Anti-NMDA and Anti-AMPA Receptor Antibodies in Central Disorders: Preclinical Approaches to Assess Their Pathological Role and Translatability to Clinic.

Autoantibodies against NMDA and AMPA receptors have been identified in the central nervous system of patients suffering from brain disorders characterized by neurological and psychiatric symptoms. It has been demonstrated that these autoantibodies can affect the functions and/or the expression of the targeted receptors, altering synaptic communication. The importance to clarify, in preclinical models, the molecular mechanisms involved in the autoantibody-mediated effects has emerged in order to understand their pathogenic role in central disorders, but also to propose new therapeutic approaches for preventing the deleterious central consequences. In this review, we describe some of the available preclinical literature concerning the impact of antibodies recognizing NMDA and AMPA receptors in neurons. This review discusses the cellular events that would support the detrimental roles of the autoantibodies, also illustrating some contrasting findings that in our opinion deserve attention and further investigations before translating the preclinical observations to clinic.

Healthy Properties of a New Formulation of Pomegranate-Peel Extract in Mice Suffering from Experimental Autoimmune Encephalomyelitis.

A new formulation of a pomegranate-peel extract (PEm) obtained by PUAE (Pulsed Ultrasound-Assisted Extraction) and titrated in both ellagic acid (EA) and punicalagin is proposed, characterized and then analyzed for potential health properties in mice suffering from the experimental autoimmune encephalomyelitis (EAE). PEm effects were compared to those elicited by a formulation containing EA (EAm). Control and EAE mice were chronically administered EAm and Pem dissolved in the drinking water, starting from the day 10 post-immunization (d.p.i.), with a "therapeutic" protocol to deliver daily 50 mg/kg of EA. Treated EAE mice did not limit their daily access to the beverage, nor did they show changes in body weight, but they displayed a significant amelioration of "in vivo" clinical symptoms. "Ex vivo" histochemical analysis showed that spinal-cord demyelination and inflammation in PEm and EAm-treated EAE mice at 23 ± 1 d.p.i. were comparable to those in the untreated EAE animals, while microglia activation (measured as Ionized Calcium Binding Adaptor 1, Iba1 staining) and astrocytosis (quantified as glial fibrillar acid protein, GFAP immunopositivity) significantly recovered, particularly in the gray matter. EAm and PEm displayed comparable efficiencies in controlling the spinal pathological cellular hallmarks in EAE mice, and this would support their delivery as dietary supplementation in patients suffering from multiple sclerosis (MS).

Somatostatin, a Presynaptic Modulator of Glutamatergic Signal in the Central Nervous System.

Somatostatin is widely diffused in the central nervous system, where it participates to control the efficiency of synaptic transmission. This peptide mainly colocalizes with GABA, in inhibitory, GABA-containing interneurons from which it is actively released in a Ca2+ dependent manner upon application of depolarizing stimuli. Once released in the synaptic cleft, somatostatin acts locally, or it diffuses in the extracellular space through "volume diffusion", a mechanism(s) of distribution which mainly operates in the cerebrospinal fluid and that assures the progression of neuronal signalling from signal-secreting sender structures towards receptor-expressing targeted neurons located extrasynaptically, in a non-synaptic, inter-neuronal form of communication. Somatostatin controls the efficiency of central glutamate transmission by either modulating presynaptically the glutamate exocytosis or by metamodulating the activity of glutamate receptors colocalized and functionally coupled with somatostatin receptors in selected subpopulations of nerve terminals. Deciphering the role of somatostatin in the mechanisms of "volume diffusion" and in the "receptor-receptor interaction" unveils new perspectives in the central role of this fine tuner of synaptic strength, paving the road to new therapeutic approaches for the cure of central disorders.

Neuroinflammation in Aged Brain: Impact of the Oral Administration of Ellagic Acid Microdispersion.

