Trafficking of presynaptic AMPA receptors mediating neurotransmitter release: neuronal selectivity and relationships with sensitivity to cyclothiazide

Postsynaptic glutamate AMPA receptors (AMPARs) can recycle between plasma membrane and intracellular pools. In contrast, trafficking of presynaptic AMPARs has not been investigated. AMPAR surface expression involves interactions between the GluR2 carboxy tail and various proteins including glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), protein interacting with C kinase 1 (PICK1), N-ethyl-maleimide-sensitive fusion protein (NSF). Here, peptides known to selectively block the above interactions were entrapped into synaptosomes to study the effects on the AMPA-evoked release of [3H]noradrenaline ([3H]NA) and [3H]acetylcholine ([3H]ACh) from rat hippocampal and cortical synaptosomes, respectively. Internalization of pep2-SVKI to prevent GluR2-GRIP/ABP/PICK1 interactions potentiated the AMPA-evoked release of [3H]NA but left unmodified that of [3H]ACh. Similar potentiation was caused by pep2-AVKI, the blocker of GluR2-PICK1 interaction. Conversely, a decrease in the AMPA-evoked release of [3H]NA, but not of [3H]ACh, was caused by pep2m, a selective blocker of the GluR2-NSF interaction. In the presence of pep2-SVKI the presynaptic AMPARs on noradrenergic terminals lost sensitivity to cyclothiazide. AMPARs releasing [3H]ACh, but not those releasing [3H]NA, were sensitive to spermine, suggesting that they are GluR2-lacking AMPARs. To conclude: (i) release-regulating presynaptic AMPARs constitutively cycle in isolated nerve terminals; (ii) the process exhibits neuronal selectivity; (iii) AMPAR trafficking and desensitization may be interrelated.     

Aniracetam, 1-BCP and cyclothiazide differentially modulate the function of NMDA and AMPA receptors mediating enhancement of noradrenaline release in rat hippocampal slices

Aniracetam, 1-(1,3-benzodioxol-5-yl-carbo-nyl)piperidine (1-BCP) and cyclothiazide, three compounds considered to enhance cognition through modulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, were evaluated in the ‘kynurenate test’, a biochemical assay in which some nootropics have been shown to prevent the antagonism by kynurenic acid of the N-methyl-d-aspartate (NMDA)-evoked [³H]noradrenaline ([³H]NA) release from rat hippocampal slices. Aniracetam attenuated the kynurenate (100 μM) antagonism of the [³H]NA release elicited by 100 μM NMDA with high potency (EC₅₀≤0.1 μM). Cyclothiazide and 1-BCP were about 10 and 100 times less potent than aniracetam, respectively. The effect of aniracetam persisted in the presence of the AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) added at 5 μM, a concentration that did not affect NMDA receptors; in contrast, NBQX reduced the effect of 1-BCP and abolished that of cyclothiazide. The AMPA-evoked release of [³H]NA from hippocampal slices or synaptosomes was enhanced by cyclothiazide, less potently by 1-BCP and weakly by aniracetam. High concentrations of kynurenate (1 mM) antagonized the AMPA-evoked [³H]NA release in slices; this antagonism was attenuated by 1 μM cyclo-thiazide and reversed to an enhancement of AMPA-evoked [³H]NA release by 10 μM of the drug, but was insensitive to 1-BCP or aniracetam. It is concluded that aniracetam exerts a dual effect on glutamatergic transmission: modulation of NMDA receptor function at nanomolar concentrations, and modulation of AMPA receptors at high micromolar concentrations. As to cyclothiazide and 1-BCP, our data concur with the idea that both compounds largely act through AMPA receptors, although an NMDA component may be involved in the effect of 1-BCP. Received: 14 September 1998 / Accepted: 19 January 1999     

In vitro exposure to nicotine induces endocytosis of presynaptic AMPA receptors modulating dopamine release in rat nucleus accumbens nerve terminals

