Synaptosomal Plasma Membrane Research Articles

Thiazolium salt mimics the non-coenzyme effects of vitamin B1 in rat synaptosomes

Long-term studies have confirmed a causal relationship between the development of neurodegenerative processes and vitamin B1 (thiamine) deficiency. However, the biochemical mechanisms underlying the high neurotropic activity of thiamine are not fully understood. At the same time, there is increasing evidence that vitamin B1, in addition to its coenzyme functions, may have non-coenzyme activities that are particularly important for neurons. To elucidate which effects of vitamin B1 in neurons are due to its coenzyme function and which are due to its non-coenzyme activity, we conducted a comparative study of the effects of thiamine and its derivative, 3-decyloxycarbonylmethyl-5-(2-hydroxyethyl)-4-methyl-1,3-thiazolium chloride (DMHT), on selected processes in synaptosomes. The ability of DMHT to effectively compete with thiamine for binding to thiamine-binding sites on the plasma membrane of synaptosomes and to participate as a substrate in the thiamine pyrophosphokinase reaction was demonstrated. In experiments with rat brain synaptosomes, unidirectional effects of DMHT and thiamine on the activity of the pyruvate dehydrogenase complex (PDC) and on the incorporation of radiolabeled [2–14C]pyruvate into acetylcholine were demonstrated. The observed effects of thiamine and DMHT on the modulation of acetylcholine synthesis can be explained by suggesting that both compounds, which interact in cells with enzymes of thiamine metabolism, are phosphorylated and exert an inhibitory/activating effect (concentration-dependent) on PDC activity by affecting the regulatory enzymes of the complex. Such effects were not observed in the presence of structural analogues of thiamine and DMHT without a 2-hydroxyethyl substituent at position 5 of the thiazolium cycle. The effect of DMHT on the plasma membrane Ca-ATPase was similar to that of thiamine. At the same time, DMHT showed high cytostatic activity against neuroblastoma cells.

Prevention of amyloid β fibril deposition on the synaptic membrane in the precuneus by ganglioside nanocluster-targeting inhibitors.

Alzheimer's disease (AD), a progressive neurodegenerative condition, is one of the most common causes of dementia. Senile plaques, a hallmark of AD, are formed by the accumulation of amyloid β protein (Aβ), which starts to aggregate before the onset of the disease. Gangliosides, sialic acid-containing glycosphingolipids, play a key role in the formation of toxic Aβ aggregates. In membrane rafts, ganglioside-bound complexes (GAβ) act as nuclei for Aβ assembly, suggesting that GAβ is a promising target for AD therapy. The formation of GAβ-induced Aβ assemblies has been evaluated using reconstituted planar lipid membranes composed of synaptosomal plasma membrane (SPM) lipids extracted from human and mouse brains. Although the effects of gangliosides on Aβ accumulation in the precuneus have been established, effects on Aβ fibrils have not been determined. In this study, Aβ42 fibrils on reconstituted membranes composed of SPM lipids prepared from the precuneus cortex of human autopsied brains were evaluated by atomic force microscopy. In particular, Aβ42 accumulation, as well as the fibril number and size were higher for membranes with precuneus lipids than for membranes with calcarine cortex lipids. In addition, artificial peptide inhibitors targeting Aβ-sensitive ganglioside nanoclusters cleared Aβ assemblies on synaptic membranes in the brain, providing a novel therapeutic strategy for AD.

Neuro-Immune Modulation of Cholinergic Signaling in an Addiction Vulnerability Trait.