The immune system and the central nervous system message each other to preserving central homeostasis. Both systems undergo changes during aging that determine central age-related defects. Ellagic acid (EA) is a natural product which is beneficial in both peripheral and central diseases, including aging. We analyzed the impact of the oral administration of a new oral ellagic acid micro-dispersion (EAm), that largely increased the EA solubility, in young and old mice. Oral EAm did not modify animal weight and behavioral skills in young and old mice, but significantly recovered changes in "ex-vivo, in vitro" parameters in old animals. Cortical noradrenaline exocytosis decreased in aged mice. EAm administration did not modify noradrenaline overflow in young animals, but recovered it in old mice. Furthermore, GFAP staining was increased in the cortex of aged mice, while IBA-1 and CD45 immunopositivities were unchanged when compared to young ones. EAm treatment significantly reduced CD45 signal in both young and old cortical lysates; it diminished GFAP immunopositivity in young mice, but failed to affect IBA-1 expression in both young and old animals. Finally, EAm treatment significantly reduced IL1beta expression in old mice. These results suggest that EAm is beneficial to aging and represents a nutraceutical ingredient for elders.

Complement tunes glutamate release and supports synaptic impairments in an animal model of multiple sclerosis.

BACKGROUND AND PURPOSE: To deepen our knowledge of the role of complement in synaptic impairment in experimental autoimmune encephalomyelitis (EAE) mice, we investigated the distribution of C1q and C3 proteins and the role of complement as a promoter of glutamate release in purified nerve endings (synaptosomes) and astrocytic processes (gliosomes) isolated from the cortex of EAE mice at the acute stage of the disease (21 ± 1 day post-immunization). EXPERIMENTAL APPROACH: EAE cortical synaptosomes and gliosomes were analysed for glutamate release efficiency (measured as release of preloaded [3H]D-aspartate ([3H]D-ASP)), C1q and C3 protein density, and for viability and ongoing apoptosis. KEY RESULTS: In healthy mice, complement releases [3H]D-ASP from gliosomes more efficiently than from synaptosomes. The releasing activity occurs in a dilution-dependent manner and involves the reversal of the excitatory amino acid transporters (EAATs). In EAE mice, the complement-induced releasing activity is significantly reduced in cortical synaptosomes but amplified in cortical gliosomes. These adaptations are paralleled by decreased density of the EAAT2 protein in synaptosomes and increased EAAT1 staining in gliosomes. Concomitantly, PSD95, GFAP, and CD11b, but not SNAP25, proteins are overexpressed in the cortex of the EAE mice. Similarly, C1q and C3 protein immunostaining is increased in EAE cortical synaptosomes and gliosomes, although signs of ongoing apoptosis or altered viability are not detectable. CONCLUSION AND IMPLICATIONS: Our results unveil a new noncanonical role of complement in the CNS of EAE mice relevant to disease progression and central synaptopathy that suggests new therapeutic targets for the management of MS.

Progress in metamodulation and receptor-receptor interaction: From physiology to pathology and therapy.

The organization and the role of receptor-receptor interaction (RRI) and metamodulation in physiological conditions have been extensively analyzed and discussed. In this Special Issue of Neuropharmacology, we review recent advances in the understanding of the RRI and the mechanisms underlying its adaptation that could be relevant to the etiopathogenesis of central neuropsychiatric disorders, as well as to the development of new therapeutic approaches to control the activity and to restore the physiological functions, posing the basis for new targeted pharmacological interventions.

Metamodulation of presynaptic NMDA receptors: New perspectives for pharmacological interventions.

Metamodulation shifted the scenario of the central neuromodulation from a simplified unimodal model to a multimodal one. It involves different receptors/membrane proteins physically associated or merely colocalized that act in concert to control the neuronal functions influencing each other. Defects or maladaptation of metamodulation would subserve neuropsychiatric disorders or even synaptic adaptations relevant to drug dependence. Therefore, this "vulnerability" represents a main issue to be deeply analyzed to predict its aetiopathogenesis, but also to propose targeted pharmaceutical interventions. The review focusses on presynaptic release-regulating NMDA receptors and on some of the mechanisms of their metamodulation described in the literature. Attention is paid to the interactors, including both ionotropic and metabotropic receptors, transporters and intracellular proteins, which metamodulate their responsiveness in physiological conditions but also undergo adaptation that are relevant to neurological dysfunctions. All these structures are attracting more and more the interest as promising druggable targets for the treatment of NMDA receptor-related central diseases: these substances would not exert on-off control of the colocalized NMDA receptors (as usually observed with NMDA receptor full agonists/antagonists), but rather modulate their functions, with the promise of limiting side effects that would favor their translation from preclinic to clinic. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Presynaptic Release-Regulating Sphingosine 1-Phosphate 1/3 Receptors in Cortical Glutamatergic Terminals: Adaptations in EAE Mice and Impact of Therapeutic FTY720.