Here we provide functional and immunocytochemical evidence supporting the presence on Nucleus Accumbens (NAc) dopaminergic terminals of cyclothiazide-sensitive, alfa-amino-3-hydroxy-5-methyl-4-isoxazolone propionate (AMPA) receptors, which activation causes Ca²⁺-dependent [³H]dopamine ([³H]DA) exocytosis. These AMPA receptors cross-talk with co-localized nicotinic receptors (nAChRs), as suggested by the finding that in vitro short-term pre-exposure of synaptosomes to 30 μM nicotine caused a significant reduction of both the 30 μM nicotine and the 100 μM AMPA-evoked [³H]DA overflow. Entrapping pep2-SVKI, a peptide known to compete for the binding of GluA2 subunit to scaffolding proteins involved in AMPA receptor endocytosis, in NAC synaptosomes prevented the nicotine-induced reduction of AMPA-mediated [³H]DA exocytosis, while pep2-SVKE, used as negative control, was inefficacious. Immunocytochemical studies showed that a significant percentage of NAc terminals were dopaminergic and that most of these terminals also posses GluA2 receptor subunits. Western blot analysis of GluA2 immunoreactivity showed that presynaptic GluA2 proteins in NAc terminals were reduced in nicotine-pretreated synaptosomes when compared to the control. The nACh-AMPA receptor–receptor interaction was not limited to dopaminergic terminals since nicotine pre-exposure also affected the presynaptic AMPA receptors controlling hippocampal noradrenaline release, but not the presynaptic AMPA receptors controlling GABA and acetylcholine release. These observations could be relevant to the comprehension of the molecular mechanisms at the basis of nicotine rewarding.     

Antidepressant treatments and function of glutamate ionotropic receptors mediating amine release in hippocampus

Previous evidences showed that, besides noradrenaline (NA) and 5-hydroxytryptamine (5-HT), glutamate transmission is involved in the mechanism of action of antidepressants (ADs), although the relations between aminergic and glutamatergic systems are poorly understood. The aims of this investigation were to evaluate changes in the function of glutamate AMPA and NMDA receptors produced by acute and chronic administration of the two ADs reboxetine and fluoxetine, selective inhibitors of NA and 5-HT uptake, respectively. Rats were treated acutely (intraperitoneal injection) or chronically (osmotic minipump infusion) with reboxetine or fluoxetine. Isolated hippocampal nerve endings (synaptosomes) prepared following acute/chronic treatments were labelled with [(3)H]NA or [(3)H]5-HT and [(3)H]amine release was monitored during exposure in superfusion to NMDA/glycine, AMPA or K(+)-depolarization. Acute and chronic reboxetine reduced the release of [(3)H]NA evoked by NMDA/glycine or by AMPA. The NMDA/glycine-evoked release of [(3)H]NA was also down-regulated by chronic fluoxetine. Only acute, but not chronic, fluoxetine inhibited the AMPA-evoked release of [(3)H]5-HT. The release of [(3)H]NA and [(3)H]5-HT elicited by K(+)-depolarization was almost abolished by acute reboxetine or fluoxetine, respectively, but recovered during chronic ADs administration. ADs reduced NMDA receptor-mediated releasing effects in noradrenergic terminals after acute and chronic administration, although by different mechanisms. Chronic treatments markedly reduced the expression level of NR1 subunit in synaptic membranes. The noradrenergic and serotonergic release systems seem to be partly functionally interconnected and interact with glutamatergic transmission to down-regulate its function. The results obtained support the view that glutamate plays a major role in AD activity.     

