitization experiments between ADPβS or UTP and ATP suggested that ATP acts on the one hand via P2Y_1,2 receptors and on the other hand by additional signaling mechanisms. These mechanisms may involve the release of glutamate (which in consequence activates ionotropic glutamate receptors) and the entry of Ca²⁺ via store‐operated Ca²⁺ channels. Evidence for the presence of functional P2X receptors, in particular P2X₇, remains elusive. J. Comp. Neurol. 516:343–359, 2009. © 2009 Wiley‐Liss, Inc.     

Excitotoxicity Through NMDA Receptors Mediates Cerebellar Granule Neuron Apoptosis Induced by Prion Protein 90-231 Fragment

Prion diseases recognize, as a unique molecular trait, the misfolding of CNS-enriched prion protein (PrP^C) into an aberrant isoform (PrP^Sc). In this work, we characterize the in vitro toxicity of amino-terminally truncated recombinant PrP fragment (amino acids 90-231, PrP90-231), on rat cerebellar granule neurons (CGN), focusing on glutamatergic receptor activation and Ca²⁺ homeostasis impairment. This recombinant fragment assumes a toxic conformation (PrP90-231^TOX) after controlled thermal denaturation (1 h at 53 °C) acquiring structural characteristics identified in PrP^Sc (enrichment in β-structures, increased hydrophobicity, partial resistance to proteinase K, and aggregation in amyloid fibrils). By annexin-V binding assay, and evaluation of the percentage of fragmented and condensed nuclei, we show that treatment with PrP90-231^TOX, used in pre-fibrillar aggregation state, induces CGN apoptosis. This effect was associated with a delayed, but sustained elevation of [Ca²⁺]_i. Both CGN apoptosis and [Ca²⁺]_i increase were not observed using PrP90-231 in PrP^C-like conformation. PrP90-231^TOX effects were significantly reduced in the presence of ionotropic glutamate receptor antagonists. In particular, CGN apoptosis and [Ca²⁺]_i increase were largely reduced, although not fully abolished, by pre-treatment with the NMDA antagonists APV and memantine, while the AMPA antagonist CNQX produced a lower, although still significant, effect. In conclusion, we report that CGN apoptosis induced by PrP90-231^TOX correlates with a sustained elevation of [Ca²⁺]_i mediated by the activation of NMDA and AMPA receptors.     

NMDA induces a biphasic change in intracellular pH in rat hippocampal slices

As alterations in intracellular pH (pH_i) tend to exert a profound effect on the properties of cells, this study was undertaken to examine NMDA-induced changes in pH_i in rat hippocampal slices using the BCECF fluorescent technique. The ‘resting' pH_i in the CA1 pyramidal cell layers was 6.93±0.07 (mean±S.D., n=72 slices) in 25 mM HCO₃⁻/5% CO₂-buffered solution at 37°C. Exposure of hippocampal slices to NMDA in the range of 10–1000 μM produced a biphasic change in pH_i: an initial transient alkaline shift was followed by a long-lasting acid shift. Dizocilpine (10 μM) but not CNQX (40 μM) blocked the NMDA-induced changes in pH_i. In 0 Ca medium (0 mM Ca²⁺ supplemented 1 mM EGTA, referred to as 0 Ca), pH_i acid shift caused by NMDA (20 μM) declined by about 11%, whereas the initial alkaline shift almost completely disappeared. In an independent experiment, the NMDA-induced increase in intracellular Ca²⁺ ([Ca²⁺]_i) was reduced by more than 80% in 0 Ca medium. Glucose substitution using equimolar pyruvate (as an energy-yielding substrate) suppressed this NMDA-induced pH_i acid shift by two-thirds, while the NMDA-induced pH_i alkaline shift was enhanced. Fluoride (10 mM), a glycolytic inhibitor, abolished NMDA-induced pH_i acid shift. Furthermore, the lactate content of hippocampal slices was markedly increased following exposure to NMDA. In conclusion, activation of NMDA receptors in rat hippocampal slices evokes a biphasic change in pH_i. The initial alkaline shift is suggested to be associated with calcium influx, and the following acid shift may be caused by an increase in lactate production through the acceleration of glycolysis, as well as the increased [Ca²⁺]_i. The pH_i acid shift produced by the increased lactate may contribute to proton modulation of the NMDA receptor and NMDA-induced cell injury or death.     

