Correction: Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

[This corrects the article DOI: 10.1371/journal.pone.0170825.].

Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

Fingolimod, the first oral, disease-modifying therapy for MS, has been recently proposed to modulate glutamate transmission in the central nervous system (CNS) of mice suffering from Experimental Autoimmune Encephalomyelitis (EAE) and in MS patients. Our study aims at investigating whether oral fingolimod recovers presynaptic defects that occur at different stages of disease in the CNS of EAE mice. In vivo prophylactic (0.3 mg/kg for 14 days, from the 7th day post immunization, d.p.i, the drug dissolved in the drinking water) fingolimod significantly reduced the clinical symptoms and the anxiety-related behaviour in EAE mice. Spinal cord inflammation, demyelination and glial cell activation are markers of EAE progression. These signs were ameliorated following oral fingolimod administration. Glutamate exocytosis was shown to be impaired in cortical and spinal cord terminals isolated from EAE mice at 21 ± 1 d.p.i., while GABA alteration emerged only at the spinal cord level. Prophylactic fingolimod recovered these presynaptic defects, restoring altered glutamate and GABA release efficiency. The beneficial effect occurred in a dose-dependent, region-specific manner, since lower (0.1-0.03 mg/kg) doses restored, although to a different extent, synaptic defects in cortical but not spinal cord terminals. A delayed reduction of glutamate, but not of GABA, exocytosis was observed in hippocampal terminals of EAE mice at 35 d.p.i. Therapeutic (0.3 mg/kg, from 21 d.p.i. for 14 days) fingolimod restored glutamate exocytosis in the cortex and in the hippocampus of EAE mice at 35 ± 1 d.p.i. but not in the spinal cord, where also GABAergic defects remained unmodified. These results improve our knowledge of the molecular events accounting for the beneficial effects elicited by fingolimod in demyelinating disorders.

CXCR4 and NMDA Receptors Are Functionally Coupled in Rat Hippocampal Noradrenergic and Glutamatergic Nerve Endings.

Previous studies had shown that the HIV-1 capsidic glycoprotein gp120 (strain IIIB) modulates presynaptic release-regulating NMDA receptors on noradrenergic and glutamatergic terminals. This study aims to assess whether the chemokine CXC4 receptors (CXCR4s) has a role in the gp120-mediated effects. The effect of CXCL12, the endogenous ligand at CXCR4, on the NMDA-mediated releasing activity was therefore investigated. Rat hippocampal synaptosomes were preloaded with [3H]noradrenaline ([3H]NA) or [3H]D-aspartate ([3H]D-Asp) and acutely exposed to CXCL12, to NMDA or to both agonists. CXCL12, inactive on its own, facilitated the NMDA-evoked tritium release. The NMDA antagonist MK-801 abolished the NMDA/CXCL12-evoked tritium release of both radiolabelled tracers, while the CXCR4 antagonist AMD 3100 halved it, suggesting that rat hippocampal nerve endings possess presynaptic release-regulating CXCR4 receptors colocalized with NMDA receptors. Accordingly, Western blot analysis confirmed the presence of CXCR4 proteins in synaptosomal plasmamembranes. In both synaptosomal preparations, CXCL12-induced facilitation of NMDA-mediated release was dependent upon PLC-mediated src-induced events leading to mobilization of Ca2+ from intraterminal IP3-sensitive stores Finally, the gp120-induced facilitation of NMDA-mediated release of [3H]NA and [3H]D-Asp was prevented by AMD 3100. We propose that CXCR4s are functionally coupled to NMDA receptors in rat hippocampal noradrenergic and glutamatergic terminals and account for the gp120-induced modulation of the NMDA-mediated central effects. The NMDA/CXCR4 cross-talk could have a role in the neuropsychiatric symptoms often observed in HIV-1 positive patients.

Presynaptic, release-regulating mGlu2 -preferring and mGlu3 -preferring autoreceptors in CNS: pharmacological profiles and functional roles in demyelinating disease.