Sign-tracking (ST) describes the propensity to approach and contact a Pavlovian reward cue. By contrast, goal-trackers (GTs) respond to such a cue by retrieving the reward. These behaviors index the presence of opponent cognitive-motivational traits, with STs exhibiting attentional control deficits, behavior dominated by incentive motivational processes, and vulnerability for addictive drug taking. Attentional control deficits in STs were previously attributed to attenuated cholinergic signaling, resulting from deficient translocation of intracellular choline transporters (CHTs) into synaptosomal plasma membrane. Here, we investigated a posttranslational modification of CHTs, poly-ubiquitination, and tested the hypothesis that elevated cytokine signaling in STs contributes to CHT modification. We demonstrated that intracellular CHTs, but not plasma membrane CHTs, are highly ubiquitinated in male and female sign-tracking rats when compared with GTs. Moreover, levels of cytokines measured in cortex and striatum, but not spleen, were higher in STs than in GTs. Activation of the innate immune system by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) elevated ubiquitinated CHT levels in cortex and striatum of GTs only, suggesting ceiling effects in STs. In spleen, LPS increased levels of most cytokines in both phenotypes. In cortex, LPS particularly robustly increased levels of the chemokines CCL2 and CXCL10. Phenotype-specific increases were restricted to GTs, again suggesting ceiling effects in STs. These results indicate that interactions between elevated brain immune modulator signaling and CHT regulation are essential components of the neuronal underpinnings of the addiction vulnerability trait indexed by sign-tracking.

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.

α2A- and α2C-adrenoceptor expression and functionality in postmortem prefrontal cortex of schizophrenia subjects

Previous evidence suggests that α2-adrenoceptors (α2-AR) may be involved in the pathophysiology of schizophrenia. However, postmortem brain studies on α2-AR expression and functionality in schizophrenia are scarce. The aim of our work was to evaluate α2A-AR and α2C-AR expression in different subcellular fractions of prefrontal cortex postmortem tissue from antipsychotic-free (absence of antipsychotics in blood at the time of death) (n = 12) and antipsychotic-treated (n = 12) subjects with schizophrenia, and matched controls (n = 24). Functional coupling of α2-AR to Gα proteins induced by the agonist UK14304 was also tested. Additionally, Gα protein expression was also evaluated. In antipsychotic-free schizophrenia subjects, α2A-AR and α2C-AR protein expression was similar to controls in all the subcellular fractions. Conversely, in antipsychotic-treated schizophrenia subjects, increased α2A-AR expression was found in synaptosomal plasma membrane and postsynaptic density (PSD) fractions (+60% and +79% vs controls, respectively) with no significant changes in α2C-AR. [35S]GTPγS SPA experiments showed a significant lower stimulation of Gαi2 and Gαi3 proteins by UK14304 in antipsychotic-treated schizophrenia subjects, whereas stimulation in antipsychotic-free schizophrenia subjects remained unchanged. Gαo protein stimulation was significantly decreased in both antipsychotic-free and antipsychotic-treated schizophrenia subjects compared to controls. Expression of Gαi3 protein did not differ between groups, whereas Gαi2 levels were increased in PSD of schizophrenia subjects, both antipsychotic-free and antipsychotic-treated. Gαo protein expression was increased in PSD of antipsychotic-treated subjects and in the presynaptic fraction of antipsychotic-free schizophrenia subjects. The present results suggest that antipsychotic treatment is able to modify in opposite directions both the protein expression and the functionality of α2A-AR in the cortex of schizophrenia patients.

GABAA receptor agonist cinazepam and its active metabolite 3-hydroxyphenazepam act differently at the presynaptic site

Cinazepam C19H14BrClN2O5, (“LevanaⓇ ІC”) a partial GABAA receptor agonist, and its active metabolite 3-hydroxyphenazepam C15H10BrClN2O2 were comparatively assessed in vitro using nerve terminals isolated from rat cortex (synaptosomes). At the presynaptic site, cinazepam (100 and 200 µM) facilitated synaptosomal transporter-mediated [3H]GABA uptake by enhancing both the initial rate and accumulation, and decreased the ambient level and transporter-mediated release of [3H]GABA. Whereas, 3-hydroxyphenazepam decreased the uptake and did not change the ambient synaptosomal level and transporter-mediated release of [3H]GABA. To exclude GABA transporter influence, NO-711, the transporter blocker, was applied and it was found that exocytotic release of [3H]GABA decreased, whereas tonic release of [3H]GABA was not changed in the presence of both cinazepam or 3-hydroxyphenazepam after treatment of synaptosomes with NO-711. In fluorimetric studies using potential- and pH-sensitive dyes rhodamine 6G and acridine orange, respectively, it was found that cinazepam hyperpolarized the synaptosomal plasma membrane, and increased synaptic vesicle acidification, whereas, 3-hydroxyphenazepam demonstrated opposite effects on these parameters. Therefore, action of cinazepam and its active metabolite 3-hydroxyphenazepam on GABAergic neurotransmission was different. Therapeutic effects of cinazepam can be associated with its ability to hyperpolarize the plasma membrane, to increase synaptic vesicle acidification and capacity of its active metabolite 3-hydroxyphenazepam to inhibit GABA transporter functioning.

