Hippocampal AMPA autoreceptors positively coupled to NMDA autoreceptors traffic in a constitutive manner and undergo adaptative changes following enriched environment training

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) autoreceptors exist on glutamate hippocampal terminals. Aimed at investigating whether these autoreceptors traffic constitutively, (S)AMPA-evoked [³H]D-ASP release from synaptosomes enriched with peptides that impede the interaction of GluA2 subunits with cytosolic proteins involved in receptor movements [namely Glutamate Receptor-Interacting Protein (GRIP), Protein Interacting with C kinase 1 (PICK1), N-ethyl-maleimide-Sensitive Fusion protein NSF proteins] was monitored. (S)AMPA alone had no effect on the spontaneous release of [³H]D-ASP from control synaptosomes, but became efficacious in the presence of cyclothiazide or when preventing GluA2/GRIP/PICK1, but not GluA2/NSF, interaction. Hippocampal glutamatergic terminals also possess NMDA autoreceptors. 10 μM NMDA/1 μM glycine-induced [³H]D-ASP release was concentration-dependently increased by (S)AMPA. Cyclothiazide potentiated the 10 μM NMDA/1 μM glycine/50 μM (S)AMPA-induced [³H]D-ASP overflow, while NBQX halved and MK-801 abolished it, suggesting NMDA-AMPA autoreceptor cross-talk. Western Blot analysis of sub-synaptic fractions confirmed presynaptic GluN2B-GluA2/3 co-localization. Impeding GluA2/GRIP/PICK1 interaction facilitated the NMDA/glycine/(S)AMPA-induced release of [³H]D-ASP, while competing for GluA2/NSF interaction reduced it, indicating that NMDA receptor favours AMPA receptor insertion in synaptosomal plasmamembranes. Finally, rearing mice in enriched environment unveiled the (S)AMPA-induced release of [³H]D-ASP, but leaved unmodified that caused by NMDA/glycine. The NBQX-sensitive, 50 μM (S)AMPA-evoked release of [³H]D-ASP was insensitive to cyclothiazide and to peptide interfering with GluA2/GRIP/PICK1 interaction but was addictive to that caused by NMDA/glycine. Presynaptic GluA2/3 immunoreactivity in EE hippocampal terminals was increased, while GluN2B was unchanged. We conclude that hippocampal AMPA autoreceptors positively coupled to NMDA autoreceptors traffic in a constitutive manner and undergo functional up-regulation in EE animals.     

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.     

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.     

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     

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     

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.     

Differential redistribution of native AMPA receptor complexes following LTD induction in acute hippocampal slices

AMPAR trafficking is crucial for the expression of certain forms of synaptic plasticity. Here, using surface biotinylation of hippocampal slices and subsequent synaptosome isolation we assessed AMPAR surface expression in synaptosomes following NMDA-evoked long-term depression (NMDA-LTD). Surface levels of GluR1, GluR2 and GluR3 in synaptosomes were markedly reduced 90 min after NMDA-LTD induction. Consistent with endocytosis and degradation, whole-cell surface and total expression levels of GluR2 and GluR3 were also reduced. In contrast, whole-cell surface levels of GluR1 were unaltered at 90 min suggesting that AMPARs with different subunit composition are redistributed to different non-synaptic compartments following LTD induction in acute hippocampal slices.     

Characterization of two central AMPA-preferring receptors having distinct location,function and pharmacology

The existence of a pharmacological heterogeneity among the glutamate receptors sensitive to (RS)--amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) has been investigated in the adult rat central nervous system (CNS). AMPA stimulated [³H]noradrenaline ([³H]NA) release from hippocampal synaptosomes (pD₂ = 4.58) and the production of cGMP in cerebellar slices (pD₂ = 7.75). The AMPA effects in the two systems were tested against several glutamate receptor antagonists including 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 6,7-dinitro-quinoxaline-2,3-dione (DNQX),l-glutamate diethylester (GDEE), -d-glutamyl-glycine (GDGG) and -d-glutamyl-aminomethylsulphonate (GAMS). In both systems the AMPA effect was equally sensitive to CNQX or DNQX. However, while the AMPA-evoked increase of [³H]NA release from presynaptic terminals was not affected by GAMS, GDGG or GDEE, the postsynaptic cGMP response was prevented by GDGG and GDEE. It is concluded that rat hippocampus and cerebellum possess, respectively, presynaptic and post-synaptic AMPA-sensitive receptors involved in different functions and endowed with diverse pharmacological properties. Correspondence to: M. Raiteri at the above address     

Functional interactions between presynaptic NMDA receptors and metabotropic glutamate receptors co-expressed on rat and human noradrenergic terminals