BACKGROUND AND PURPOSE: Presynaptic, release-regulating metabotropic glutamate 2 and 3 (mGlu2/3) autoreceptors exist in the CNS. They represent suitable targets for therapeutic approaches to central diseases that are typified by hyperglutamatergicity. The availability of specific ligands able to differentiate between mGlu2 and mGlu3 subunits allows us to further characterize these autoreceptors. In this study we investigated the pharmacological profile of mGlu2/3 receptors in selected CNS regions and evaluated their functions in mice with experimental autoimmune encephalomyelitis (EAE). EXPERIMENTAL APPROACH: The comparative analysis of presynaptic mGlu2/3 autoreceptors was performed by determining the effect of selective mGlu2/3 receptor agonist(s) and antagonist(s) on the release of [(3)H]-D-aspartate from cortical and spinal cord synaptosomes in superfusion. In EAE mice, mGlu2/3 autoreceptor-mediated release functions were investigated and effects of in vivo LY379268 administration on impaired glutamate release examined ex vivo. KEY RESULTS: Western blot analysis and confocal microscopy confirmed the presence of presynaptic mGlu2/3 receptor proteins. Cortical synaptosomes possessed LY541850-sensitive, NAAG-insensitive autoreceptors having low affinity for LY379268, while LY541850-insensitive, NAAG-sensitive autoreceptors with high affinity for LY379268 existed in spinal cord terminals. In EAE mice, mGlu2/3 autoreceptors completely lost their inhibitory activity in cortical, but not in spinal cord synaptosomes. In vivo LY379268 administration restored the glutamate exocytosis capability in spinal cord but not in cortical terminals in EAE mice. CONCLUSIONS AND IMPLICATIONS: We propose the existence of mGlu2-preferring and mGlu3-preferring autoreceptors in mouse cortex and spinal cord respectively. The mGlu3 -preferring autoreceptors could represent a target for new pharmacological approaches for treating demyelinating diseases.

Inhibition of hippocampal plasticity in rats performing contrafreeloading for water under repeated administrations of pramipexole.

RATIONALE: Compulsive symptoms develop in patients exposed to pramipexole (PPX), a dopaminergic agonist with high selectivity for the D3 receptor. Consistently, we demonstrated that PPX produces an exaggerated increase in contrafreeloading (CFL) for water, a repetitive and highly inflexible behavior that models core aspects of compulsive disorders. OBJECTIVES: Given the role of the hippocampus in behavioral flexibility, motivational control, and visuospatial working memory, we investigated the role of hippocampus in the expression of PPX-induced CFL. To this aim, rats were subjected to CFL under chronic PPX, and then examined for the electrophysiological, structural, and molecular properties of their hippocampus. METHODS: We measured long-term potentiation (LTP) at CA1 Schaffer collaterals, dendritic spine density in CA1 pyramidal neurons, and then glutamate release and expression of pre and postsynaptic proteins in hippocampal synaptosomes. The effects of PPX on hippocampal-dependent working memory were assessed through the novel object recognition (NOR) test. RESULTS: We found that PPX-treated rats showing CFL exhibited a significant decrease in hippocampal LTP and failed to exhibit the expected increase in hippocampal spine density. Glutamate release and PSD-95 expression were decreased, while pSYN expression was increased in hippocampal synaptosomes of PPX-treated rats showing CFL. Despite a general impairment of hippocampal synaptic function, working memory was unaffected by PPX treatment. CONCLUSIONS: Our findings demonstrate that chronic PPX affects synaptic function in the hippocampus, an area that is critically involved in the expression of flexible, goal-centered behaviors. We suggest that the hippocampus is a promising target in the pharmacotherapy of compulsive disorders.

Presynaptic c-Jun N-terminal Kinase 2 regulates NMDA receptor-dependent glutamate release.

Activation of c-Jun N-terminal kinase (JNK) signaling pathway is a critical step for neuronal death occurring in several neurological conditions. JNKs can be activated via receptor tyrosine kinases, cytokine receptors, G-protein coupled receptors and ligand-gated ion channels, including the NMDA glutamate receptors. While JNK has been generally associated with postsynaptic NMDA receptors, its presynaptic role remains largely unexplored. Here, by means of biochemical, morphological and functional approaches, we demonstrate that JNK and its scaffold protein JIP1 are also expressed at the presynaptic level and that the NMDA-evoked glutamate release is controlled by presynaptic JNK-JIP1 interaction. Moreover, using knockout mice for single JNK isoforms, we proved that JNK2 is the essential isoform in mediating this presynaptic event. Overall the present findings unveil a novel JNK2 localization and function, which is likely to play a role in different physiological and pathological conditions.