Typhaneoside Suppresses Glutamate Release Through Inhibition of Voltage-Dependent Calcium Entry in Rat Cerebrocortical Nerve Terminals.

Glutamate is the major excitatory neurotransmitter in the brain and is involved in many brain functions. In this study, we investigated whether typhaneoside, a flavonoid from Typhae angustifolia pollen, affects endogenous glutamate release from rat cortical synaptosomes. Using a one-line enzyme-coupled fluorometric assay, glutamate release stimulated by the K+ channel blocker 4-aminopyridine was monitored to explore the possible underlying mechanisms. The vesicular transporter inhibitor bafilomycin A1 and chelation of extracellular Ca2+ ions with EGTA suppressed the effect of typhaneoside on the induced glutamate release. Nevertheless, the typhaneoside activity has not been affected by the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate. The synaptosomal plasma membrane potential was assayed using a membrane potential-sensitive dye DiSC3(5), and cytosolic Ca2+ concentrations ([Ca2+]C) was monitored by a Ca2+ indicator Fura-2. Results showed that typhaneoside did not alter the synaptosomal membrane potential but lowered 4-aminopyridine-induced increases in [Ca2+]C. Furthermore, the Cav2.2 (N-type) channel blocker ω-conotoxin GVIA blocked Ca2+ entry and inhibited the effect of typhaneoside on 4-aminopyridine-induced glutamate release. However, the inhibitor of intracellular Ca2+ release dantrolene and the mitochondrial Na+/Ca2+ exchanger blocker 7-chloro-5-(2-chloropheny)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one have no effect on the suppression of glutamate release mediated by typhaneoside. Moreover, inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) prevented the inhibitory effect of typhaneoside on induced glutamate release. Typhaneoside reduced 4-aminopyridine-induced phosphorylation of ERK1/2 and the major presynaptic ERK target synapsin I, which is a synaptic vesicle-associated protein. In conclusion, these findings suggest a role for typhaneoside in modulating glutamate release by suppressing voltage-dependent Ca2+ channel mediated presynaptic Ca2+ influx and the MAPK/ERK/synapsin I signaling cascade.

High Concentration of Ketone Body β-Hydroxybutyrate Modifies Synaptic Vesicle Cycle and Depolarizes Plasma Membrane of Rat Brain Synaptosomes

Ketoacidosis is a dangerous complication of diabetes mellitus in which plasma levels of ketone bodies can reach 20-25mM. This condition is life-threatening. In contrast, a ketogenic diet, achieving plasma levels of ketone bodies of about 4-5mM, can be used for treating different brain diseases. However, the factors leading to the conversion of the neuroprotective ketone bodies' action to the neurotoxic action during ketoacidosis are still unknown. We investigated the influence of high concentration (25mM) of the main ketone body, β-hydroxybutyrate (BHB), on intrasynaptosomal pH (pHi), synaptic vesicle cycle, plasma membrane, and mitochondrial potentials. Using the fluorescent dye BCECF-AM, it was shown that BHB at concentrations of 8 and 25mM did not influence pHi in synaptosomes. By means of the fluorescent dye acridine orange, it was demonstrated that 25mM of BHB had no effect on exocytosis but inhibited compensatory endocytosis by 5-fold. Increasing buffer capacity with 25mM HEPES did not affect endocytosis. Glucose abolished BHB-induced endocytosis inhibition. Using the fluorescent dye DiSC3(5), it was shown that 25mM of BHB induced a significant plasma membrane depolarization. This effect was not impacted by glucose. Using the fluorescent dye rhodamine-123, it was shown that BHB alone (25mМ) did not alter the potential of intrasynaptosomal mitochondria.Importantly, the high concentration of BHB (25mМ) causes the depolarization of the plasma membrane and stronger inhibition of endocytosis compared with the intermediate concentration (8mM).