BACKGROUND AND PURPOSE: Electrophysiological studies described potentiation of NMDA receptor function by metabotropic glutamate receptors (mGluRs) of group I occurring postsynaptically. Since release-enhancing NMDA receptors exist on noradrenergic terminals and group I mGluRs have recently been identified on these nerve endings, we have investigated if NMDA receptor-mGluR interactions also can occur at the presynaptic level. EXPERIMENTAL APPROACH: Rat hippocampus and human neocortex synaptosomes were labelled with [(3)H]noradrenaline and superfused with mGluR agonists and antagonists. NMDA-evoked [(3)H]noradrenaline release was produced by removal of external Mg(2+) or by simultaneous application of NMDA and AMPA in Mg(2+)-containing solutions. KEY RESULTS: The mGluR1/5 agonist 3,5-DHPG, inactive on its own, potentiated both the release of [(3)H]noradrenaline elicited by AMPA/NMDA/glycine and that evoked by NMDA/glycine following Mg(2+) removal. The effect of 3,5-DHPG on the AMPA/NMDA/glycine-induced release was insensitive to the mGluR1 antagonist CPCCOEt, but it was abolished by the mGluR5 antagonist MPEP; moreover, it was potentiated by the mGluR5 positive allosteric modulator DFB. When NMDA receptors were activated by Mg(2+) removal, both mGluR5 and mGluR1 contributed to the evoked release, the mGluR-mediated release being blocked only by CPCCOEt and MPEP in combination. Experiments with human neocortex synaptosomes show NMDA receptor-mGluR interactions qualitatively similar to those observed in rodents. CONCLUSIONS AND IMPLICATIONS: Group I mGluRs, both of the mGluR1 and mGluR5 subtypes, co-localize with NMDA receptors on noradrenergic terminals of rat hippocampus and human neocortex. Depending on the mode of activation, NMDA receptors exert differential permissive roles on the activation of presynaptic mGluR1 and mGluR5.     

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.     

Pharmacological characterization of presynaptic alpha-adrenoceptors in the nucleus tractus solitarii and the cerebral cortex of the rat

The presynaptic alpha-adrenoceptors mediating inhibition of K+-induced release of [3H]noradrenaline (NA) from superfused slices and synaptosomes obtained from the nucleus tractus solitarii (NTS) region were characterized pharmacologically and compared to those in the cerebral cortex. Clonidine concentration-dependently (pD2 7.80) reduced the K+-induced [3H]NA release from slices and synaptosomes from the nucleus tractus solitarii of both normotensive and spontaneously hypertensive rats; 10(-6) M clonidine inhibited the [3H]NA release induced by 10 mM K+ to 40% of control. This inhibitory effect of clonidine was antagonized by the alpha2-adrenoceptor antagonist yohimbine (10(-7) M), but not by the alpha1-adrenoceptor antagonist prazosin (10(-7) M). The rank order of apparent affinities of agonists for the presynaptic alpha-adrenoceptors mediating inhibition of [3H]NA release from slices of nucleus tractus solitarii induced by 20 mM K+, was: oxymetazoline greater than clonidine greater than adrenaline greater than alpha-methylnoradrenaline greater than noradrenaline much greater than methoxamine. A similar rank order was found for cerebral cortex slices. The rank order of intrinsic activities in both brain regions was found to be: noradrenaline approximately equal to alpha-methylnoradrenaline approximately equal to adrenaline greater than clonidine greater than oxymetazoline much greater than methoxamine. The data indicate that presynaptic alpha-adrenoceptors mediating inhibition of NA release both in the nucleus tractus solitarii and the cerebral cortex belong to the alpha2-type. The presynaptic alpha2-adrenoceptors in the two brain regions appear to be similar both functionally and pharmacologically. It is suggested that these receptors in the nucleus tractus solitarii may play a role in the hypotensive effect of clonidine.     

Chronic antidepressant treatment increases the membrane expression of AMPA receptors in rat hippocampus

It has been proposed that potentiation of AMPA receptor (AMPAR) function may be useful in the treatment of depression. Here we studied the acute and chronic effect of the antidepressants desipramine and paroxetine, which differentially affect monoamine reuptake, on the expression of the AMPAR subunits GluR1 and GluR2/3, analyzed by Western blot, both in total and in membrane-enriched extracts from rat hippocampus. Acute antidepressant treatment did not produce any change in the expression of AMPAR subunits. In chronic treatments, rats were daily treated with the antidepressants (10 mg/kg/day) for 7, 14, or 21 days. In rats receiving either of the two antidepressant treatments for 21 consecutive days and killed 24 h after the last injection, an increase in GluR1 and GluR2/3 levels was found in the membrane fraction, with no significant change in the total extract, suggesting a trafficking of the AMPAR subunits from intracellular pools to synaptic sites in the hippocampus. This appeared to be a region-specific effect since no change in AMPAR subunit expression was found in the frontal cortex. Previously reported modifications in phosphorylating enzymes by chronic antidepressants could perhaps play a role in hippocampal membrane insertion of AMPAR subunits. When the survival time after the 21-day-treatment was longer — 72 instead of 24 h — the hippocampal membrane expression of GluR1, but not of GluR2/3 subunits, was still increased, as could be expected from the distinct mechanisms operating in synaptic delivery of GluR1 and GluR2/3 subunits. The antidepressant-induced increase in the number of GluR1- and GluR2/3-containing AMPARs at the synapses may indicate an enhanced AMPAR-mediated synaptic transmission which could help to counteract the alterations in neuronal connectivity which appear to underlie the pathophysiology of mood disorders.     