Functional adaptation of presynaptic chemokine receptors in EAE mouse central nervous system.

In cortical synaptosomes of Experimental Autoimmune Encephalomyelitis (EAE) mice at the early stage of disease (13 days post immunization, d.p.i.), the Regulated upon Activation Normal T cell Expressed and Secreted (RANTES, CCL5)-mediated control of [3H]D-aspartate ([3H]D-ASP) exocytosis elicited by a mild depolarizing stimulus (12 mM KCl) shifted from inhibition to facilitation. By using selective antagonists for the chemokine receptor (CCR) 1, 3, and 5 subtypes, we found that the pharmacological profile of the receptor(s) accounting for CCL5-mediated effect was unaltered when compared to control. Inasmuch, CCR protein expression was unaltered. This studies was not extended at 21 d.p.i. since, at this stage, CCL5 failed to affect [3H]D-ASP exocytosis. At 13 d.p.i., the expression of CCR proteins was largely conserved when compared to control. In spinal cord synaptosomes of EAE mice at 21 d.p.i., when presynaptic defects became evident, the [3H]D-ASP exocytosis elicited by 15 mM KCl was significantly increased when compared to control and it was significantly potentiated by 1 nM CCL5. The antagonist pharmacological profile and the western blot analysis of the CCR proteins unveiled that the receptor repertoire involved was unmodified. Differently from controls, however, the CCR1 antagonist BX513 efficiently inhibited on its own [3H]D-ASP exocytosis suggesting that this receptor could have adopted an active conformation. Altogether, our observations favor the use of CCR antagonists to the cure of neurological symptoms in patients suffering from demyelinating syndrome.

Acute desipramine restores presynaptic cortical defects in murine experimental autoimmune encephalomyelitis by suppressing central CCL5 overproduction.

BACKGROUND AND PURPOSE: Altered glutamate exocytosis and cAMP production in cortical terminals of experimental autoimmune encephalomyelitis (EAE) mice occur at the early stage of disease (13 days post-immunization, d.p.i.). Neuronal defects were paralleled by overexpression of the central chemokine CCL5 (also known as RANTES), suggesting it has a role in presynaptic impairments. We propose that drugs able to restore CCL5 content to physiological levels could also restore presynaptic defects. Because of its efficacy in controlling CCL5 overexpression, desipramine (DMI) appeared to be a suitable candidate to test our hypothesis. EXPERIMENTAL APPROACH: Control and EAE mice at 13 d.p.i. were acutely or chronically administered DMI and monitored for behaviour and clinical scores. Noradrenaline and glutamate release, cAMP, CCL5 and TNF-α production were quantified in cortical synaptosomes and homogenates. Peripheral cytokine production was also determined. KEY RESULTS: Noradrenaline exocytosis and α2 -adrenoeceptor-mediated activity were unmodified in EAE mice at 13 d.p.i. when compared with control. Acute, but not chronic, DMI reduced CCL5 levels in cortical homogenates of EAE mice at 13 d.p.i., but did not affect peripheral IL-17 and TNF-α contents or CCL5 plasma levels. Acute DMI caused a long-lasting restoration of glutamate exocytosis, restored endogenous cAMP production and impeded the shift from inhibition to facilitation of the CCL5-mediated control of glutamate exocytosis. Finally, DMI ameliorated anxiety-related behaviour but not motor activity or severity of clinical signs. CONCLUSIONS: We propose DMI as an add-on therapy to normalize neuropsychiatric symptoms in multiple sclerosis patients at the early stage of the disease.

The effects of antidepressant treatment in prenatally stressed rats support the glutamatergic hypothesis of stress-related disorders.

