Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (\ub1)baclofen (3 \u3bcM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1\u3b1 with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 \u3bcM (\ub1)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03\u20131 \u3bcM), inactive on its own on GABA exocytosis, amplified the 3 \u3bcM (\ub1)baclofen-induced inhibition of [3H]GABA overflow. The ( \ub1 )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (\ub1)baclofen (1 \u3bcM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 \u3bcM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (\ub1)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor\u2013receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity

the 3rs reduction and refinement through a multivariate statistical analysis approach in a behavioural study to unveil anxiolytic effects of natural extracts of tilia tomentosa

We propose a multivariate statistical approach based on Principal Component Analysis (PCA) as an useful instrument to improve the Rules of Refinement and Reduction in in vivo animal experimentation. We analysed with PCA the preliminary data from a study on the effects of the oral administration of Tilia tomentosa bud extracts (TTBEs) on the behavioural skills of adult and aged male and female mice. PCA allows to rationalize the data set information and to dissect the results, showing connections among variables under study (behavioural parameters) and different trends in the experimental groups (control and TTBEs-administered animals). Our results show that PCA can give some important information that can be useful for the refinement of the experimental protocol, in order to reduce the number of the animals used in the experiments and/or the behavioural tests to get reliable information

Presynaptic mGlu1 Receptors Control GABAB Receptors in an Antagonist-Like Manner in Mouse Cortical GABAergic and Glutamatergic Nerve Endings

Mouse cortical GABAergic synaptosomes possess presynaptic inhibitory GABAB autoreceptors. Accordingly, (±)baclofen (3 μM) inhibits in a CGP53423-sensitive manner the 12 mM KCl-evoked release of preloaded [3H]GABA. Differently, the existence of presynaptic release-regulating metabotropic glutamate type 1 (mGlu1) heteroreceptors in these terminals is still matter of discussion, although confocal microscopy unveiled the existence of mGlu1α with GABAB1 or GABAB2 proteins in cortical VGAT-positive synaptosomes. The group I mGlu agonist 3,5-DHPG failed to modify on its own the 12 mM KCl-evoked [3H]GABA exocytosis from cortical nerve endings, but, when added concomitantly to the GABAB agonist, it significantly reduced the 3 μM (±)baclofen-induced inhibition of [3H]GABA exocytosis. Conversely, the mGlu1 antagonist LY367385 (0.03–1 μM), inactive on its own on GABA exocytosis, amplified the 3 μM (±)baclofen-induced inhibition of [3H]GABA overflow. The ( ± )baclofen-induced inhibition of [3H]GABA exocytosis was more pronounced in cortical synaptosomes from Grm1crv4/crv4 mice, which bear a spontaneous mutation of the Grm1 gene leading to the functional inactivation of the mGlu1 receptor. Inasmuch, the expression of GABAB2 receptor protein in cortical synaptosomal lysates from Grm1crv4/crv4 mice was increased when compared to controls. Altogether, these observations seem best interpreted by assuming that mGlu1 coexist with GABAB receptors in GABAergic cortical synaptosomes, where they control GABA receptors in an antagonist-like manner. We then asked whether the mGlu1-mediated control of GABAB receptors is restricted to GABAergic terminals, or if it occurs also in other subpopulations of nerve endings. Release-regulating GABAB receptors also exist in glutamatergic nerve endings. (±)baclofen (1 μM) diminished the 12 mM KCl-evoked [3H]D-aspartate overflow. Also in these terminals, the concomitant presence of 1 μM LY367385, inactive on its own, significantly amplified the inhibitory effect exerted by (±)baclofen on [3H]D-aspartate exocytosis. Confocal microscopy confirmed the colocalization of mGlu1 with GABAB1 and GABAB2 labeling in vesicular glutamate type1 transporter-positive particles. Our results support the conclusion that mGlu1 receptors modulate in an antagonist-like manner presynaptic release-regulating GABAB receptors. This receptor–receptor interaction could be neuroprotective in central disease typified by hyperglutamatergicity

Pharmacological activation of mGlu5 receptors with the positive allosteric modulator, VU0360172 modulates thalamic GABAergic transmission

Previous studies have shown that injection of the mGlu5 receptor positive allosteric modulator (PAM) VU0360172 into either the thalamus or somatosensory cortex markedly reduces the frequency of spike-and-wave discharges (SWDs) in the WAG/Rij model of absence epilepsy. Here we have investigated the effects of VU0360172 on GABA transport in the thalamus and somatosensory cortex, as possible modes of action underlying the suppression of SWDs. Systemic VU0360172 injections increase GABA uptake in thalamic synaptosomes from epileptic WAG/Rij rats. Consistent with this observation, VU0360172 could also enhance thalamic GAT-1 protein expression, depending on the dosing regimen. This increase in GAT-1 expression was also observed in the thalamus from non-epileptic rats (presymptomatic WAG/Rij and Wistar) and appeared to occur selectively in neurons. The tonic GABAA receptor current present in ventrobasal thalamocortical neurons was significantly reduced by VU0360172 consistent with changes in GAT-1 and GABA uptake. The in vivo effects of VU0360172 (reduction in tonic GABA current and increase in GAT-1 expression) could be reproduced in vitro by treating thalamic slices with VU0360172 for at least 1 hour and appeared to be dependent on the activation of PLC. Thus, the effects of VU0360172 do not require an intact thalamocortical circuit. In the somatosensory cortex, VU0360172 reduced GABA uptake but did not cause significant changes in GAT-1 protein levels. These findings reveal a novel mechanism of regulation mediated by mGlu5 receptors, which could underlie the powerful anti-absence effect of mGlu5 receptor enhancers in animal models

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 [ 3 H]D-aspartate ([ 3 H]D-Asp) or [ 3 H]noradrenaline ([ 3 H]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

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 \u3bcM NMDA/1 \u3bcM glycine-evoked [ 3 H]d-aspartate ([ 3 H]d-ASP) release. The NMDA/glycine-evoked [ 3 H]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 [ 3 H]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