The Combined Effect of Glucose and β-Hydroxybutyrate on the Membrane Potential of Synaptosomal Mitochondria

Abstract—The ketogenic diet is used as a treatment for different brain diseases. It involves replacement of dietary carbohydrates with fat, which leads to production of ketone bodies, predominantly β-hydroxybutyrate. The mechanism of the protective effect of the ketogenic diet remains poorly studied. In this work, the combined influence of glucose and β-hydroxybutyrate on the potentials of the synaptosomal plasma membrane and the mitochondrial membrane was investigated. It was found that the presence of ketone bodies in a glucose-containing incubation medium did not affect the synaptosomal plasma membrane potential, in contrast to what was previously observed for the neuronal plasma membrane. It was shown that incubation of synaptosomes in the medium containing only β-hydroxybutyrate resulted in mitochondrial depolarization (ΔΨ change). At the same time, the presence of ketone bodies in the glucose-containing incubation medium led to mitochondrial hyperpolarization. The observed depolarization was partially compensated by an increase in mitochondrial ΔpH, which was induced by mitochondrial depolarization as such, but not by the presence of β-hydroxybutyrate. Thus, ketone bodies can be utilized by synapses as energy substrates, but β-hydroxybutyrate is a less effective energy source than glucose.

Allicin Inhibits Glutamate Release from Rat Cerebral Cortex Nerve Terminals Through Suppressing Ca2+ Influx and Protein Kinase C Activity.

Evidence indicates that indirect inhibitory regulation of glutamatergic transmission, via reducing glutamate release, may induce neuroprotection. The present work was designed to examine whether allicin, a major component of garlic with neuroprotective effects, affected the release of glutamate evoked by 4-aminopyridine in rat cerebrocortical nerve terminals (synaptosomes). Allicin caused a potent inhibition on the release of glutamate evoked by 4-aminopyridine, and this inhibitory effect was abolished in the presence of Ca2+-free medium and vesicular transporter inhibitor. Allicin decreased the 4-aminopyridine-evoked elevation of intrasynaptosomal Ca2+ levels, but had no effect on the synaptosomal plasma membrane potential. The allicin-mediated inhibition of glutamate release was prevented by the N- and P/Q-type channel blocker and the protein kinase C (PKC) inhibitor, but was not affected by the intracellular Ca2+-release inhibitors, mitogen-activated protein kinase inhibitor, and protein kinase A inhibitor. Western blotting data also showed that allicin significantly reduced the phosphorylation of PKC. Together, these data indicate that in rat cerebrocortical nerve terminals, allicin depresses glutamate release and appears to decrease N- and P/Q-type Ca2+ channel and PKC activity.

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.

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 30μM NMDA/1μM 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.

5-HT2A-mGlu2/3 receptor complex in rat spinal cord glutamatergic nerve endings: A 5-HT2A to mGlu2/3 signalling to amplify presynaptic mechanism of auto-control of glutamate exocytosis