This study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings (synaptosomes) of healthy mice. The conclusion relies on the findings that (i) the S1P1R agonist CS-2100 (0.1-30 nM) inhibits the 12 mM KCl-evoked glutamate exocytosis (quantified as the release of [3H]D-aspartate) while the S1P3R allosteric agonist CYM-5541 potentiates it and (ii) these effects are inhibited by the S1P1R antagonist Ex 26 (30-300 nM) and the S1P3R antagonist TY-52156 (100-1000 nM), respectively. Confocal microscopy and western blot analysis confirmed the presence of S1P1R and S1P3R proteins in cortical glutamatergic synaptosomes, which were scarcely accessible to biotin in a biotinylation study. Then, we demonstrated that S1P1R and S1P3R densities and their release activity are amplified in cortical synaptosomes of mice suffering from experimental autoimmune encephalomyelitis (EAE), despite receptors maintain their preferential internal distribution. Receptor changes recover following chronic oral therapeutic FTY720 (0.03 mg/Kg/day). These results improve our knowledge of the role of presynaptic release-regulating S1P1Rs and S1P3Rs controlling glutamate transmission in the CNS also unravelling functional adaptations during EAE that recover following chronic FTY720. In a whole, these findings provide new information on the central neuroprotectant activities of FTY720.

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.].

The Anti-Aggregative Peptide KLVFF Mimics Aß1-40 in the Modulation of Nicotinic Receptors: Implications for Peptide-Based Therapy.

In recent years, the inhibition of beta-amyloid (Aß) aggregation has emerged as a potential strategy for Alzheimer's disease. KLVFF, a small peptide corresponding to the aminoacidic sequence 16-20 of Aß, reduces Aß fibrillation dose dependently. Therefore, the toxic and functional characterization of its brain activity is fundamental for clarifying its potential therapeutic role. Accordingly, we studied the modulatory role of KLVFF on the cholinergic receptors regulating dopamine and noradrenaline release in rat synaptosomes. Nicotinic receptors on dopaminergic nerve terminals in the nucleus acccumbens are inhibited by KLVFF, which closely resembles full-length Aß1-40. Moreover, KLVFF entrapped in synaptosomes does not modify the nicotinic receptor's function, suggesting that external binding to the receptor is required for its activity. The cholinergic agent desformylflustrabromine counteracts the KLVFF effect. Remarkably, muscarinic receptors on dopaminergic terminals and nicotinic receptors regulating noradrenaline release in the hippocampus are completely insensitive to KLVFF. Based on our findings, KLVFF mimics Aß1-40 as a negative modulator of specific nicotinic receptor subtypes affecting dopamine transmission in the rat brain. Therefore, new pharmacological strategies using the anti-aggregative properties of KLVFF need to be evaluated for potential interference with nicotinic receptor-mediated transmission.

Presynaptic 5-HT2A-mGlu2/3 Receptor-Receptor Crosstalk in the Prefrontal Cortex: Metamodulation of Glutamate Exocytosis.