Glycine taken up through GLYT1 and GLYT2 heterotransporters into glutamatergic axon terminals of mouse spinal cord elicits release of glutamate by homotransporter reversal and through anion channels

Glycine concentration-dependently elicited [3H]D-aspartate ([3H]D-ASP) release from superfused mouse spinal cord synaptosomes. Glycine effect was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was prevented by the glycine transporter blocker glycyldodecylamide. Glycine also evoked release of endogenous glutamate, which was sensitive to glycyldodecylamide and abolished in low-Na+ medium. Experiments with purified synaptosomes and gliasomes show that the glycine-evoked [3H]D-ASP release largely originates from glutamatergic nerve terminals. The glycine-evoked [3H]D-ASP release was halved by NFPS, a selective blocker of GLYT1 transporters, or by Org 25543, a selective GLYT2 blocker, and almost abolished by a mixture of the two, suggesting that activation of GLYT1 and GLYT2 present on glutamatergic terminals triggers the release of [3H]D-ASP. Accordingly, confocal microscopy experiments show localization of GLYT1 and GLYT2 in purified synaptosomes immuno-stained for the vesicular glutamate transporter vGLUT1. The glycine effect was independent of extra- and intraterminal Ca2+ ions. It was partly inhibited by the glutamate transporter blocker DL-TBOA and largely prevented by the anion channel blockers niflumic acid and NPPB. To conclude, transporters for glycine (GLYT1 or/and GLYT2) and for glutamate coexist on the same spinal cord glutamatergic terminals. Activation of glycine heterotransporters elicits glutamate release partly by homotransporter reversal and largely through anion channels.     

Pharmacological actions of thymol and an analogue at GABAB autoreceptors

GABA_B autoreceptors inhibit release of GABA from GABAergic nerve terminals. Agonists of these receptors (e.g. baclofen) inhibit, whereas antagonists (e.g. (+)‐(S)‐5,5‐dimethylmorpholinyl‐2‐acetic acid; Sch 50911) enhance release of the transmitter. The actions of thymol (2‐isopropyl‐5‐methylphenol) and the structurally related compound 2‐tert‐butyl‐4‐methylphenol, (4MP) on the release of [³H]‐GABA were examined in rat neocortical slices where the GABAergic nerves had been preloaded with [³H]‐GABA and subsequently stimulated electrically on two occasions (S₁ and S₂). Test agents, baclofen and Sch 50911 were added to the superfusion medium prior to the second period of stimulation (S₂). Stimulation‐induced overflow (SIO) of [³H]‐GABA as a consequence of these stimulations (SIO₁ and SIO₂) were calculated and the effects of agents determined by comparing the SIO₂/SIO₁ ratio in the presence of each agent with that in control tissue. Thymol potentiated the release of [³H]‐GABA (EC₅₀ 170 μmol/L), an action reversed by baclofen (2 μmol/L). Baclofen alone had little effect on GABA release. Release of [³H]‐GABA was inhibited by 4MP (IC₅₀ 3 μmol/L) and this effect was blocked by Sch 50911 (10 μmol/L). Alone, Sch 50911 markedly potentiated the release of GABA. These results imply that 4MP is an agonist of GABA_B autoreceptors; however, further studies are needed to confirm that thymol is indeed a GABA_B autoreceptor antagonist. Of interest are structural differences in these agents. Thymol has a propyl group in the ortho position relative to the phenolic hydroxyl, whereas in 4MP this is a butyl group and the methyl group moves from position 5 to 4. Whether one or both of these changes was responsible for the above actions is unknown.     

In-vitro Characterization of YM872, a Selective,Potent and Highly Water-soluble α-Amino-3-hydroxy-5-methylisoxazole-4-propionate Receptor Antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-***1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL^?1 in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxa-line-2,3-dione (CNQX). YM872 potently inhibits [³H]AMPA binding with a K_i (apparent equilibrium dissociation constant) value of 0.096 ± 0.0024 μM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [³H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 ± 0.14 μm), [³H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 μM) and [³H]glycine (NMDA receptor glycine-binding site, IC50 > 100 μM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA₂ value of 6.97 ± 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-μM AMPA-induced increase of intracellular Ca²⁺ concentration with an IC50 value of 0.82 ± 0.031 μM, and blocked 300-μM kainate-induced neurotoxicity with an IC50 value of 1.02 μM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