Presynaptic mGlu2/3 autoreceptors exist in rat spinal cord nerve terminals as suggested by the finding that LY379268 inhibited the 15 mM KCl-evoked release of [3H]D-aspartate ([3H]D-Asp) in a LY341495-sensitive manner. Spinal cord glutamatergic nerve terminals also possess presynaptic release-regulating 5-HT2A heteroreceptors. Actually, the 15 mM KCl-evoked [3H]D-Asp exocytosis from spinal cord synaptosomes was reduced by the 5-HT2A agonist (±)DOI, an effect reversed by the 5-HT2A antagonists MDL11,939, MDL100907, ketanserin and trazodone (TZD). We investigated whether mGlu2/3 and 5-HT2A receptors colocalize and cross-talk in these terminals and if 5-HT2A ligands modulate the mGlu2/3-mediated control of glutamate exocytosis. Western blot analysis and confocal microscopy highlighted the presence of mGlu2/3 and 5-HT2A receptor proteins in spinal cord VGLUT1 positive synaptosomes, where mGlu2/3 and 5-HT2A receptor immunoreactivities largely colocalize. Furthermore, mGlu2/3 immunoprecipitates from spinal cord synaptosomes were also 5-HT2A immunopositive. Interestingly, the 100 pM LY379268-induced reduction of the 15 mM KCl-evoked [3H]D-Asp overflow as well as its inhibition by 100 nM (±)DOI became undetectable when the two agonists were concomitantly added. Conversely, 5-HT2A antagonists (MDL11,939, MDL100907, ketanserin and TZD) reinforced the release-regulating activity of mGlu2/3 autoreceptors. Increased expression of mGlu2/3 receptor proteins in synaptosomal plasmamembranes paralleled the gain of function of the mGlu2/3 autoreceptors elicited by 5-HT2A antagonists. Based on these results, we propose that in spinal cord glutamatergic terminals i) mGlu2/3 and 5-HT2A receptors colocalize and interact one each other in an antagonist-like manner, ii) 5-HT2A antagonists are indirect positive allosteric modulator of mGlu2/3 autoreceptors controlling glutamate exocytosis.

Effects of surface functionalization of hydrophilic NaYF4 nanocrystals doped with Eu3+ on glutamate and GABA transport in brain synaptosomes

Specific rare earth doped nanocrystals (NCs), a recent class of nanoparticles with fluorescent features, have great bioanalytical potential. Neuroactive properties of NaYF4 nanocrystals doped with Eu3+ were assessed based on the analysis of their effects on glutamate- and γ-aminobutyric acid (GABA) transport process in nerve terminals isolated from rat brain (synaptosomes). Two types of hydrophilic NCs were examined in this work: (i) coated by polyethylene glycol (PEG) and (ii) with OH groups at the surface. It was found that NaYF4:Eu3+-PEG and NaYF4:Eu3+-OH within the concentration range of 0.5–3.5 and 0.5–1.5 mg/ml, respectively, did not influence Na+-dependent transporter-dependent l-[14C]glutamate and [3H]GABA uptake and the ambient level of the neurotransmitters in the synaptosomes. An increase in NaYF4:Eu3+-PEG and NaYF4:Eu3+-OH concentrations up to 7.5 and 3.5 mg/ml, respectively, led to the (1) attenuation of the initial velocity of uptake of l-[14C]glutamate and [3H]GABA and (2) elevation of ambient neurotransmitters in the suspension of nerve terminals. In the mentioned concentrations, nanocrystals did not influence acidification of synaptic vesicles that was shown with pH-sensitive fluorescent dye acridine orange, however, decreased the potential of the plasma membrane of synaptosomes. In comparison with other nanoparticles studied with similar methodological approach, NCs start to exhibit their effects on neurotransmitter transport at concentrations several times higher than those shown for carbon dots, detonation nanodiamonds and an iron storage protein ferritin, whose activity can be registered at 0.08, 0.5 and 0.08 mg/ml, respectively. Therefore, NCs can be considered lesser neurotoxic as compared to above nanoparticles.

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.