The glutamatergic nerve endings of a rat prefrontal cortex (PFc) possess presynaptic 5-HT2A heteroreceptors and mGlu2/3 autoreceptors, whose activation inhibits glutamate exocytosis, and is measured as 15 mM KCl-evoked [3H]D-aspartate ([3H]D-asp) release (which mimics glutamate exocytosis). The concomitant activation of the two receptors nulls their inhibitory activities, whereas blockade of the 5-HT2A heteroreceptors with MDL11,939 (1 µM) strengthens the inhibitory effect elicited by the mGlu2/3 receptor agonist LY329268 (1 µM). 5-HT2A receptor antagonists (MDL11,939; ketanserin; trazodone) amplify the impact of low (3 nM) LY379268. Clozapine (0.1-10 µM) mimics the 5-HT2A agonist (±) DOI and inhibits the KCl-evoked [3H]D-asp overflow in a MDL11,939-dependent fashion, but does not modify the (±) DOI-induced effect. mGlu2 and 5-HT2A proteins do not co-immunoprecipitate from synaptosomal lysates, nor does the incubation of PFc synaptosomes with MDL11,939 (1 µM) or clozapine (10 µM) modify the insertion of mGlu2 subunits in synaptosomal plasma membranes. In conclusion, 5-HT2A and mGlu2/3 receptors colocalize, but do not physically associate, in PFc glutamatergic terminals, where they functionally interact in an antagonist-like fashion to control glutamate exocytosis. The mGlu2/3-5-HT2A metamodulation could be relevant to therapy for central neuropsychiatric disorders, including schizophrenia, but also unveil cellular events accounting for their development, which also influence the responsiveness to drugs regimens.

Efficacy of a vegetal mixture composed of Zingiber officinale, Echinacea purpurea, and Centella asiatica in a mouse model of neuroinflammation: In vivo and ex vivo analysis.

Experimental evidence suggests that neuroinflammation is a key pathological event of many diseases affecting the nervous system. It has been well recognized that these devastating illnesses (e.g., Alzheimer's, Parkinson's, depression, and chronic pain) are multifactorial, involving many pathogenic mechanisms, reason why pharmacological treatments are unsatisfactory. The purpose of this study was to evaluate the efficacy of a vegetal mixture capable of offering a multiple approach required to manage the multifactoriality of neuroinflammation. A mixture composed of Zingiber officinale (150 mg kg-1), Echinacea purpurea (20 mg kg-1), and Centella asiatica (200 mg kg-1) was tested in a mouse model of systemic neuroinflammation induced by lipopolysaccharide (LPS, 1 mg kg-1). Repeated treatment with the vegetal mixture was able to completely counteract thermal and mechanical allodynia as reported by the Cold plate and von Frey tests, respectively, and to reduce the motor impairments as demonstrated by the Rota rod test. Moreover, the mixture was capable of neutralizing the memory loss in the Passive avoidance test and reducing depressive-like behavior in the Porsolt test, while no efficacy was shown in decreasing anhedonia as demonstrated by the Sucrose preference test. Finally, LPS stimulation caused a significant increase in the activation of glial cells, of the central complement proteins and of inflammatory cytokines in selected regions of the central nervous system (CNS), which were rebalanced in animals treated with the vegetal mixture. In conclusion, the vegetal mixture tested thwarted the plethora of symptoms evoked by LPS, thus being a potential candidate for future investigations in the context of neuroinflammation.

Presynaptic Release-Regulating Alpha2 Autoreceptors: Potential Molecular Target for Ellagic Acid Nutraceutical Properties.

Polyphenol ellagic acid (EA) possesses antioxidant, anti-inflammatory, anti-carcinogenic, anti-diabetic and cardio protection activities, making it an interesting multi-targeting profile. EA also controls the central nervous system (CNS), since it was proven to reduce the immobility time of mice in both the forced swimming and the tail-suspension tests, with an efficiency comparable to that of classic antidepressants. Interestingly, the anti-depressant-like effect was almost nulled by the concomitant administration of selective antagonists of the noradrenergic receptors, suggesting the involvement of these cellular targets in the central effects elicited by EA and its derivatives. By in silico and in vitro studies, we discuss how EA engages with human α2A-ARs and α2C-AR catalytic pockets, comparing EA behaviour with that of known agonists and antagonists. Structurally, the hydrophobic residues surrounding the α2A-AR pocket confer specificity on the intermolecular interactions and hence lead to favourable binding of EA in the α2A-AR, with respect to α2C-AR. Moreover, EA seems to better accommodate within α2A-ARs into the TM5 area, close to S200 and S204, which play a crucial role for activation of aminergic GPCRs such as the α2-AR, highlighting its promising role as a partial agonist. Consistently, EA mimics clonidine in inhibiting noradrenaline exocytosis from hippocampal nerve endings in a yohimbine-sensitive fashion that confirms the engagement of naïve α2-ARs in the EA-mediated effect.

