Involvement of ionotropic glutamate receptors in low frequency electroacupuncture analgesia in rats

The present study was conducted to determine whether blockage of both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid/kainate (AMPA/KA) receptors influences the induction of low frequency electroacupuncture (EA) analgesia. Although neither intrathecal injection of NMDA antagonist D-2-amino-5-phosphonopentanoic acid (D-AP-5) or AMPA/KA antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonami-de (NBQX) disodium alone had an effect on analgesia, spinal application of D-AP-5 and NBQX disodium significantly prevented analgesia induced by 2 Hz EA. The intrathecal injection of the excitatory amino acid NMDA produced analgesia for several minutes after intrathecal injection, as did EA stimulation. These results suggest that ionotropic glutamate receptors may be involved in the induction of 2 Hz EA analgesia.     

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated stimulation of glutamate and GABA release in the rat striatum in vivo: a dual-label microdialysis study

The existence of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate autoreceptors on glutamate nerve terminals in vitro has recently been demonstrated using synaptosomal and brain slice preparations. In the present study we have used a modification of a rapid dual-label intracerebral microdialysis method, previously developed by Young and co-workers(80,81) for the study of presynaptic mechanisms of neurotransmitter release, to investigate whether presynaptic AMPA receptors also play a role in the control of striatal glutamate release in vivo. For comparative purposes, the action of locally applied AMPA on striatal GABA release in vivo was also monitored. Local application of AMPA (0.01-100 microM), by reverse dialysis, into the striatum resulted in concentration-dependent increases in the Ca(2+)-dependent efflux of both [3H]L-glutamate and [14C]GABA. Maximum responses reached 142.0+/-6.5% and 166.8+/-7.7% of basal efflux for [3H]L-glutamate and [14C]GABA, respectively. No marked behavioural changes were observed at any dose of the agonist. Unexpectedly, the AMPA-evoked responses were not potentiated by the AMPA receptor desensitization inhibitors cyclothiazide (10-100microM) or aniracetam (1mM). Consistent with this finding, AMPA-stimulated [3H]L-glutamate and [14C]GABA efflux were significantly attenuated by co-perfusion with the selective, competitive AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo(F)quinoxaline-2,3-dione (100microM) but not 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (100microM), a non-competitive AMPA receptor antagonist known to interact with the cyclothiazide site to control AMPA receptor function. The broad spectrum ionotropic glutamate receptor antagonist, kynurenic acid (100-1000microM) also markedly inhibited the AMPA-evoked responses in the striatum in vivo. None of the antagonists, when given alone, influenced basal efflux of [3H]L-glutamate suggesting a lack of tonic regulatory control of glutamate release via presynaptic AMPA-type autoreceptors in the rat striatum. These results demonstrate the presence of presynaptic AMPA receptors, of a novel cyclothiazide- and aniracetam-insensitive subtype, on presynaptic nerve terminals in the rat striatum in vivo, acting to enhance glutamate and GABA release. Our data support the concept of AMPA receptor heterogeneity in vivo, a finding which may facilitate the development of novel, more selective drugs for the treatment of a range of neurological disorders associated with abnormal cerebral glutamate release. The pharmacological profile of these novel presynaptic receptors is currently under investigation.     

Presynaptic ionotropic glutamate receptors modulate in vivo release and metabolism of striatal dopamine,noradrenaline, and 5-hydroxytryptamine: involvement of both NMDA and AMPA/kainate subtypes

In order to explore further the presynaptic modulation of monoamine release by glutamatergic nerve fibers, we investigated the effects of selective agonists for ionotropic glutamate (GLU) receptors on striatal release of dopamine (DA), noradrenaline (NA) and 5-hydroxytryptamine (5-HT). In the striatum of anesthetized Sprague-Dawley rats, in vivo microdialysis was performed to measure the release of monoamines and metabolites, and also to administer GLU agonists locally in the tissue. l-GLU and its selective agonists (N-methyl-d-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and kainate (KA)) evoked simultaneous release of striatal DA, NA and 5-HT in a dose-dependent manner. Pretreatment with MK-801 (5 mg/kg i.p.), a noncompetitive NMDA receptor antagonist, selectively suppressed NMDA-evoked monoamine release. The rank order of GLU agonist efficacy in releasing monoamines was different among DA, NA, and 5-HTergic terminals: AMPA = KA > NMDA for DA release, AMPA > NMDA = KA for NA release, and NMDA = AMPA = KA for 5-HT release. In conclusion, presynaptic ionotropic GLU receptors exist extensively on monoaminergic terminals including not only catecholaminergic (DA and NA) but also indoleaminergic (5-HT) terminals in the rat striatum. Their subtypes include both NMDA subtype and AMPA/KA subtype, and show a differential distribution among these three monoaminergic terminals and a differential contribution to facilitating monoamine release.     