The Antidepressant Metapramine is a Low-affinity Antagonist at N-methyl-d-aspartic Acid Receptors

Metapramine, a pharmacological compound with antidepressant activity in humans, was tested for possible antiglutamatergic activity, in vitro. We investigated the effects of metapramine on the N-methyl-d-aspartic acid (NMDA) receptor complex, by determining whether this compound would interfere with the binding of [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) to rat cortical membranes in the presence of either glycine NMDA, or both. Metapramine in the micromolar range inhibited the binding of [³H]TCP in the presence of both NMDA and glycine (ic₅₀ = 1.4 ± 0.2 μM). That very similar affinities were observed when either NMDA or glycine was present suggests that metapramine exerted a direct action at the PCP site. The affinity of metapramine for this site was about 25 and 350 times lower than that of PCP and MK-801, respectively. Metapramine inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (ic₅₀ = 13 μM). These results suggest that metapramine is a low-affinity antagonist of the NMDA receptor complex channel. This paper discusses the potential application of metapramine to the treatment of diseases linked to excessive stimulation of glutamatergic NMDA receptors. © 1997 Elsevier Science Ltd. All rights reserved.     

Aryl‐ and heteroaryl‐substituted phenylalanines as AMPA receptor ligands

A series of racemic unnatural amino acids was rationally designed on the basis of recently published X‐ray structures of the GluA2 LBD with bound phenylalanine‐based antagonists. Twelve new diaryl‐ or aryl/heteroaryl‐substituted phenylalanine derivatives were synthesized and evaluated in vitro in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)‐2‐amino‐3‐(3′‐hydroxy‐5‐(1H‐pyrazol‐4‐yl)‐[1,1′‐biphenyl]‐3‐yl)propanoic acid 7e , showed the binding affinity of 4.6 μm for native AMPA receptors and over fourfold lower affinity for kainic acid receptors. Furthermore, 7e was evaluated at recombinant homomeric rat GluA2 and GluA3 receptors. Recently reported X‐ray structures 5CBR and 5CBS, representing two distinct antagonist binding modes, were used as templates for molecular docking of the synthesized series. Binding data supported with molecular modeling confirmed that aryl/heteroaryl‐substituted phenylalanine analogues effectively bind to AMPA receptors with low micromolar affinity and high selectivity over native NMDA and kainate receptors. These properties make 7e a promising lead for the further development of new AMPA receptor ligands.     

The Antidepressant Metapramine is a Low-affinity Antagonist at N-methyl- -aspartic Acid Receptors

Metapramine, a pharmacological compound with antidepressant activity in humans, was tested for possible antiglutamatergic activity, in vitro. We investigated the effects of metapramine on the N-methyl- -aspartic acid (NMDA) receptor complex, by determining whether this compound would interfere with the binding of [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) to rat cortical membranes in the presence of either glycine NMDA, or both. Metapramine in the micromolar range inhibited the binding of [³H]TCP in the presence of both NMDA and glycine ( ₅₀ = 1.4 ± 0.2 μM). That very similar affinities were observed when either NMDA or glycine was present suggests that metapramine exerted a direct action at the PCP site. The affinity of metapramine for this site was about 25 and 350 times lower than that of PCP and MK-801, respectively. Metapramine inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices ( ₅₀ = 13 μM). These results suggest that metapramine is a low-affinity antagonist of the NMDA receptor complex channel. This paper discusses the potential application of metapramine to the treatment of diseases linked to excessive stimulation of glutamatergic NMDA receptors. © 1997 Elsevier Science Ltd. All rights reserved.     