N-methyl-d-aspartate receptor-mediated,calcium-induced calcium release in rat dentate gyrus/CA4 in vivo

Previously, by using in vivo microdialysis, we demonstrated a huge release of ⁴⁵Ca²⁺ from prelabeled tissues to dialysate that was evoked by application of N-methyl-d-aspartate (NMDA) to the rat dentate gyrus (DG) and sector 4 of the cornu ammonis. To establish the mechanism of this phenomenon, in the present study, we characterized its NMDA receptor dependence, investigated the mechanism of ⁴⁵Ca²⁺ removal from the cells, and evaluated the possible involvement of calcium-binding protein calbindin D_28k and of ryanodine receptors. Microdialysis experiments demonstrated a dose-response relation between NMDA and ⁴⁵Ca²⁺ release and sensitivity of this phenomenon to inhibition by 10 μMK-801 and 5 mm 5-(N,N-dimethyl)-amiloride, thus indicating the NMDA receptor dependence and a role of Na⁺/Ca²⁺ exchanger in mediating ⁴⁵Ca²⁺ release from cells. Immunocytochemical experiments confirmed that DG granule cells in the investigated inbred rat strain are strongly calbindin D_28k-immunopositive, indicating probable involvement of this protein. However, micro-dialysis studies demonstrated that NMDA-evoked ⁴⁵Ca²⁺ release was suppressed by 100 μdantrolene and 250 μryanodine, whereas 50 μryanodine stimulated this effect. This points to a key role in the investigated phenomenon of calcium-induced calcium release (CICR) via ryanodine receptors. To our knowledge, this is the first in vivo demonstration of NMDA-evoked CICR. We postulate the usefulness of microdialysis in such studies.     

Novel glycine-dependent inactivation of NMDA receptors in cultured hippocampal neurons

Objective Glycine acts as a co-agonist for the activation of N-methyl-D-aspartate receptors (NMDARs) by binding to glycine sites, thus potentiating glutamate-elicited responses and inhibiting NMDAR desensitization in a dose-dependent manner. The present study aimed to characterize the glycine-dependent inactivation of NMDARs and to explore its pathophysiological significance. Methods Primary hippocampal cell cultures from embryonic days 17-18 rats were treated with NMDA or NMDA plus glycine. Patch-clamp recording and intracellular Ca 2+ imaging were performed to test the effects of glycine on NMDA-activated currents and increase of intracellular free Ca 2+ respectively. Immunofluorescence staining was conducted to examine NR1 internalization. Cell damage was tested with MTT method and lactate dehydrogenase leakage. Results Glycine reduced the peak current and Ca 2+ influx elicited by NMDA application at concentrations ≥300 μmol/L. This is a novel suppressive influence of glycine on NMDAR function, since it occurs via the NMDAR glycine-binding site, in contrast to the classic suppression, which occurs through the binding of glycine to glycine receptors. The level of membrane NMDARs was measured to evaluate whether internalization was involved. Immunohistochemical labeling showed that incubation with high concentrations of NMDA plus glycine did not change the expression of NMDARs on the cell surface when compared to the expression without glycine; hence the possibility of NMDAR internalization primed by glycine binding was excluded. Conclusion In summary, the novel suppressive effect of glycine on NMDARs was mediated via binding to the glycine site of the NMDAR and not by activation of the strychnine-sensitive glycine-receptor-gated chloride channel or by the internalization of NMDARs. The inhibitory influence of glycine on NMDARs adds a new insight to our knowledge of the complexity of synaptic transmission.     

Selective enhancement by basic fibroblast growth factor of NMDA receptor-mediated increase of intracellular Ca concentration in hippocampal neurons

The short-term effect of bFGF on intracellular Ca²⁺ concentration ([Ca²⁺]_i) of hippocampal neurons was investigated using dissociated cell cultures. Changes in [Ca²⁺]_i were measured by microfluorometrically monitoring the fluorescence intesities from indivudual neurons loaded with fura-2. Perfusion of bFGF (20 ng/ml) alone did not affect the basal level of [Ca²⁺]_i in hippocampal neurons, but clearly enhanced the [Ca²⁺]_i increase induced by NMDA. Quisqualate or KCl-induced [Ca²⁺]_i increase was not influenced by bFGF. These results suggest that bFGF selectively enhances the NMDA receptor-mediated response in hippocampal neurons.     