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes)

Ionotropic glutamate receptor agonists, kainate, -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl- -aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release.

We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.     

7-硝基吲唑对大鼠大脑皮质离子型谷氨酸受体和GABAA受体的影响

目的　探讨一氧化氮 (NO)对大鼠脑皮质离子型谷氨酸受体 (NMDA、AMPA、KA受体 )和 GABA受体的影响。　方法　将 Wistar大鼠腹腔注射神经细胞结构型一氧化氮合酶抑制剂 7-硝基吲唑 ,以氚标配体分别标记 NMDA、AMPA、KA和 GABAA 受体 ,用图像分析仪对大鼠额区、顶区、后肢区、梨状区、压部后区和味觉区皮质内标记受体进行定量分析。　结果　实验组大鼠额区、后肢区和梨状区内 NMDA、AMPA、KA受体和 GABAA 受体含量均显著增加 ;顶区皮质内 NMDA、KA受体和 GABAA 受体增加显著 ;味觉区皮质内 KA受体增加显著。　结论　 NO可能参与大鼠脑皮质离子型谷氨酸受体和 GABA受体水平的调节     

Properties of miniature glutamatergic EPSCs in neurons of the locomotor regions of the developing zebrafish

As a first step in understanding the development of synaptic activation in the locomotor network of the zebrafish, we examined the properties of spontaneous, glutamatergic miniature excitatory postsynaptic currents (mEPSCs). Whole cell patch-clamp recordings were obtained from visually identified hindbrain reticulospinal neurons and spinal motoneurons of curarized zebrafish 1-5 days postfertilization (larvae hatch after the 2nd day of embryogenesis). In the presence of tetrodotoxin (TTX) and blockers of inhibitory receptors (strychnine and picrotoxin), we detected fast glutamatergic mEPSCs that were blocked by the AMPA/kainate receptor-selective antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At positive voltages or in the absence of Mg(2+), a second, slower component of the mEPSCs was revealed that the N-methyl-D-aspartate (NMDA) receptor-selective antagonist DL-2-amino-5-phosphonovalerate (AP-5) abolished. In the presence of both CNQX and AP-5, all mEPSCs were eliminated. The NMDA component of reticulospinal mEPSCs had a large single-channel conductance estimated to be 48 pS. Larval AMPA/kainate and NMDA components of the mEPSCs decayed with biexponential time courses that changed little during development. At all stages examined, approximately one-half of synapses had only NMDA responses (lacking AMPA/kainate receptors), whereas the remainder of the synapses were composed of a mixture of AMPA/kainate and NMDA receptors. There was an overall increase in the frequency and amplitude of mEPSCs with an NMDA component in reticulospinal (but not motoneurons) during development. These results indicate that glutamate is a prominent excitatory transmitter in the locomotor regions of the developing zebrafish and that it activates either NMDA receptors alone at functionally silent synapses or together with AMPA/kainate receptors.     

Dynorphin A (1-17) induces apoptosis in striatal neurons in vitro through alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor-mediated cytochrome c release and caspase-3 activation