Glycine-evoked release of [3H]acetylcholine from rat striatal slices is independent of the NMDA receptor

Summary KCl-, NMDA-, and glycine-evoked release of [³H]acetylcholine was studied in superfused rat striatal slices. KCl-evoked release of [³H]acetylcholine was inhibited by 1.2 mM MgC12 and 100 M lidocaine. Similarly, NMDA-evoked release was inhibited by MgCl₂ and lidocaine as well as 10 M CGS 19755, a competitive antagonist at NMDA receptors, and 10 nM MK-801, a noncompetitive antagonist of NMDA-induced responses. Glycine-evoked release was calcium-dependent and was inhibited by 0.1 M strychnine whereas KCl- and NMDA-evoked release were resistant to strychnine. In addition, lidocaine inhibited the glycine-induced response. Cross-tachyphylaxis was not observed between NMDA- and glycine-evoked release. These results indicate that the strychnine-sensitive, glycine-evoked release of [³H]acetylcholine is independent of the NMDA receptor.     

Phenoxybenzamine blocks dopamine autoreceptors irreversibly: Implications for multiple dopamine receptor hypotheses

Phenoxybenzamine in μM concentrations increased the electrically evoked overflow of recently taken up [³H]dopamine from superfused slices of cat caudate nucleus, an effect which is also observed for dopamine receptor antagonists. The magnitude of the increase in electrically evoked [³H]dopamine release caused by 1 μM phenoxybenzamine was equal to that elicited by maximally effective concentrations of the specific dopamine receptor antagonist, S-sulpiride. Phenoxybenzamine (1 μM) completely antagonized the inhibition of [³H]dopamine release caused by the dopamine receptor agonist pergolide (10 nM). The α-adrenoceptor antagonist phentolamine (1 μM) had no effect on the electrically evoked overflow of [³H]dopamine, ruling out the possibility that the effect of phenoxybenzamine could be attributed to α-adrenoceptor blockade. A 20 min exposure to 1 μM phenoxybenzamine increased the electrically evoked [³H]dopamine overflow even after the tissue had been washed for two and a half hours. When the caudate slices were exposed for 30 min to the reversible dopamine receptor antagonist S-sulpiride (1 μM) and washed for two and a half hours, no similar increase in stimulation-evoked [³H]dopamine overflow was observed. When sulpiride (1 μM) was present during the exposure to phenoxybenzamine (1 μM), no increase in electrically evoked [³H]dopamine overflow was observed after the washout period. Thus phenoxybenzamine at 1 μM appears to block irreversibly the dopamine autoreceptor in the caudate nucleus. Phenoxybenzamine has been previously reported to block irreversibly dopamine-stimulated adenylate cyclase (D₁) and neuroleptic receptor binding (D₂). The present demonstration that phenoxybenzamine also blocks the dopamine autoreceptor irreversibly thus supports the view that all currently well-established dopamine receptors are sensitive to phenoxybenzamine.     

Electrophysiological characterisation of the actions of kynurenic acid at ligand-gated ion channels

To better understand the effects of the tryptophan metabolite kynurenic acid (kynA) in the brain, we characterised its actions at five ligand-gated ion channels: NMDA, AMPA, GABA_A, glycine and α7 nicotinic acetylcholine receptors. Using whole-cell patch-clamp recordings, we found that kynA was a more potent antagonist at human NR1a/NR2A compared with NR1a/NR2B receptors (IC₅₀: 158 μM and 681 μM, respectively; in 30 μM glycine). KynA inhibited AMPA-evoked currents to a similar degree in cultured hippocampal neurons and a human GluR2(flip/unedited) cell line (IC₅₀: 433 and 596 μM, respectively) and at higher concentrations, kynA also inhibited the strychnine-sensitive glycine receptor (∼35% inhibition by 3 mM kynA). Interestingly, kynA inhibited the peak amplitude (IC₅₀: 2.9 mM for 10 μM GABA) and slowed the decay kinetics of GABA-evoked currents in cultured neurons. In contrast, we found that kynA (1-3 mM) had no effect on ACh-evoked, methyllycaconitine (MLA)-sensitive currents in a human α7 nicotinic receptor (nAChR) cell line, rat hippocampal neurons in primary culture or CA1 stratum radiatum interneurons in rat brain slices. However, DMSO (>1%) did inhibit α7 nAChR-mediated currents. In conclusion, kynA is an antagonist at NMDA, AMPA and glycine receptors and a modulator of GABA_A receptors, but we find no evidence for any effect of kynA at the α7 nAChR.     