Reduced expressions of calmodulin genes and protein and reduced ability of calmodulin to activate plasma membrane Ca2+‐ATPase in the brain of protein undernourished rats: modulatory roles of selenium and zinc supplementation

The roles of protein undernutrition as well as selenium (Se) and zinc (Zn) supplementation on the ability of calmodulin (CaM) to activate erythrocyte ghost membrane (EGM) Ca(2+)-ATPase and the calmodulin genes and protein expressions in rat's cortex and cerebellum were investigated. Rats on adequate protein diet and protein-undernourished (PU) rats were fed with diet containing 16% and 5% casein, respectively, for a period of 10 weeks. The rats were then supplemented with Se and Zn at a concentration of 0.15 and 227 mg l(-1), respectively, in drinking water for 3 weeks. The results obtained from the study showed significant reductions in synaptosomal plasma membrane Ca(2+)-ATPase (PMCA) activity, Ca(2+)/CaM activated EGM Ca(2+) ATPase activity and calmodulin genes and protein expressions in PU rats. Se or Zn supplementation improved the ability of Ca(2+)/CaM to activate EGM Ca(2+)-ATPase and protein expressions. Se or Zn supplementation improved gene expression in the cerebellum but not in the cortex. Also, the activity of PMCA was significantly improved by Zn. In conclusion, it is postulated that Se and Zn might be beneficial antioxidants in protecting against neuronal dysfunction resulting from reduced level of calmodulin such as present in protein undernutrition.

Antibody/receptor protein immunocomplex in human and mouse cortical nerve endings amplifies complement-induced glutamate release

Previous studies have demonstrated that complement alone releases glutamate from human and mouse cortical terminals in an antibody-independent manner. In order to expand our knowledge on complement-mediated effects, we investigated whether the presence of an antigen-antibody complex in synaptosomal plasmamembranes could also trigger complement-induced functional responses that might affect neurotransmitter release. To this aim, we focused on the chemokine 5 receptor (CCR5) expressed in human and mouse cortical glutamate terminals, whose activation by CCL5 elicits [3H]D-aspartate ([3H]D-ASP) release. Preincubating synaptosomes with an antibody recognizing the NH2 terminus of the CCR5 protein (anti-NH2-CCR5 antibody) abolished the CCL5-induced [3H]D-ASP release. Similarly, enriching synaptosomes with an antibody recognizing the COOH terminus of CCR5 (anti-COOH-CCR5 antibody) prevented the CCL5-induced [3H]D-ASP release. The antagonist-like activity of the anti-NH2-CCR5 antibody turned to facilitation when anti-NH2-CCR5-treated synaptosomes were exposed to complement. In these terminals, the releasing effect was significantly higher than that elicited by complement in untreated synaptosomes. On the contrary, the complement-induced [3H]D-ASP release from anti-COOH-CCR5 antibody-entrapped synaptosomes did not differ from that from untreated synaptosomes. Preincubating synaptosomes with anti-beta tubulin III antibody, used as negative control, neither prevented the CCL5-induced releasing effect nor it amplified the complement-induced [3H]D-ASP release. Finally, serum lacking the C1q protein, i.e. the protein essential to promote the antibody-mediated activation of complement, elicited a comparable [3H]D-ASP release from both untreated and anti-NH2-CCR5 antibody-treated synaptosomes. Thus, we propose that antibodies raised against the outer sequence of a receptor protein can trigger the activation of the complement through the classic, C1q-mediated antibody-dependent pathway, which results in an abnormal release of glutamate that could be deleterious to central nervous system.

Diabetes-induced impairments of the exocytosis process and the effect of gabapentin: the link with cholesterol level in neuronal plasma membranes.