Dynorphin A (1-17), an endogenous opioid neuropeptide, can have pathophysiological consequences at high concentrations through actions involving glutamate receptors. Despite evidence of excitotoxicity, the basic mechanisms underlying dynorphin-induced cell death have not been explored. To address this question, we examined the role of caspase-dependent apoptotic events in mediating dynorphin A (1-17) toxicity in embryonic mouse striatal neuron cultures. In addition, the role of opioid and/or glutamate receptors were assessed pharmacologically using dizocilpine maleate (MK(+)801), a non-equilibrium N-methyl-D-aspartate (NMDA) antagonist; 6-cyano-7-nitroquinoxaline-2,3-dione, a competitive alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate antagonist; or (-)-naloxone, a general opioid antagonist. The results show that dynorphin A (1-17) (>or=10 nM) caused concentration-dependent increases in caspase-3 activity that were accompanied by mitochondrial release of cytochrome c and the subsequent death of cultured mouse striatal neurons. Moreover, dynorphin A-induced neurotoxicity and caspase-3 activation were significantly attenuated by the cell permeable caspase inhibitor, caspase-3 inhibitor-II (z-DEVD-FMK), further suggesting an apoptotic cascade involving caspase-3. AMPA/kainate receptor blockade significantly attenuated dynorphin A-induced cytochrome c release and/or caspase-3 activity, while NMDA or opioid receptor blockade typically failed to prevent the apoptotic response. Last, dynorphin-induced caspase-3 activation was mimicked by the ampakine CX546 [1-(1,4-benzodioxan-6-ylcarbonyl)piperidine], which suggests that the activation of AMPA receptor subunits may be sufficient to mediate toxicity in striatal neurons. These findings provide novel evidence that dynorphin-induced striatal neurotoxicity is mediated by a caspase-dependent apoptotic mechanism that largely involves AMPA/kainate receptors.     

Activation of ionotropic glutamate receptors on peripheral axons of primary motoneurons mediates transmitter release at the zebrafish NMJ

The development and function of the vertebrate neuromuscular junction (NMJ) is continually being redefined. Previous studies have indicated that glutamate may play a role in the development or function of the NMJ by associating with presynaptic receptors. We have used larval zebrafish (Danio rerio) to investigate the presence of presynaptic ionotropic glutamate receptors (iGluRs) at the NMJ in vivo. In whole-mount zebrafish larvae, antibody staining directed to NR2A subunits colocalized with specific staining of motoneuron axon tracts. Whole cell voltage-clamp recordings of miniature endplate currents (mEPCs) from axial white muscle were performed during application of iGluR agonists and antagonists. Local perfusion of the NMJ with iGluR agonists resulted in significant increases in the frequency of spontaneous acetylcholine (ACh) release. These increases were blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (50 microM) and by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxalene-2,3-dione (50 microM). Further pharmacological investigation revealed no effect of the kainate receptor-specific antagonist (2S,4R)-4-methylglutamate (10 microM) on kainate-induced rises in the frequency of spontaneous ACh release. However, these were blocked with the AMPA receptor-specific antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (50 microM). Application of glutamate (1 mM) in the presence of the glutamate uptake inhibitor d-threo-beta-benzyloxyaspartate(200 microM) resulted in a significant increase in the frequency of mEPCs. These results suggest the presence of AMPA and NMDA receptors in association with motoneuron axons of larval zebrafish.     

近交系小鼠海马结构兴奋性氨基酸受体密度的变化(英文)

应用定量放射自显影技术，以氚标配体MK－801、AMPA和KA分别标记NMDA、AMPA和KA受体，定量观察了七种近交系小鼠海马结构内兴奋性氨基酸受体的区域分布类型和受体密度变化．结果表明，海马CA1区含有高密度NMDA和AMPA受体，CA3和齿状回含高密度KA受体。三种受体在海马各区的分布类型无明显系间差异，而其受体密度存在着明显的系间差别。NMDA和AMPA受体在BALB.c鼠海马显示最高密度，而CPB-K鼠以上两种受作密度最低。KA受体在NMRI鼠海马显示最高密度，BA鼠其受体密度最低．这种受体密度的系间差异可能与近交系小鼠合成兴奋性氨基酸受体的基因调控不同有关。     

Transient postnatal increases in excitatory amino acid binding sites in rat ventral mesencephalon.

The developmental profile of three sub-types of excitatory amino acid (EAA) binding sites was determined in the ventral mesencephalon and the striatum of rats from prenatal day 19 to adult (3 months) using membrane binding assays. In the ventral mesencephalon, there was a transient increase of EAA receptor binding sites beyond adult levels, which peaked at postnatal day 7 (P7) for [3H]glutamate binding to NMDA receptors and at P14 for [3H]AMPA and [3H]kainate binding. In the striatum, [3H]glutamate/NMDA and [3H]kainate binding reached adult levels during the early postnatal period, stabilizing at this level with no transient overexpression beyond adult levels. [3H]AMPA binding also showed an increase above adult levels at P14 in the striatum. These results raise the possibility that the transient overexpression of EAA receptors in the ventral mesencephalon may affect the developmental fate of dopaminergic and other neurons in this region.     