Abnormalities of synaptic transmission in the hippocampus represent an integral part of the altered programming triggered by early life stress, which enhances the vulnerability to stress-related disorders in the adult life. Rats exposed to prenatal restraint stress (PRS) develop enduring biochemical and behavioral changes characteristic of an anxious/depressive-like phenotype. Most neurochemical abnormalities in PRS rats are found in the ventral hippocampus, a region that encodes memories related to stress and emotions. We have recently demonstrated a causal link between the reduction of glutamate release in the ventral hippocampus and anxiety-like behavior in PRS rats. To confer pharmacological validity to the glutamatergic hypothesis of stress-related disorders, we examined whether chronic treatment with two antidepressants with different mechanisms of action could correct the defect in glutamate release and associated behavioral abnormalities in PRS rats. Adult unstressed or PRS rats were treated daily with either agomelatine (40 mg/kg, i.p.) or fluoxetine (5 mg/kg, i.p.) for 21 d. Both treatments reversed the reduction in depolarization-evoked glutamate release and in the expression of synaptic vesicle-associated proteins in the ventral hippocampus of PRS rats. Antidepressant treatment also corrected abnormalities in anxiety-/depression-like behavior and social memory performance in PRS rats. The effect on glutamate release was strongly correlated with the improvement of anxiety-like behavior and social memory. These data offer the pharmacological demonstration that glutamatergic hypofunction in the ventral hippocampus lies at the core of the pathological phenotype caused by early life stress and represents an attractive pharmacological target for novel therapeutic strategies.

Complement selectively elicits glutamate release from nerve endings in different regions of mammal central nervous system.

Our study was aimed at investigating whether complement, a complex of soluble and membrane-associated serum proteins, could, in addition to its well-documented post-synaptic activity, also pre-synaptically affect the release of classic neurotransmitters in central nervous system (CNS). Complement (dilution 1 : 10 to 1 : 10000) elicited the release of preloaded [(3) H]-d-aspartate ([(3) H]d-ASP) and endogenous glutamate from mouse cortical synaptosomes in a dilution-dependent manner. It also evoked [(3) H]d-ASP release from mouse hippocampal, cerebellar, and spinal cord synaptosomes, as well as from rat and human cortical nerve endings, but left unaltered the release of GABA, [(3) H]noradrenaline or [(3) H]acetylcholine. Lowering external Na(+) (from 140 to 40 mM) or Ca(2+) (from 1.2 to 0.1 mM) ions prevented the 1 : 300 complement-evoked [(3) H]d-ASP release from mouse cortical synaptosomes. Complement-induced releasing effect was unaltered in synaptosomes entrapped with the Ca(2+) ions chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N', tetra-acetic acid or with pertussis toxin. Nifedipine,/ω-conotoxin GVIA/ω-conotoxin MVIIC mixture as well as the vesicular ATPase blocker bafilomycin A1 were also inefficacious. The excitatory amino acid transporter blocker DL-threo-ß-benzyloxyaspartic acid, on the contrary, reduced the complement-evoked releasing effect in a concentration-dependent manner. We concluded that complement-induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier-mediated release. Our observations afford new insights into the molecular events accounting for immune and CNS crosstalk. We investigated whether complement, a complex of soluble and membrane-associated serum proteins, could pre-synaptically affect the release of classic neurotransmitters in the central nervous system (CNS). Our data provide evidence that complement-induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier-mediated release. Our observations add new insights to the knowledge of the molecular events accounting for immune and CNS crosstalk. EAAT = excitatory amino acid transporter.

CCL5-glutamate interaction in central nervous system: Early and acute presynaptic defects in EAE mice.