AMPAR exocytosis through NO modulation of PICK1

The activation of NMDA receptors (NMDARs) triggers long-term changes in AMPA receptor-mediated synaptic transmission in the CNS. These long-lasting changes occur via the addition or removal of AMPA receptors (AMPARs) at the synaptic membrane and are mediated by a number of regulatory proteins including the GluR2 AMPAR-interacting proteins n-ethylmaleimide sensitive factor (NSF) and Protein Interacting with C Kinase (PICK1). We have shown that the potent activation of NMDARs drives unclustering of PICK1 and PICK1-GluR2 dissociation in dendrites resulting in increased surface delivery of AMPARs. Here we show that the dispersal of PICK1 is mediated by the actions of NSF. We find that elevated NMDAR signaling leads to the S-nitrosylation of NSF and increased NSF-GluR2 association. Both NMDAR-dependent unclustering of PICK1 and the delivery of surface AMPARs are dependent on release of nitric oxide (NO). Our data suggest that NMDAR activation can drive the surface delivery of AMPARs from a pool of intracellular AMPARs retained by PICK1 through the NO-dependent modification of NSF.     

Presynaptic mGlu1 and mGlu5 autoreceptors facilitate glutamate exocytosis from mouse cortical nerve endings

The effects of mGlu1 and mGlu5 receptor activation on the depolarization-evoked release of [(3)H]d-aspartate ([(3)H]d-ASP) from mouse cortical synaptosomes were investigated. The mGlu1/5 receptor agonist 3,5-DHPG (0.1-100muM) potentiated the K(+)(12mM)-evoked [(3)H]d-ASP overflow. The potentiation occurred in a concentration-dependent manner showing a biphasic pattern. The agonist potentiated [(3)H]d-ASP exocytosis when applied at 0.3muM; the efficacy of 3,5-DHPG then rapidly declined and reappeared at 30-100muM. The fall of efficacy of agonist at intermediate concentration may be consistent with 3,5-DHPG-induced receptor desensitization. Facilitation of [(3)H]d-ASP exocytosis caused by 0.3muM 3,5-DHPG was prevented by the selective mGlu5 receptor antagonist MPEP, but was insensitive to the selective mGlu1 receptor antagonist CPCCOEt. In contrast, CPCCOEt prevented the potentiation by 50muM 3,5-DHPG, while MPEP had minimal effect. Unexpectedly, LY 367385 antagonized both the 3,5-DHPG-induced effects. A total of 0.3muM 3,5-DHPG failed to facilitate the K(+)-evoked [(3)H]d-ASP overflow from mGlu5 receptor knockout (mGlu5(-/-)) cortical synaptosomes, but not from nerve terminals prepared from the cortex of animals lacking the mGlu1 receptors, the crv4/crv4 mice. On the contrary, 50muM 3,5-DHPG failed to affect the [(3)H]d-ASP exocytosis from cortical synaptosomes obtained from crv4/crv4 and mGlu5(-/-)mice. Western blot analyses in subsynaptic fractions support the existence of both mGlu1 and mGlu5 autoreceptors located presynaptically, while immunocytochemistry revealed their presence at glutamatergic terminals. We propose that mGlu1 and mGlu5 autoreceptors exist on mouse glutamatergic cortical terminals; mGlu5 receptors may represent the "high affinity" binding sites for 3,5-DHPG, while mGlu1 autoreceptors represent the "low affinity" binding sites.     

Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells

The effects of ethanol and/or glycine on NMDA-induced enhancement of cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]_i), ⁴⁵Ca²⁺ influx, 4-b-[³H]phorbol-12,13-dibutyrate ([³H]PDBu) binding, and neuronal necrosis in cultured rat cortical and cerebellar granule neurons were examined. Using microfluorimetric techniques in combination with rapid perfusion of single brain neurons, we found that glycine (10 M) was a necessary co-agonist for NMDA-induced depolarization in cerebellar granule cells. In contrast, depolarization with NMDA in cortical cells was observed even without the addition of exogenous glycine as well as in the absence or presence of 1 mM MgCl₂. Ethanol (50 mM) inhibited the effects of NMDA in some, but not all, neurons indicative of the existence of ethanol-sensitive and ethanol-insensitive cortical and cerebellar granule neurons. In studies performed in monolayers of cortical and cerebellar granule cells, we observed that the presence of glycine (10 M) was a necessary prerequisite to unmask inhibitory actions of ethanol on ⁴⁵Ca²⁺ influx induced by NMDA. In another set of experiments, we noted that NMDA-induced stimulation of [³H]PDBu binding to monolayers of intact cerebellar granule cells was inhibited by ethanol (50 mM). Finally, we report that ethanol caused a concentration-dependent inhibition of NMDA-induced necrotic cell death, assessed by measuring the ability of cerebellar granule cells to transform 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MIT) into formazan. In none of the four assays used to demonstrate the inhibitory effects of ethanol on NMDA receptor activity, the ethanol-induced inhibition was reversed by glycine (up to 100 M). Thus, in contrast to earlier reports, our data suggest that ethanol and glycine produce their effects by acting at different regulatory sites within the NMDA receptor system in brain neurons.     

Excessive and precocious glutamate release in a mouse model of amyotrophic lateral sclerosis

The release of [3H]D-aspartate ([3H]D-ASP) or [3H]GABA evoked by glycine and that of [3H]D-ASP or [3H]glycine evoked by GABA from spinal cord synaptosomes were studied in SOD1-G93A(+) mice, a transgenic model of amyotrophic lateral sclerosis, SOD1(+) mice and SOD1(-)/G93A(-) animals. Mutant mice were killed at advanced phase of pathology or during the presymptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice glycine evoked [(3)H]d-ASP and [(3)H]GABA release, while GABA caused [3H]D-ASP, but not [3H]glycine, release. The glycine-evoked release of [3H]D-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]D-ASP release, but not that of [3H]glycine, were more pronounced in SOD1-G93A(+) than in SOD1(+) mice. Furthermore, these potentiations were already present in asymptomatic 30- to 40-day-old mice. Basal [3H]D-ASP release was also higher in SOD1-G93A(+) than SOD1(+) or SOD1(-)/G93A(-) mice. The release of endogenous glutamate and GABA was also enhanced in asymptomatic animals; the glycine-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in SOD1(+) animals. The effects of glycine and GABA were insensitive to receptor blockers, but sensitive to transporter inhibitors, indicating coexistence of glutamate and glycine transporters and of glutamate and GABA transporters on glutamate-releasing terminals. The glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.     