Localization of ionotropic glutamate receptors in peripheral axons of human skin

Recent observations suggest that glutamate is important in sensory transduction in the periphery, contributing to peripheral sensitization of nociceptors and the hyperalgesia that accompanies inflammation. This study examined the presence of ionotropic glutamate receptors N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazolone-4-propionic acid (AMPA) and kainate (KA) in normal human hairy skin (n=6) using immunohistochemistry at the electron microscopic level. Analysis of labeled axons at the dermal-epidermal junction demonstrated that 26. 9+/-2, 19.5+/-3 and 18.5+/-1% of the axons analyzed were labeled for subunits of the NMDA, AMPA or KA receptors, respectively. An occasional Schwann cell process was labeled for either NMDA or KA receptors. The findings support the hypothesis that glutamate and its ionotropic receptors may play a role in the periphery in sensory processing in humans.     

Endogenous glutamatergic synaptic activity elicits acetylcholine release from rat cultured septal cells

We tested the characteristics of acetylcholine (ACh) release from cultured rat septal cells. The spontaneous release was inhibited by treatment with tetrodotoxin (TTX) and omega-conotoxin (GVIA), indicating that the release was elicited by synaptic activity. The release was also inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor blocker, in both the absence and presence of nerve growth factor (NGF), suggesting that endogenously released glutamate produced the ACh release by stimulating AMPA receptors.This is the first report of detection of the release of ACh by endogenous spontaneous synaptic activity conducted by glutamate AMPA receptor activation in cultured septal cells. This in vitro experimental system is useful for the study of cholinergic functions.     

L-glutamate-evoked release of dopamine from synaptosomes of the rat striatum: involvement of AMPA and N-methyl-D-aspartate receptors.

Previously, using purified synaptosomes from the rat striatum, we have shown that agonists of D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors stimulate the release of [3H]dopamine continuously synthesized from [3H]tyrosine. Similar results were obtained with N-methyl-D-aspartate in the absence of magnesium. In the present study, using the same approach, attempts were made to determine whether in the presence of magnesium, the combined stimulation of AMPA receptors allows us to demonstrate the presynaptic facilitation of [3H]dopamine release through N-methyl-D-aspartate receptors. L-Glutamate (10(-3) M) markedly stimulated the release of [3H]dopamine from synaptosomes, this effect being about twice that found with AMPA (10(-3) M) while N-methyl-D-aspartate (10(-3) M) even in the presence of glycine (10(-6) M) was ineffective. In agreement with previous results, a stimulatory effect of N-methyl-D-aspartate and glycine was only observed in the absence of magnesium. This response was blocked by 6,7-dinitro-quinoxaline-2,3-dione (3 x 10(-5) M), confirming that this compound, generally used as an AMPA antagonist, also blocks N-methyl-D-aspartate receptors. The AMPA (10(-3) M)-evoked release of [3H]dopamine was markedly potentiated by the combined application of N-methyl-D-aspartate (10(-3) M) and glycine (10(-6) M) in the presence of strychnine, indicating that the concomitant activation of AMPA receptors removes the voltage-dependent magnesium block of N-methyl-D-aspartate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synaptic pathways that shape the excitatory drive in an OFF retinal ganglion cell

Different types of retinal ganglion cells represent distinct spatiotemporal filters that respond selectively to specific features in the visual input. Much about the circuitry and synaptic mechanisms that underlie such specificity remains to be determined. This study examines how N-methyl-d-aspartate (NMDA) receptor signaling combines with other excitatory and inhibitory mechanisms to shape the output of small-field OFF brisk-sustained ganglion cells (OFF-BSGCs) in the rabbit retina. We used voltage clamp to separately resolve NMDA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and inhibitory inputs elicited by stimulation of the receptive field center. Three converging circuits were identified. First is a direct glutamatergic input, arising from OFF cone bipolar cells (CBCs), which is mediated by synaptic NMDA and AMPA receptors. The NMDA input was saturated at 10% contrast, whereas the AMPA input increased monotonically up to 60% contrast. We propose that NMDA inputs selectively enhance sensitivity to low contrasts. The OFF bipolar cells, mediating this direct excitatory input, express dendritic kainate (KA) receptors, which are resistant to the nonselective AMPA/KA receptor antagonist, 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt (NBQX), but are suppressed by a GluK1- and GluK3-selective antagonist, (S)-1-(2-amino-2-carboxyethyl)-3-(2-carboxy-thiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione (UBP-310). The second circuit entails glycinergic crossover inhibition, arising from ON-CBCs and mediated by AII amacrine cells, which modulate glutamate release from the OFF-CBC terminals. The third circuit also comprises glycinergic crossover inhibition, which is driven by the ON pathway; however, this inhibition impinges directly on the OFF-BSGCs and is mediated by an unknown glycinergic amacrine cell that expresses AMPA but not KA receptors.     