The Depolarization-Evoked, Ca2+-Dependent Release of Exosomes From Mouse Cortical Nerve Endings: New Insights Into Synaptic Transmission.

Whether exosomes can be actively released from presynaptic nerve terminals is a matter of debate. To address the point, mouse cortical synaptosomes were incubated under basal and depolarizing (25 mM KCl-enriched medium) conditions, and extracellular vesicles were isolated from the synaptosomal supernatants to be characterized by dynamic light scattering, transmission electron microscopy, Western blot, and flow cytometry analyses. The structural and biochemical analysis unveiled that supernatants contain vesicles that have the size and the shape of exosomes, which were immunopositive for the exosomal markers TSG101, flotillin-1, CD63, and CD9. The marker content increased upon the exposure of nerve terminals to the high-KCl stimulus, consistent with an active release of the exosomes from the depolarized synaptosomes. High KCl-induced depolarization elicits the Ca2+-dependent exocytosis of glutamate. Interestingly, the depolarization-evoked release of exosomes from cortical synaptosomes also occurred in a Ca2+-dependent fashion, since the TSG101, CD63, and CD9 contents in the exosomal fraction isolated from supernatants of depolarized synaptosomes were significantly reduced when omitting external Ca2+ ions. Differently, (±)-baclofen (10 µM), which significantly reduced the glutamate exocytosis, did not affect the amount of exosomal markers, suggesting that the GABAB-mediated mechanism does not control the exosome release. Our findings suggest that the exposure of synaptosomes to a depolarizing stimulus elicits a presynaptic release of exosomes that occurs in a Ca2+-dependent fashion. The insensitivity to the presynaptic GABAB receptors, however, leaves open the question on whether the release of exosomes could be a druggable target for new therapeutic intervention for the cure of synaptopathies.

Antibodies Against the NH2-Terminus of the GluA Subunits Affect the AMPA-Evoked Releasing Activity: The Role of Complement.

Antibodies recognizing the amino-terminal domain of receptor subunit proteins modify the receptor efficiency to controlling transmitter release in isolated nerve endings (e.g., synaptosomes) indirectly confirming their presence in these particles but also allowing to speculate on their subunit composition. Western blot analysis and confocal microscopy unveiled the presence of the GluA1, GluA2, GluA3, and GluA4 receptor subunits in cortical synaptosomes. Functional studies confirmed the presence of presynaptic release-regulating AMPA autoreceptors in these terminals, whose activation releases [3H]D-aspartate ([3H]D-Asp, here used as a marker of glutamate) in a NBQX-dependent manner. The AMPA autoreceptors traffic in a constitutive manner, since entrapping synaptosomes with the pep2-SVKI peptide (which interferes with the GluA2-GRIP1/PICK1 interaction) amplified the AMPA-evoked releasing activity, while the inactive pep2-SVKE peptide was devoid of activity. Incubation of synaptosomes with antibodies recognizing the NH2 terminus of the GluA2 and the GluA3 subunits increased, although to a different extent, the GluA2 and 3 densities in synaptosomal membranes, also amplifying the AMPA-evoked glutamate release in a NBQX-dependent fashion. We then analyzed the releasing activity of complement (1:300) from both treated and untreated synaptosomes and found that the complement-induced overflow occurred in a DL-t-BOA-sensitive, NBQX-insensitive fashion. We hypothesized that anti-GluA/GluA complexes in neuronal membranes could trigger the classic pathway of activation of the complement, modifying its releasing activity. Accordingly, the complement-evoked release of [3H]D-Asp from antiGluA2 and anti-GluA3 antibody treated synaptosomes was significantly increased when compared to untreated terminals and facilitation was prevented by omitting the C1q component of the immunocomplex. Antibodies recognizing the NH2 terminus of the GluA1 or the GluA4 subunits failed to affect both the AMPA and the complement-evoked tritium overflow. Our results suggest the presence of GluA2/GluA3-containing release-regulating AMPA autoreceptors in cortical synaptosomes. Incubation of synaptosomes with commercial anti-GluA2 or anti-GluA3 antibodies amplifies the AMPA-evoked exocytosis of glutamate through a complement-independent pathway, involving an excessive insertion of AMPA autoreceptors in plasma membranes but also affects the complement-dependent releasing activity, by promoting the classic pathway of activation of the immunocomplex. Both events could be relevant to the development of autoimmune diseases typified by an overproduction of anti-GluA subunits.