Facilitation of [3H]acetylcholine and [3H]5-hydroxytryptamine release from rat cerebral cortex synaptosomes by a factor extracted from the skin of the Australian frog Pseudophryne coriacea

A partly purified extract of the skin of the Australian frog Pseudophryne coriacea (PsC) evoked the release of [3H]acetylcholine [( 3H]ACh) and of [3H]5-hydroxytryptamine [( 3H]5-HT) from superfused rat cerebral cortex synaptosomes prelabeled with [3H]choline or [3H]5-HT, respectively. The PsC-evoked release of both transmitters was sensitive to tetrodotoxin and was strictly Ca2+-dependent. The release of [3H]5-HT caused by PsC was unaffected by the 5-HT uptake inhibitor citalopram. Activation of muscarinic autoreceptors by ACh or of serotonin autoreceptors by 5-HT depressed the PsC-evoked release of [3H]ACh or of [3H]5-HT, respectively. It is concluded that PsC elicits a Ca2+-dependent exocytotic-like transmitter release, possibly by opening Na+ channels in the presynaptic membrane.     

Heterogeneity of muscarinic autoreceptors and heteroreceptors in the rat brain: effects of a novel M1 agonist, AF102B

The effects of oxotremorine and AF102B (cis-2-methylspiro-(1,3-oxathiolane-5,3')-quinuclidine), a novel M1-selective muscarinic agonist, on acetylcholine (ACh) and dopamine (DA) release from superfused rat hippocampal and striatal synaptosomes were investigated. Synaptosomes that had been prelabeled with [3H]choline or [3H]DA were depolarized by high K+. Oxotremorine and AF102B decreased the K+-evoked [3H]ACh release from hippocampal synaptosomes and increased the K+-evoked [3H]DA release from striatal synaptosomes. The dose-response curves showed that AF102B was far less potent than oxotremorine at the hippocampal presynaptic muscarinic receptors (autoreceptors). On the other hand, AF102B was more potent than oxotremorine at the muscarinic receptors on the striatal dopaminergic terminals (heteroreceptors). Pirenzepine, a selective M1 antagonist, counteracted the effects of oxotremorine on [3H]DA release more potently than it did the effects of oxotremorine on [3H]ACh release. Our results suggest that AF102B and pirenzepine discriminate pharmacologically between muscarinic autoreceptors and heteroreceptors.     

Cyclothiazide unmasks AMPA-evoked stimulation of [3H]-L-glutamate release from rat hippocampal synaptosomes.

The effect of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) on Ca(2+)-sensitive, tetrodotoxin (TTX)-insensitive K(+)-stimulated [3H]-L-glutamate release from rat hippocampal synaptosomes was determined. AMPA in the presence, but not in the absence of cyclothiazide, a drug which blocks AMPA receptor desensitization, elicited a dose-dependent increase in K(+)-stimulated [3H]-L-glutamate release but had no effect on basal release. The AMPA/cyclothiazide stimulation was blocked by CNQX and by GYKI 52466, an antagonist at the cyclothiazide site. These results indicate that AMPA receptors are present on presynaptic terminals and suggest that they may play a role in the regulation of neurotransmitter release.     

Postsynaptic silent synapses: evidence and mechanisms

In this review I discuss the evidence that some glutamatergic synapses exist that lack surface-expressed postsynaptic AMPA receptors (AMPARs) but contain NMDA receptors opposed to a functional release site. I have summarised the electrophysiological, anatomical and cell biological evidence for such postsynaptically silent synapses, and data that support the idea of rapid AMPAR insertion at silent synapses during long-term potentiation (LTP). I also discuss recent findings suggesting that developmental and activity-dependent alteration in the postsynaptic glutamate receptor composition is a general principle that occurs for other receptor subtypes. This review is not intended to provide a full discussion of possible presynaptic mechanisms for silent synapses; these are covered in the accompanying recent article [Voronin and Cherubini (this issue)].     

Pharmacological heterogeneity among calcium channels that subserve acetylcholine release in vertebrate forebrain.