We investigated the CCL5-glutamate interaction in the cortex and in the spinal cord from mice with Experimental Autoimmune Encephalomyelitis (EAE) at 13 and 21/30 days post immunization (d.p.i.), representing the onset and the peak of the disease, respectively. An early reduction of the KCl-evoked glutamate release was observed in cortical terminals from EAE mice at 13 d.p.i., persisting until 21/30 d.p.i. A concomitant reduction of the depolarization-evoked cyclic adenosine monophosphate (cAMP), but not of the inositol 1,4,5-trisphosphate (IP3) cortical production also occurred at 13 d.p.i, that still was detectable at the acute stage of disease (21 dp.i.). Inasmuch, the CCL5-mediated inhibition of glutamate exocytosis observed in control mice turned to facilitation in EAE mouse cortex at 13 d.p.i., then becoming undetectable at 21/30 d.p.i. Differently, glutamate exocytosis, as well as IP3 and cAMP productions were unaltered in spinal cord synaptosomes from EAE mice at 13 d.p.i., but significantly increased at 21/30 d.p.i., while the presynaptic CCL5-mediated facilitation of glutamate exocytosis observed in control mice remained unchanged. In both CNS regions, the presynaptic defects were parallelled by increased CCL5 availability. Inasmuch, the presynaptic defects so far described in EAE mice were reminiscent of the effects acute CCL5 exerts in control conditions. Based on these observations we propose that increased CCL5 bioavailability could have a role in determining the abovedescribed impaired presynaptic impairments in both CNS regions. These presynaptic defects could be relevant to the onset of early cognitive impairments and acute neuroinflammation and demyelinating processes observed in multiple sclerosis patients.

Effects of the neoclerodane Hardwickiic acid on the presynaptic opioid receptors which modulate noradrenaline and dopamine release in mouse central nervous system.

We have comparatively investigated the effects of Hardwickiic acid and Salvinorin A on the K(+)-evoked overflow of [(3)H]noradrenaline ([(3)H]NA) and [(3)H]dopamine ([(3)H]DA) from mouse hippocampal and striatal nerve terminals, respectively. The K(+)-evoked overflow of [(3)H]DA was inhibited in presence of Salvinorin A (100 nM) but not in presence of Hardwickiic acid (100 nM). Hardwickiic acid (100 nM) mimicked Salvinorin A (100 nM) in facilitating K(+)-evoked hippocampal [(3)H]NA overflow and the two compounds were almost equipotent. Facilitation of [(3)H]NA overflow caused by 100 nM Hardwickiic acid was prevented by the κ-opioid receptor (KOR) antagonist norbinaltorphimine (norBNI, 100 nM) and by the selective δ-opioid receptor (DOR) antagonist naltrindole (100 nM), but was not altered by 100 nM D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective µ-opioid receptor (MOR) antagonist. We conclude that Hardwickiic acid modulates hippocampal [(3)H]NA overflow evoked by a mild depolarizing stimulus by acting at presynaptic opioid receptor subtypes.

Presynaptic mGlu7 receptors control GABA release in mouse hippocampus.

The functional role of presynaptic release-regulating metabotropic glutamate type 7 (mGlu7) receptors in hippocampal GABAergic terminals was investigated. Mouse hippocampal synaptosomes were preloaded with [(3)H]D-γ-aminobutyric acid ([(3)H]GABA) and then exposed in superfusion to 12 mM KCl. The K(+)-evoked [(3)H]GABA release was inhibited by the mGlu7 allosteric agonist N,N'-dibenzyhydryl-ethane-1,2-diamine dihydrochloride (AMN082, 0.001-10 µM), as well as by the group III mGlu receptor agonist l-(+)-2-amino-4-phosphonobutyric acid [(l)-AP4, 0.01-1 mM]. The mGlu8 receptor agonist (S)-3,4-dicarboxyphenylglycine [(S)-3,4-DCPG, 10-100 nM] was ineffective. AMN082 and (l)-AP4-induced effects were recovered by the mGlu7 negative allosteric modulator (NAM) 6-(4-methoxyphenyl)-5-methyl-3-(4-pyridinyl)-isoxazolo[4,5-c]pyridin-4(5H)-one hydrochloride (MMPIP). AMN082 also inhibited in a MMPIP-sensitive manner the K(+)-evoked release of endogenous GABA. AMN082 and the adenylyl cyclase (AC) inhibitor MDL-12,330A reduced [(3)H]GABA exocytosis in a 8-Br-cAMP-sensitive. AMN082-inhibitory effect was additive to that caused by (-)baclofen, but insensitive to the GABA(B) antagonist 3-[[(3,4-Dichlorophenyl)methyl]amino]propyl] diethoxymethyl) phosphinic acid (CGP52432). Conversely, (-)baclofen-induced inhibition of GABA exocytosis was insensitive to MMPIP. Finally, the forskolin-evoked [(3)H]GABA release was reduced by AMN082 or (-)baclofen but abolished when the two agonists were added concomitantly. Mouse hippocampal synaptosomal plasmamembranes posses mGlu7 receptor proteins; confocal microscopy analysis unveiled that mGlu7 proteins colocalize with syntaxin-1A (Stx-1A), with vesicular GABA transporter (VGAT)-proteins and with GABA(B) receptor subunit proteins. We propose that presynaptic inhibitory mGlu7 heteroreceptors, negatively coupled to AC-dependent intraterminal pathway, exist in mouse hippocampal GABA-containing terminals, where they colocalize, but do not functionally cross-talk, with GABA(B) autoreceptors. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Group I metabotropic glutamate autoreceptors induce abnormal glutamate exocytosis in a mouse model of amyotrophic lateral sclerosis.