Presynaptic Release-regulating Metabotropic Glutamate Receptors: An Update.

Metabotropic glutamate (mGlu) receptors represent the largest family of glutamate receptors in mammals and act as fine tuners of the chemical transmission in central nervous system (CNS). In the last decade, results concerning the expression and the subcellular localization of mGlu receptors further clarified their role in physio-pathological conditions. Concomitantly, their pharmacological characterization largely improved thanks to the identification of new compounds (chemical ligands and antibodies recognizing epitopic sequences of the receptor proteins) that allowed to decipher the protein compositions of the naive receptors. mGlu receptors are expressed at the presynaptic site of chemical synapses. Here, they modulate intraterminal enzymatic pathways controlling the migration and the fusion of vesicles to synaptic membranes as well as the phosphorylation of colocalized receptors. Both the control of transmitter exocytosis and the phosphorylation of colocalized receptors elicited by mGlu receptors are relevant events that dictate the plasticity of nerve terminals, and account for the main role of presynaptic mGlu receptors as modulators of neuronal signalling. The role of the presynaptic mGlu receptors in the CNS has been the matter of several studies and this review aims at briefly summarizing the recent observations obtained with isolated nerve endings (we refer to as synaptosomes). We focus on the pharmacological characterization of these receptors and on their receptor-receptor interaction / oligo-dimerization in nerve endings that could be relevant to the development of new therapeutic approaches for the cure of central pathologies.

Acute Low Dose of Trazodone Recovers Glutamate Release Efficiency and mGlu2/3 Autoreceptor Impairments in the Spinal Cord of Rats Suffering From Chronic Sciatic Ligation.

We investigated whether chronic sciatic ligation modifies the glutamate release in spinal cord nerve endings (synaptosomes) as well as the expression and the function of presynaptic release-regulating mGlu2/3 autoreceptors and 5-HT2A heteroreceptors in these particles. Synaptosomes were from the spinal cord of animals suffering from the sciatic ligation that developed on day 6 post-surgery a significant decrease of the force inducing paw-withdrawal in the lesioned paw. The exocytosis of glutamate (quantified as release of preloaded [3H]D-aspartate, [3H]D-Asp) elicited by a mild depolarizing stimulus (15 mM KCl) was significantly increased in synaptosomes from injured rats when compared to controls (uninjured rats). The mGlu2/3 agonist LY379268 (1000 pM) significantly inhibited the 15 mM KCl-evoked [3H]D-Asp overflow from control synaptosomes, but not in terminals isolated from injured animals. Differently, a low concentration (10 nM) of (±) DOI, unable to modify the 15 mM KCl-evoked [3H]D-Asp overflow in control spinal cord synaptosomes, significantly reduced the glutamate exocytosis in nerve endings isolated from the injured rats. Acute oral trazodone (TZD, 0.3 mg/kg on day 7 post-surgery) efficiently recovered glutamate exocytosis as well as the efficiency of LY379268 in inhibiting this event in spinal cord synaptosomes from injured animals. The sciatic ligation significantly reduced the expression of mGlu2/3, but not of 5-HT2A, receptor proteins in spinal cord synaptosomal lysates. Acute TZD recovered this parameter. Our results support the use of 5-HT2A antagonists for restoring altered spinal cord glutamate plasticity in rats suffering from sciatic ligation.

Environmental Training and Synaptic Functions in Young and Old Brain: A Presynaptic Perspective.