Synchronized overproduction of AMPA,kainate, and NMDA glutamate receptors during human spinal cord development

Quantitative receptor autoradiography was used to map the distribution in the developing human spinal cord of the three types of ionotropic glutamate receptors. N-methyl-D-Aspartate (NMDA) receptors were labeled with [³H]glutamate, kainic acid (KA) receptors were labeled with [³H]KA, and α-amino-3-hydroxy-5-methyl-4-isoxazole proprionate (AMPA) receptors were labeled with [³H]AMPA. In the adult, labeling of all three receptor subtypes is largely restricted to the substantia gelatinosa (SG) in the dorsal horn, with very low level labeling elsewhere in the spinal gray matter. In marked distinction, in late fetal life, high level ligand binding is seen throughout the spinal gray matter. In early postnatal life, binding sites diminish in all regions, but least so in the SG, until the adult pattern emerges. Thus a coordinated transient high level of ionotropic glutamate receptor expression occurs within the developing spinal cord. Saturation analysis of ligand binding shows that the affinity of [³H]KA and [³H]AMPA binding is not developmentally regulated. In contrast, the affinity of [³H]glutamate binding to the NMDA receptor in the fetal ventral horn is three-fold greater than in the adult ventral horn. Thus, in addition to quantitative changes in glutamate receptor expression, qualitative changes occur in the expression of NMDA receptors during development. The distinct glutamate receptor phenotype of fetal and early postnatal spinal cord cells suggests that alterations in the excitable properties of these cells plays an important role in activity-dependent development and in susceptibility to excitotoxic injury. J. Comp. Neurol. 384:200-210, 1997. © 1997 Wiley-Liss, Inc.     

Localization of ionotropic glutamate receptors in caudate-putamen and nucleus accumbens septi of rat brain: Comparison of NMDA,AMPA, and kainate receptors

Changes in binding of selective radioligands at NMDA ([³H]MK-801), AMPA ([³H]CNQX), and kainate ([³H]kainic acid) glutamate (GLU) ionotropic receptors in rat caudate-putamen (CPu) and nucleus accumbens (NAc) were examined by quantitative autoradiography following: 1) unilateral surgical ablation of frontal cerebral cortex to remove descending corticostriatal GLU projections, 2) unilateral injection of kainic acid (KA) into CPu or NAc to degenerate local intrinsic neurons, or 3) unilateral injections of 6-hydroxydopamine (6-OH-DA) into substantia nigra to degenerate ascending nigrostriatal dopamine (DA) projections. Cortical ablation significantly decreased NMDA receptor binding in ipsilateral medial CPu (20%), and NAc (16%), similar to previously reported losses of DA D₄ receptors. KA lesions produced large losses of NMDA receptor labeling in CPu and NAc (both by 52%), AMPA (41% and 45%, respectively), and kainate receptors (40% and 45%, respectively) that were similar to the loss of D₂ receptors in CPu and NAc after KA injections. Nigral 6-OH-DA lesions yielded smaller but significant losses in NMDA (17%), AMPA (12%), and kainate (11%) receptor binding in CPu. The results indicate that most NMDA, AMPA, and kainate receptors in rat CPu and NAc occur on intrinsic postsynaptic neurons. Also, some NMDA, but not AMPA or kainate, receptors are also found on corticostriatal projections in association with D₄ receptors; these may, respectively, represent excitatory presynaptic NMDA autoreceptors and inhibitory D₄ heteroceptors that regulate GLU release from corticostriatal axons in medial CPu and NAc. Conversely, the loss of all three GLU receptor subtypes after lesioning DA neurons supports their role as excitatory heteroceptors promoting DA release from nigrostriatal neurons. Synapse 30:227–235, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Regulation of GABA release via NMDA and 5-HT1A receptors in guinea pig dentate gyrus