Trans-2-carboxy-3-pyrrolidineacetic acid (CPAA), a novel agonist at NMDA-type receptors

Although 2-carboxy-3-pyrrolidineacetic acid (CPAA) analogs are associated with activity as agonists at kainate-type receptors, here we report that CPAA is an agonist at N-methyl-D-aspartic acid-type receptors. CPAA evoked the release of [3H]acetylcholine (ACh) from striatal slices with the same efficacy as NMDA, and an EC50 of 20.0 microM, compared to an EC50 of 45.8 microM for NMDA. CPAA-evoked [3H]ACh release was inhibited by CPP (IC50 = 5.1 microM), tiletamine (IC50 = 0.53 microM), MK-801 (IC50 = 0.12 microM), and MgCl2 (IC50 = 26 microM). CPAA produced a tachyphylaxis when applied continuously for 18 min or more, and a cross-tachyphylaxis to NMDA. Similarly, NMDA generated a cross-tachyphylaxis to CPAA. All of these data suggest that CPAA is an agonist at NMDA-type receptors.     

Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex

The atypical antipsychotic drug clozapine effectively alleviates both negative and positive symptoms of schizophrenia via unclear cellular mechanisms. Clozapine may modulate both glutamatergic and dopaminergic transmission in the prefrontal cortex (PFC) to achieve part of its therapeutic actions. Using whole cell patch-clamp techniques, current-clamp recordings in layers V-VI pyramidal neurons from rat PFC slices showed that stimulation of local afferents (in 2 microM bicuculline) evoked mixed [AMPA/kainate and N-methyl-D-aspartate (NMDA) receptors] glutamate receptor-mediated excitatory postsynaptic potentials (EPSPs). Clozapine (1 microM) potentiated polysynaptically mediated evoked EPSPs (V(Hold) = -65 mV), or reversed EPSPs (rEPSP, V(Hold) = +20 mV) for >30 min. The potentiated EPSPs or rEPSPs were attenuated by elevating [Ca(2+)](O) (7 mM), by application of NMDA receptor antagonist 2-amino5-phosphonovaleric acid (50 microM), or by pretreatment with dopamine D1/D5 receptor antagonist SCH23390 (1 microM) but could be further enhanced by a dopamine reuptake inhibitor bupropion (1 microM). Clozapine had no significant effect on pharmacologically isolated evoked NMDA-rEPSP or AMPA-rEPSPs but increased spontaneous EPSPs without changing the steady-state resting membrane potential. Under voltage clamp, clozapine (1 microM) enhanced the frequency, and the number of low-amplitude (5-10 pA) AMPA receptor-mediated spontaneous EPSCs, while there was no such changes with the mini-EPSCs (in 1 microM TTX). Taken together these data suggest that acute clozapine can increase spike-dependent presynaptic release of glutamate and dopamine. The glutamate stimulates distal dendritic AMPA receptors to increase spontaneous EPSCs and enabled a voltage-dependent activation of neuronal NMDA receptors. The dopamine released stimulates postsynaptic D1 receptor to modulate a lasting potentiation of the NMDA receptor component of the glutamatergic synaptic responses in the PFC neuronal network. This sequence of early synaptic events induced by acute clozapine may comprise part of the activity that leads to later cognitive improvement in schizophrenia.     

Noradrenaline- and glutamate-induced taurine release from bulk isolated adult rat astrocytes.