BACKGROUND: Aging is an unavoidable, physiological process that reduces the complexity and the plasticity of the synaptic contacts in Central Nervous System (CNS), having profound implications for human well-being. The term "cognitive reserve" refers to central cellular adaptations that augment the resilience of human brain to damage and aging. The term "Cognitive training" indicates the cultural, social and physical stimulations proposed as add-on therapy for the cure of central neurological diseases. "Cognitive training" reinforces the "cognitive reserve" permitting to counteract brain impairments and rejuvenating synaptic complexity. The research has begun investigating the clinical impact of the "cognitive training" in aged people, but additional work is needed to definitively assess its effectiveness. In particular, there is a need to understand, from a preclinical point of view, whether "cognitive training" promotes compensatory effects or, alternatively, if it elicits genuine recovery of neuronal defects. Although the translation from rodent studies to the clinical situation could be difficult, the results from pre-clinical models are of high clinical relevance, since they should allow a better understanding of the effects of environmental interventions in aging-associated chronic derangements in mammals. CONCLUSION: Data in literature and the recent results obtained in our laboratory concerning the impact of environmental stimulation on the presynaptic release of noradrenaline, glutamate and gamma amino butyric acid (GABA) suggest that these neurotransmitters undergo different adaptations during aging and that they are differently tuned by "cognitive training". The impact of "cognitive training" on neurotransmitter exocytosis might account for the cellular events involved in reinforcement of "cognitive reserve" in young and old animals.

Immuno-Pharmacological Characterization of Presynaptic GluN3A-Containing NMDA Autoreceptors: Relevance to Anti-NMDA Receptor Autoimmune Diseases.

Mouse hippocampal glutamatergic nerve endings express presynaptic release-regulating NMDA autoreceptors (NMDARs). The presence of GluN1, GluN2A, GluN2B, and GluN3A subunits in hippocampal vesicular glutamate transporter type 1-positive synaptosomes was confirmed with confocal microscopy. GluN2C, GluN2D, and GluN3B immunopositivity was scarcely present. Incubation of synaptosomes with the anti-GluN1, the anti-GluN2A, the anti-GluN2B, or the anti-GluN3A antibody prevented the 30 µM NMDA/1 µM glycine-evoked [3H]D-aspartate ([3H]D-ASP) release. The NMDA/glycine-evoked [3H]D-ASP release was reduced by increasing the external protons, consistent with the participation of GluN1 subunits lacking the N1 cassette to the receptor assembly. The result also excludes the involvement of GluN1/GluN3A dimers into the NMDA-evoked overflow. Complement (1:300) released [3H]D-ASP in a dizocilpine-sensitive manner, suggesting the participation of a NMDAR-mediated component in the releasing activity. Accordingly, the complement-evoked glutamate overflow was reduced in anti-GluN-treated synaptosomes when compared to the control. We speculated that incubation with antibodies had favored the internalization of NMDA receptors. Indeed, a significant reduction of the GluN1 and GluN2B proteins in the plasma membranes of anti-GluN1 or anti-GluN2B antibody-treated synaptosomes emerged in biotinylation studies. Altogether, our findings confirm the existence of presynaptic GluN3A-containing release-regulating NMDARs in mouse hippocampal glutamatergic nerve endings. Furthermore, they unveil presynaptic alteration of the GluN subunit insertion in synaptosomal plasma membranes elicited by anti-GluN antibodies that might be relevant to the central alterations occurring in patients suffering from autoimmune anti-NMDA diseases.

Prolonged activation of CXCR4 hampers the release-regulating activity of presynaptic NMDA receptors in rat hippocampal synaptosomes.

We investigated the impact of the prolonged exposure of rat hippocampal synaptosomes to CXCL12 (3 nM) on the NMDA-mediated release of [3H]D-aspartate ([3H]D-Asp) or [3H]noradrenaline ([3H]NA). Synaptosomes were stimulated twice with NMDA/CXCL12 and the amount of the NMDA-evoked tritium release (S1 and S2) quantified to calculate the S2/S1 ratio. The S2/S1 ratio for both transmitters was drastically decreased by 3 nM CXCL12 between the two stimuli (CXCL12-treated synaptosomes) in a AMD3100-sensitive manner. The phosphorylation of the GluN1 subunit in Ser 896 was reduced in CXCL12-treated synaptosomes, while the overall amount of GluN1 and GluN2B proteins as well as the GluN2B insertion in synaptosomal plasmamembranes were unchanged. We conclude that the CXCR4/NMDA cross-talk is dynamically regulated by the time of activation of the CXCR4s. Our results unveil a functional cross-talk that might account for the severe impairments of central transmission that develop in pathological conditions characterized by CXCL12 overproduction.