The regulation by N-methyl-d-aspartate (NMDA) and 5-HT_1A receptors of the endogenous γ-aminobutyric acid (GABA) release was investigated in slices of the guinea pig dentate gyrus. The release of GABA was increased in a concentration-dependent fashion by NMDA. The release of GABA evoked by NMDA was Ca²⁺-dependent, tetrodotoxin-resistant, Mg²⁺-sensitive and inhibited by MK-801, a selective non-competitive NMDA receptor antagonist. These results suggest that the NMDA receptor present on GABAergic neurons is involved in the stimulatory regulation of GABA release. The release of GABA was increased concentration-dependently by NAN-190, a 5-HT_1A receptor antagonist, but was not affected by 8-OH-DPAT, a 5-HT_1A receptor agonist. The release of GABA evoked by NAN-190 was Ca²⁺-dependent, tetrodotoxin-resistant and inhibited by 8-OH-DPAT. These results suggest that the 5-HT_1A receptor present on GABAergic neurons is involved in the inhibitory regulation of GABA release. The release of GABA evoked by NMDA from the dentate gyrus was inhibited by pretreatment with 8-OH-DPAT. The release of GABA evoked by NAN-190 was inhibited by pretreatment with MK-801. The release of GABA evoked by NMDA from the dentate gyrus was augmented by the concurrent application of NAN-190. Taken together, the results indicate that the NMDA receptor and the 5-HT_1A receptor, which are both located on GABAergic neurons in the guinea pig dentate gyrus, exert stimulatory and inhibitory regulation of neuronal GABA release, respectively.     

β-Amyloid-Induced Cholinergic Denervation Correlates with Enhanced Nitric Oxide Synthase Activity in Rat Cerebral Cortex: Reversal by NMDA Receptor Blockade

Ample experimental evidence indicates that acute β-amyloid infusion into the nucleus basalis of rats elicits abrupt degeneration of the magnocellular cholinergic neurons projecting to the cerebral cortex. In fact, involvement of a permanent Ca²⁺ overload, partially via N-methyl-

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-aspartate (NMDA) receptors, was proposed as a pivotal mechanism in β-amyloid-induced neurodegeneration. A definite measure of NMDA receptor-mediated processes and subsequent Ca²⁺ entry is the induction of Ca²⁺/calmodulin-activated neuronal nitric oxide synthase (nNOS) in nerve cells. In the present account we therefore assessed activation of nNOS in correlation with cholinergic decline after β-amyloid_(1–42) or β-amyloid_(25–35) infusion into the rat nucleus basalis. The results demonstrate the β-amyloid conformation-dependent enhancement of cortical nitric oxide synthase (NOS) activity. Furthermore, chronic application of the polyamine site NMDA receptor blocker ifenprodil effectively attenuated β-amyloid neurotoxicity. We propose that nNOS activation reflects the degree of β-amyloid-induced excitotoxic injury in a proportional manner. Moreover, Ca²⁺-mediated processes via NMDA receptors, or direct binding of β-amyloid to this receptor may be a critical step in the neurotoxic mechanisms in vivo.     

Stimulation of NMDA and AMPA glutamate receptors elicits distinct concentration dynamics of nitric oxide in rat hippocampal slices

Nitric oxide (^?NO) is an intercellular messenger implicated in memory formation and neurodegeneration in the hippocampus. Owing to its physical and chemical properties, the concentration dynamics of ^?NO is a critical issue in determining its bioactivity as a signaling molecule. Its production is closely related to glutamate N‐methyl‐D ‐aspartate (NMDA) receptors, following a rise in intracellular calcium levels. However, that dependent on α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) receptors remains elusive and controversial, despite reports describing a role for these receptors in other brain regions, largely because of lack of quantitative and dynamic measurements of ^?NO. Using a ^?NO‐selective microsensor inserted in the diffusional spread of ^?NO in the CA1 region of rat hippocampal slices, we measured its real‐time endogenous production, following activation of ionotropic glutamate receptors and under tissue physiological oxygen tension. Both NMDA and AMPA stimulation resulted in a concentration‐dependent ^?NO production but encompassing distinct kinetics for lag phases and slower rates of ^?NO production were observed for AMPA stimulation. Robustness of the results was achieved instrumentally and pharmacologically, by means of nitric oxide synthase (NOS) inhibitors and antagonists of NMDA (D ‐(?)‐2‐amino‐5‐phosphonopentanoic acid, AP5) and AMPA (2,3‐dioxo‐6‐nitro‐1,2,3,4‐tetrahydrobenzo[f]quinoxaline‐7‐sulfonamide, NBQX) receptors. When using glutamate as a stimulus, ^?NO production was of lower magnitude in the presence of AP5 plus NBQX than with AP5 alone, suggesting that even when NMDA receptors are inhibited Ca²⁺ rises to levels to induce a peak of ^?NO from the background. Whereas extracellular Ca²⁺ was required for the ^?NO signals, Philanthotoxin‐4,3,3 (PhTX‐4,3,3) a toxin used to target Ca²⁺‐permeable AMPA receptors, attenuated ^?NO production. These observations are interpreted on basis of a distinct coupling between the glutamate receptors and neuronal NOS. A role for Ca²⁺‐permeable AMPA receptors in the Ca²⁺ activation of neuronal NOS is suggested. © 2008 Wiley‐Liss, Inc.     