Evidence for strychnine-sensitive glycine receptors in human amygdala

Recent studies suggested the existence of strychnine-sensitive glycine-receptors in mammalian amygdala. In the present study, we investigated the amino acid concentrations as well as immunocytochemical and pharmacological properties of glycine-receptors in fresh human amygdala tissue obtained from epilepsy surgery.High pressure liquid chromatography revealed a considerable amount of glycine and its precursors and glycine-receptors agonists L-serine and taurine in this tissue. Immunohistochemistry using the monoclonal antibody mAb4a, recognizing an epitope common to all -subunit variants of glycine receptors, displayed a specific labeling at the soma and on proximal dendrites of mostly tripolar, large-sized neurons of irregular distribution and arrangement. To elucidate the pharmacological properties of the glycine-receptors found slices of human amygdala were preloaded with [³H]-choline and superfused. Glycine induced an overflow of [³H]-acetylcholine, which was inhibited by strychnine in a concentration-dependent manner. Furthermore, the glycine-induced release of [³H]-acetylcholine was significantly inhibited by furosemide, indicating glycine-induced actions to be attributed to chloride channels. These actions of glycine were not influenced by MK-801, D-CPPene or bicuculline. Thus, the effects of glycine did not seem to be mediated through NMDA or GABA receptors.These observations indicate that strychnine-sensitive, chloride-conducting glycine receptors, which elicit the release of [³H]-acetylcholine, are present at the soma and on proximal dendrites of neurons in human amygdala. It is hypothesized that glycine may display a regulatory role in amygdaloid functions, probably via cholinergic interneurons.Abbreviations ABC Avidin-biotin-peroxidase complex - ACh Acetylcholine - CI₉₅ 95% Confidence interval - DAB Diaminobenzidine tetrahydrochloride - D-CPPene D-3-(2-carboxypiperazine-4-yl)-1-propenyl-1-phosphonic acid - HPLC High pressure liquid chromatography - GABA -Aminobutyric acid - Gly Glycine - GlyR Glycine receptor - mAb Monoclonal antibody - MK-801 (+)-5-Methyl-10,11-dihydro-5,4-dibenzo[a,d]cyclohepten-5,10-imine maleate - NMDA N-methyl-D-aspartate - NHS Normal horse serum - OPA o-Phthalaldehyde mercaptoethanol - PBS Phosphate buffer saline - TLE Temporal lobe epilepsy     

Extracellular protons differentially potentiate the responses of native AMPA receptor subtypes regulating neurotransmitter release

1. The effects of pH changes on the basal and evoked release of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]5-hydrohytryptamine ([(3)H]5-HT) from hippocampal synaptosomes and of [(3)H]dopamine ([(3)H]DA) and [(3)H]acetylcholine ([(3)H]ACh) from striatal and cortical synaptosomes were investigated in rat brain. 2. Changing pH between 6.4 and 8.0 did not affect the spontaneous release of the four [(3)H]neurotransmitters; alkalinization to pH 8.8 significantly enhanced release. Acidification to pH 6.4 augmented the AMPA-evoked overflows of [(3)H]NA, [(3)H]5-HT and [(3)H]DA, but not that of [(3)H]ACh. In contrast, lowering pH to 6.4 decreased the K(+)-evoked overflows of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 3. AMPA released transmitters in a Ca(2+)-dependent, exocytotic manner since its effects, at pH 7.4 or 6.4, were abolished by omitting external Ca(2+) or by depleting vesicular transmitter stores with bafilomycin A1. AMPA did not evoke carrier-mediated release because the uptake blockers nisoxetine, 6-nitroquipazine, GBR12909 and hemicholinium-3 could not inhibit the AMPA-induced release of [(3)H]NA, [(3)H]5-HT, [(3)H]DA and [(3)H]ACh. 4. Extraterminal acidification to pH 6.4 prevented the potentiating effect of cyclothiazide on the AMPA-evoked release of [(3)H]NA, [(3)H]DA and [(3)H]5-HT, whereas the proton-insensitive AMPA-evoked release of [(3)H]ACh, previously found to be cyclothiazide-insensitive at pH 7.4 was cyclothiazide-resistant also at pH 6.4. 5. To conclude, the cyclothiazide-sensitive AMPA receptors mediating release of NA, 5-HT and DA, but not the cyclothiazide-insensitive AMPA receptors mediating the release of ACh, become more responsive when external pH is lowered to pathophysiologically relevant values. The results with cyclothiazide suggest that H(+) ions may prevent desensitization of some AMPA receptor subtypes.