Glutamate-mediated excitotoxicity plays a major role in ALS and reduced astrocytic glutamate transport was suggested as a cause. Based on previous work we have proposed that abnormal release may represent another source of excessive glutamate. In this line, here we studied the modulation of glutamate release in ALS by Group I metabotropic glutamate (mGlu) receptors, that comprise mGlu1 and mGlu5 members. Synaptosomes from the lumbar spinal cord of SOD1/G93A mice, a widely used murine model for human ALS, and controls were used in release, confocal or electron microscopy and Western blot experiments. Concentrations of the mGlu1/5 receptor agonist 3,5-DHPG >0.3 µM stimulated the release of [(3)H]d- aspartate, used to label the releasing pools of glutamate, both in control and SOD1/G93A mice. At variance, ≤0.3 µM 3,5-DHPG increased [(3)H]d-aspartate release in SOD1/G93A mice only. Experiments with selective antagonists indicated the involvement of both mGlu1 and mGlu5 receptors, mGlu5 being preferentially involved in the high potency effects of 3,5-DHPG. High 3,5-DHPG concentrations increased IP3 formation in both mouse strains, whereas low 3,5-DHPG did it in SOD1/G93A mice only. Release experiments confirmed that 3,5-DHPG elicited [(3)H]d-aspartate exocytosis involving intra-terminal Ca(2+) release through IP3-sensitive channels. Confocal microscopy indicated the co-existence of both receptors presynaptically in the same glutamatergic nerve terminal in SOD1/G93A mice. To conclude, activation of mGlu1/5 receptors produced abnormal glutamate release in SOD1/G93A mice, suggesting that these receptors are implicated in ALS and that selective antagonists may be predicted for new therapeutic approaches. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

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 Ca²âº-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.

RANTES-mediated control of excitatory amino acid release in mouse spinal cord.

The impact of Regulated upon Activation Normal T cells Expressed and Secreted (RANTES) on the release of pre-loaded [³H]D-aspartate ([³H]D-ASP) from mouse spinal cord synaptosomes was investigated. RANTES (0.01-1 nM) failed to affect the spontaneous release, but facilitated the 15 mM K⁺-evoked overflow of [³H]D-ASP. Incubation of synaptosomes with antibodies raised against the chemokine receptor (CCR)1 and CCR5 proteins prevented RANTES-induced facilitation of glutamate exocytosis, whereas anti-CCR3 antibody was inefficacious. Accordingly, BX513 and D-Ala-peptide T-amide (DAPTA) CCR1 and CCR5 antagonists, respectively, prevented RANTES-induced effect, whereas the CCR3 antagonist SB 328437 was inactive. To compare these findings to previous results, we quantified the effects of CCR antagonists on the RANTES-induced modifications of the spontaneous and the K⁺-evoked [³H]D-ASP release in the mouse cortex. Here, CCR1 and CCR5, but not CCR3, antagonists prevented the RANTES-mediated [³H]D-ASP release, whereas RANTES-induced inhibition of the 12 mM K⁺-evoked [³H]D-ASP exocytosis was also antagonized by SB 328437. Facilitation of glutamate exocytosis in spinal cord relied on PLC-dependent mobilization of Ca²âº from IP3-sensitive stores; adenylyl cyclase was not involved. CCR1, CCR3 and CCR5 receptor proteins were present in spinal cord synaptosomal and gliosomal lysates, although RANTES-induced changes to glutamate release could not be observed in gliosomes. Our results confirm the role of RANTES as modulator of glutamate transmission.