Role of nonsynaptic GluN2B-containing NMDA receptors in excitotoxicity: Evidence that fluoxetine selectively inhibits these receptors and may have neuroprotective effects

In acute ischaemic brain injury and chronic neurodegeneration, the primary step leading to excitotoxicity and cell death is the excessive and/or prolonged activation of glutamate (Glu) receptors, followed by intracellular calcium (Ca²⁺) overload. These steps lead to several effects: a persistent depolarisation of neurons, mitochondrial dysfunction resulting in energy failure, an increased production of reactive oxygen species (ROS), an increase in the concentration of cytosolic Ca²⁺ [Ca²⁺]_i, increased mitochondrial Ca²⁺ uptake, and the activation of self-destructing enzymatic mechanisms. Antagonists for NMDA receptors (NMDARs) are expected to display neuroprotective effects, but no evidence to support this hypothesis has yet been reported. A number of clinical trials using NMDAR antagonists have failed to demonstrate neuroprotective effects, either by reducing brain injury or by preventing neurodegeneration. Recent advances in NMDAR research have provided an explanation for this phenomenon. Synaptic and extrasynaptic NMDARs are composed of different subunits (GluN2A and GluN2B) that demonstrate opposing effects. Synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs have different co-agonists: d-serine for synaptic NMDARs and glycine for extrasynaptic NMDARs. Both co-agonists are of glial origin.The mechanisms of cell destruction or cell survival in response to the activation of NMDAR receptors depend in part on [Ca²⁺]_i and the route of entry of this ion and more significantly on the subunit composition and localisation of the NMDARs. While synaptic NMDAR activation is involved in neuroprotection, the stimulation of extrasynaptic NMDARs, which are composed of GluN2B subunits, triggers cell destruction pathways and may play a key role in the neurodegeneration associated with Glu-induced excitotoxicity. In addition, it has been found that synaptic and extrasynaptic NMDA receptors have opposing effects in determining the fate of neurons. This result has led to the targeting of nonsynaptic GluN2B-containing NMDARs as promising candidates for drug research. Under hypoxic conditions, it is likely that the failure of synaptic glutamatergic transmission, the impairment of the GluN2A-activated neuroprotective cascade, and the persistent over-activation of extrasynaptic GluN2B-containing NMDARs lead to excitotoxicity. Fluoxetine, a drug widely used in clinical practice as an antidepressant, has been found to selectively block GluNR2B-containing NMDARs. Therefore, it seems to be a potential candidate for neuroprotection.     

GFAP-positive hippocampal astrocytes in situ respond to glutamatergic neuroligands with increases in [Ca2+]i

It is becoming increasingly clear that astrocytes play very dynamic and interactive roles that are important for the normal functioning of the central nervous system. In culture, astrocytes express many receptors coupled to increases in intracellular calcium ([Ca²⁺]_i). In vivo, it is likely that these receptors are important for the modulation of astrocytic functions such as the uptake of neurotransmitters and ions. Currently, however, very little is known about the expression or stimulation of such astrocytic receptors in vivo. To address this issue, confocal microscopy and calcium sensitive fluorescent dyes were used to examine the dynamic changes in astrocytic [Ca²⁺]_i, within acutely isolated hippocampal slices. Astrocytes were subsequently identified by immunocytochemistry for glial fibrillary acidic protein. In this paper, we present data indicating that hippocampal astrocytes in situ respond to glutamate, kainate, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), N-methyl-D-aspartate (NMDA), and depolarization with increases in [Ca²⁺]_i. The increases in [Ca²⁺]_i occurred in both the astrocytic cell bodies and the processes. Temporally the changes in [Ca²⁺]_i were very dynamic, and various patterns ranging from sustained elevations to oscillations of [Ca²⁺]_i were observed. Individual astrocytes responded to neuroligands selective for both ionotropic and metabotropic glutamate receptors with increases in [Ca²⁺]_i. These findings indicate that astrocytes in vivo contain glutamatergic receptors coupled to increases in [C²⁺]_i and are able to respond to neuronally released neurotransmitters. (c) 1995 Wiley-Liss, Inc.