The influence of noradrenaline and various agonists of glutamatergic receptors on preloaded [3H]taurine release from bulk isolated adult rat brain astrocytes was investigated by a superfusion technique. In the presence of 1 mM noradrenaline a stimulation of taurine release, resembling that observed in astroglial cultures, was preceded by an inhibition of the efflux, thus demonstrating different dynamics of noradrenaline-evoked taurine release from that observed with beta-agonists on cultured astroglia. Application of 1 mM glutamate and kainate produced stimulation of the release, while 1 mM N-methyl-D-aspartate (NMDA) and 1 mM NMDA together with 65 mM K+ had no effect on the [3H]taurine release. These data suggest the presence of kainate-sensitive and the absence of NMDA-sensitive glutamate receptors on bulk isolated astrocytes, which is consistent with previous observations on astrocytes in culture.     

Decreased dendrite growth from cultured mouse cortical neurons surviving excitotoxic activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptors

During cerebral ischemia, massive glutamate release leads to cell death through ionotropic glutamate receptor activation. An early consequence of this excitotoxicity is dendrite injury, which can precede cell death. We therefore tested whether cells that survived an excitotoxic insult triggered by overactivation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate (KA) subtype of ionotropic glutamate receptors displayed altered dendrite growth. We demonstrate that 24 h exposure of cultured cortical neurons to AMPA or KA dramatically reduced dendrite growth from surviving neurons. AMPA or KA exposure decreased primary dendrite number and length, and also reduced dendritic branching. The AMPA/KA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked the deleterious effect of AMPA and KA on dendrite growth. These results suggest that AMPA/KA receptor overactivation may contribute to dendritic injury from neurons that survive an ischemic insult.     

Autoradiographic distribution of binding sites for the non-NMDA receptor antagonist CNQX in chick brain

The anatomical distribution of binding sites for the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor antagonist [3H]6-cyano-7-nitroquinoxaline-2,3-dione ([3H]CNQX) in 1-day-old chick brain was investigated. Specific [3H]CNQX binding sites were widely distributed but were particularly densely localised in the molecular layer of the cerebellum. In the midbrain, the binding was comparatively low, except for relatively high levels in the nucleus isthmi of the optic lobe. The distribution of [3H]CNQX binding was markedly different to that of the NMDA receptor ligand [3H]MK-801. Overall, the localisation of [3H]CNQX binding was similar to the combined distributions of [3H]AMPA and [3H]kainate binding sites. Kainate inhibited [3H]CNQX binding throughout the brain. AMPA also inhibited [3H]CNQX binding in the fore- and midbrain, but the dense binding in the molecular layer of the cerebellum was notable in being AMPA-insensitive.     

GABAA and strychnine-sensitive glycine receptors modulate N-methyl-D-aspartate-evoked acetylcholine release from rat spinal motoneurons: a possible role in neuroprotection

Increasing experimental and clinical evidence suggests that abnormal glutamate transmission might play a major role in a vast number of neurological disorders. As a measure of glutamatergic excitation, we have studied the acetylcholine (ACh) release induced by N-methyl-d-aspartate (NMDA) receptor stimulation in primary cultured rat ventral horn spinal neurons and we have evaluated the possibility to limit the consequences of the hyperactivation of glutamatergic receptors, by recruiting the inhibitory transmission mediated by GABA and glycine. For this purpose, we have exposed cell cultures, previously loaded with [(3)H]choline, to NMDA, which increased the spontaneous tritium efflux in a concentration-dependent manner. Tritium release is dependent upon external Ca(2+), tetrodotoxin, Cd(2+) ions and omega-conotoxin GVIA, but not on omega-conotoxin MVIIC nor nifedipine, suggesting the involvement of N-type voltage-sensitive calcium channels. NMDA-mediated [(3)H]ACh release was completely prevented by MK-801, 5,7-diclorokynurenic acid and ifenprodil, while it was strongly inhibited by a lower external pH, suggesting that the involved NMDA receptors contain NR1 and NR2B subunits. Muscimol inhibited NMDA-evoked [(3)H]ACh release and its effect was antagonized by SR95531 and potentiated by diazepam, indicating the involvement of benzodiazepine-sensitive GABA(A) receptors. Also glycine, via strychnine-sensitive receptors, inhibited the effect of NMDA. It is concluded that glutamate acts on the NMDA receptors situated on spinal motoneurons to evoke ACh release, which can be inhibited through the activation of GABA(A) and glycine receptors present on the same neurons. These data suggest that glutamatergic overload of receptors located onto spinal cord motoneurons might be decreased by activating GABA(A) and glycine receptors.