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 [(3)H]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 [(3)H]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 [(3)H]D-ASP release was concentration-dependently increased by (S)AMPA. Cyclothiazide potentiated the 10 µM NMDA/1 µM glycine/50 µM (S)AMPA-induced [(3)H]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 [(3)H]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 [(3)H]D-ASP, but leaved unmodified that caused by NMDA/glycine. The NBQX-sensitive, 50 µM (S)AMPA-evoked release of [(3)H]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.

Glycine release provoked by disturbed Na⁺, Na⁺ and Ca²âº homeostasis in cerebellar nerve endings: roles of Ca²âº channels, Na⁺/Ca²âº exchangers and GlyT2 transporter reversal.

Glycine release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in cerebellar synaptosomes selectively pre-labelled with [³H]glycine through GlyT2 transporters and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were largely external Ca²âº-dependent. Higher concentrations of KCl (50 mM) or veratridine (10 µM), but not of 4-AP (1 mM), involved also external Ca²âº-independent mechanisms. GlyT1-mediated release could not be observed; only the external Ca²âº-independent veratridine-evoked overflow occurred significantly by GlyT2 reversal. None of the three depolarizing agents activated store-operated or transient receptor potential or L-type Ca²âº channels. The overflows caused by KCl or 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca²âº-dependent overflow evoked by KCl or 4-AP (and all that caused by veratridine) were mediated by reverse plasmalemmal Na⁺/Ca²âº exchangers. Significant contribution to the overflows evoked by KCl or veratridine came from Ca²âº originated through mitochondrial Na⁺/Ca²âº exchangers. Ca²âº-induced Ca²âº release (CICR) mediated by inositoltrisphosphate receptors (InsP3Rs) represents the final trigger of the glycine release evoked by high KCl. The overflows evoked by 4-AP or, less so, by veratridine also involved InsP3R-mediated CICR and, in part, CICR mediated by ryanodine receptors. To conclude, ionic dysregulations typical of ischemia and epilepsy caused glycine release in cerebellum by multiple differential mechanisms that may represent potential therapeutic targets.

Ionic dysregulations typical of ischemia provoke release of glycine and GABA by multiple mechanisms.

Energy deprivation during ischemia causes dysregulations of ions, particularly sodium, potassium and calcium. Under these conditions, release of neurotransmitters is often enhanced and can occur by multiple mechanisms. The aim of this work was to characterize the modes of exit of glycine and GABA from nerve endings exposed to stimuli known to reproduce some of the ionic changes typical of ischemic conditions. Their approach was chosen instead of application of ischemic conditions because the release evoked during ischemia is mechanistically too heterogeneous. Mouse hippocampus and spinal cord synaptosomes, pre-labeled with [(3)H]glycine or [(3)H]GABA, were exposed in superfusion to 50 mM KCl or to 10 microM veratridine. The evoked overflows differed greatly between the two transmitters and between the two regions examined. Significant portions of the K(+)- and the veratridine-evoked overflows occurred by classical exocytosis. Carrier-mediated release of GABA, but not of glycine, was evoked by high K(+); GABA and, less so, glycine were released through transporter reversal by veratridine. External calcium-dependent overflows were only in part sensitive to omega-conotoxins; significant portions occurred following reversal of the plasmalemmal Na(+)/Ca(2+) exchanger. Finally, a relevant contribution to the overall transmitter overflows came from cytosolic calcium originating through the mitochondrial Na(+)/Ca(2+) exchanger. To conclude, ionic dysregulations typical of ischemia cause neurotransmitter release by heterogeneous mechanisms that differ depending on the transmitters and the CNS regions examined.