Diabetic neuropathy represents one of the most prevalent complications of diabetes mellitus. The aim of this study was to investigate the effect of diabetes-induced disturbances in neurons on the Ca(2+)-triggered membrane fusion process in cell-free system in relation to plasmalemma cholesterol level. The gabapentin therapy on the exocytosis process was also studied. The diabetes in rats was induced by streptozotocin (60 mg/kg of body weight, i.p.). After 4 weeks of diabetes induction the one group of diabetic rats was treated with gabapentin (50 mg/kg, i.p.) during 1 month. Fusion experiments were performed in the cell-free model system using fluorescent dye octadecylrhodamine B. The [2-(14)C]serotonin preloaded synaptosomes were used for assay of stimulated neurotransmitter release. The synaptosomal plasma membrane cholesterol level in diabetic rats was on 12 % higher than in control and was decreased on 5 % after gabapentin therapy. The rate of synaptic vesicles fusion with plasma membranes in the presence of Ca(2+) and synaptosomal cytosolic proteins was decreased to 14.5 % in diabetic rats as compared to control (23 %) and after gabapentin administration to diabetic rats was raised to 18 %. At diabetes the stimulated synaptosomal serotonin release was increased in 1.7-2 folds and was partially normalized by gabapentin therapy. Together, these findings suggest that elevated cholesterol content in neuronal plasma membranes at diabetes impairs the membrane fusion process in neurons that can induce the development of neuropathy. Diabetes-evoked impairments of the exocytotic process can be attenuated by gabapentin therapy.

Myricetin Inhibits the Release of Glutamate in Rat Cerebrocortical Nerve Terminals

The excessive release of glutamate is a critical element in the neuropathology of acute and chronic brain disorders. The purpose of the present study was to investigate the effect and possible mechanism of myricetin, a naturally occurring flavonoid with a neuroprotective profile, on endogenous glutamate release in the nerve terminals (synaptosomes) of the rat cerebral cortex. The release of glutamate was evoked by the K(+) channel blocker 4-aminopyridine (4-AP) and measured by one-line enzyme-coupled fluorometric assay. We also used a membrane potential-sensitive dye to assay the synaptosomal plasma membrane potential, and a Ca(2+) indicator Fura-2 to monitor cytosolic Ca(2+) concentrations ([Ca(2+)]C). Results show that myricetin inhibited 4-AP-evoked glutamate release, and this effect was prevented by chelating extracellular Ca(2+) ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate had no effect on myricetin action. Myricetin did not alter the synaptosomal membrane potential, but decreased 4-AP-induced increases in the cytosolic free Ca(2+) concentration. Furthermore, the myricetin effect on 4-AP-evoked glutamate release was prevented by blocking the Cav2.2 (N-type) and Cav2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release. These results suggest that myricetin inhibits glutamate release from cerebrocortical synaptosomes by attenuating voltage-dependent Ca(2+) entry. This implies that the inhibition of glutamate release is an important pharmacological activity of myricetin that may play a critical role in the apparent clinical efficacy of this compound.

Thiamine Metabolism in Neurons and Their Vital Capacity Upon the Action of Ethanol and Acetaldehyde

We studied the sensitivity of a few reactions of thiamine metabolism in nerve cells upon the action of ethanol and acetaldehyde, as well as effects of the above-mentioned agents on vital capacity of cultured cells of different origins. Experiments were carried out on preparations of isolated nerve endings (synaptosomes), preparations of synaptosomal plasma membranes (SPMs), preparations of enzymes, and cell cultures. We estimated the ІС50 for the effects of ethanol and acetaldehyde on reactions that are the components of metabolism of the mobile thiamine pool in the cells. Ethanol in physiological concentrations inhibited the thiamine-binding activity of SPMs (ІС50 = 3.9 mM). At the same time, this agent used in the above concentrations practically did not influence the thiamine phosphate hydrolase activity of SPMs (estimated by the thiamine triphosphatase activity) and thiamine pyrophosphate kinase (TPK) activity. A product of ethanol metabolism, acetaldehyde, inhibited the thiamine triphosphatase activity with K і = 6.2 μM and the TPK activity with K і = 1.2 μM. The survival indices of cultured cells were estimated under conditions of the addition of ethanol and acetaldehyde to the medium. The cells of an astrocytic origin (line strain 1321 N1) and blood cells (line strain U937) practically did not respond to the presence of ethanol or acetaldehyde even in rather high concentrations in the culture medium. Under conditions of primary culture, neuron-like (differentiated) cultured РС-12 cells and also cerebellar granular neurons responded by significant decreases in the index of vital capacity to the addition of the above agents.