Inhibition of calcium-evoked [3H]ACh release by different classes of calcium channel blockers was compared among calcium-naive synaptosomes from chick, frog and rat forebrain tissues. In all three species, [3H]ACh release was insensitive to nifedipine (0.03-3 microM) but was completely inhibited by cadmium (IC50 range = 0.7-1.7 microM) or cobalt (190-350 microM). In contrast, the peptide omega-conotoxin revealed marked species heterogeneity in that [3H]ACh release was potently blocked in chick and rat synaptosomes (IC50 congruent to 1 nM), whereas frog tissue was notably resistant (IC50 greater than 100 nM). Together, these data provide functional evidence for pharmacological heterogeneity among presynaptic calcium channels that subserve [3H]ACh release.     

Functional development of the synaptic transmission in the rat CNS and its interaction with drugs (author's transl)

The ontogenesis of the synaptic function was investigated in the central noradrenaline (NA), dopamine (DA), serotonin (5-HT), acetylcholine (ACh) and GABA nervous systems of developing rats. Monoamines histochemically first appeared on days 12 to 14 in the fetal brain and may exert a trophic action on target neurons as a "differentiation signal". The presynaptic functions such as the high affinity uptake and depolarization-induced, Ca2+-dependent release of L-[3H]DA, and [2H]DA, and [3H]DA, and [3H]5-HT were observed in synaptosomes and slices from the fetus (18 days of gestation) and brain of newborn rats. Monoamine-stimulated activity of adenylate cyclase and specific binding of ligands in NA(alpha 1, alpha 2, beta 1, beta 2), DA, 5-HT, ACh (muscarinic and nicotinic) and GABA receptors indicated dynamic changes through postnatal development. Behavioral findings suggest when neurotransmitter receptors become sensitive and reach functional maturity (NA and DA, already at birth; 5-HT and muscarinic ACh, 15 to 20 days; GABA, 12 to 13 days (mouse)). In addition, differences in behavioral responsiveness to drugs were observed in rats at various developing stages, probably due to the interaction between plural neuronal systems. Finally, the brain undergoing rapid differentiation seems to be most sensitive to hormones and centrally acting drugs. Thus, permanent alteration in central functions may occur when some classes of drugs, dose-dependently, are administrated to animals at the perinatal stage.     

Subunit dependencies of N-methyl-D-aspartate (NMDA) receptor-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor internalization

N-Methyl-D-aspartate (NMDA) receptor (NMDAR) activity regulates the net number of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPAR) at the cell surface by modulating the balance between AMPAR membrane insertion and endocytosis. In this study, we addressed the role of NMDAR subtypes and of NMDAR-mediated Ca2+ influx in the NMDAR-induced endocytosis of GluR2-containing AMPARs in primary murine hippocampal neurons. We found that NMDAR activation enhanced the endocytosis of AMPARs containing the GluR2 splice variants with short, but not long, cytoplasmic tails. NMDA-induced GluR2 endocytosis was completely inhibited by pharmacological block of NR2B-containing NMDARs. In turn, preferential block of NR2A-containing NMDARs did not affect NMDA-induced AMPAR endocytosis, indicating that AMPAR internalization is controlled by a restricted set of NMDARs. The NMDA-induced GluR2 internalization was also observed in the absence of extracellular Na+ ions, suggesting that membrane depolarization is not a prerequisite for this effect. Furthermore, the activation of Ca2+-impermeable NMDARs containing the mutant NR1(N598R) subunit failed to enhance AMPAR endocytosis, indicating a requirement of Ca2+ influx directly through the NMDAR channels. In summary, our findings suggest that the NMDAR-induced selective internalization of short C-terminal GluR2-containing AMPARs requires a Ca2+ signal that originates from NMDAR channels and is processed in an NMDAR subtype-restricted manner.     

Effects of adenosine on 45Ca uptake and [3H]acetylcholine release in synaptosomal preparation from guinea-pig ileum myenteric plexus

The effects of adenosine on acetylcholine (ACh) release and calcium uptake were examined in a synaptosomal fraction prepared from guinea-pig ileum myenteric plexus-longitudinal muscle. A high concentration of potassium (40 mM) and electrical pulses (ES:10Hz) caused a marked increase in the output of [3H]ACh from [3H]choline-preloaded crude synaptosomes. This [3H]ACh output was calcium- and temperature-dependent. Adenosine reduced the high potassium-induced release significantly, and the electrically stimulated release completely. When the preparation was depolarized by high potassium or electrical pulses, the 45Ca uptake by synaptosomes was significantly enhanced. The uptake of 45Ca induced by high potassium was significantly reduced and that induced by electrical stimulation was completely abolished by adenosine. From these results, it may be suggested that adenosine inhibits neurotransmitter release by suppressing the presynaptic influx of calcium ion during depolarization of the cholinergic nerve terminals in guinea-pig ileum.