Glutamate In Terminals Research Articles

Harmful impact on presynaptic glutamate and GABA transport by carbon dots synthesized from sulfur-containing carbohydrate precursor.

Carbon nanoparticles that may be potent air pollutants with adverse effects on human health often contain heteroatoms including sulfur. In order to study in detail their effects on different physiological and biochemical processes, artificially produced carbon dots (CDs) with well-controlled composition that allows fluorescence detection may be of great use. Having been prepared from different types of organic precursors, CDs expose different atoms at their surface suggesting a broad variation of functional groups. Recently, we demonstrated neurotoxic properties of CDs synthesized from the amino acid β-alanine, and it is of importance to analyze whether CDs obtained from different precursors and particularly those exposing sulfur atoms induce similar neurotoxic effects. This study focused on synthesis of CDs from the sulfur-containing precursor thiourea-CDs (TU-CDs) with a size less than 10nm, their characterization, and neuroactivity assessment. Neuroactive properties of TU-CDs were analyzed based on their effects on the key characteristics of glutamatergic and γ-aminobutyric acid (GABA) neurotransmission in isolated rat brain nerve terminals. It was observed that TU-CDs (0.5-1.0mg/ml) attenuated the initial velocity of Na+-dependent transporter-mediated uptake and accumulation of L-[14C]glutamate and [3H]GABA by nerve terminals in a dose-dependent manner and increased the ambient level of the neurotransmitters. Starting from the concentration of 0.2mg/ml, TU-CDs evoked a gradual dose-dependent depolarization of the plasma membrane of nerve terminals measured with the cationic potentiometric dye rhodamine 6G. Within the concentration range of 0.1-0.5mg/ml, TU-CDs caused an "unphysiological" step-like increase in fluorescence intensity of the рН-sensitive fluorescent dye acridine orange accumulated by synaptic vesicles. Therefore, despite different surface properties and fluorescent features of CDs prepared from different starting materials (thiourea and β-alanine), their principal neurotoxic effects are analogous but displayed at a different level of efficiency. Sulfur-containing TU-CDs exhibit lower effects (by ~30%) on glutamate and GABA transport in the nerve terminals in comparison with sulfur-free β-alanine CDs. Our results suggest considering that an uncontrolled presence of carbon-containing particulate matter in the human environment may pose a toxicity risk for the central nervous system.

Ventral tegmental area glutamate neurons co-release GABA and promote positive reinforcement.

In addition to dopamine neurons, the ventral tegmental area (VTA) contains GABA-, glutamate- and co-releasing neurons, and recent reports suggest a complex role for the glutamate neurons in behavioural reinforcement. We report that optogenetic stimulation of VTA glutamate neurons or terminals serves as a positive reinforcer on operant behavioural assays. Mice display marked preference for brief over sustained VTA glutamate neuron stimulation resulting in behavioural responses that are notably distinct from dopamine neuron stimulation and resistant to dopamine receptor antagonists. Whole-cell recordings reveal EPSCs following stimulation of VTA glutamate terminals in the nucleus accumbens or local VTA collaterals; but reveal both excitatory and monosynaptic inhibitory currents in the ventral pallidum and lateral habenula, though the net effects on postsynaptic firing in each region are consistent with the observed rewarding behavioural effects. These data indicate that VTA glutamate neurons co-release GABA in a projection-target-dependent manner and that their transient activation drives positive reinforcement.

Effect of O-methyl-β-cyclodextrin-modified magnetic nanoparticles on the uptake and extracellular level of l-glutamate in brain nerve terminals

Changes in cholesterol concentration in the plasma membrane of presynaptic nerve terminals nonspecifically modulate glutamate transport and homeostasis in the central nervous system. Reduction of the cholesterol content in isolated rat brain nerve terminals (synaptosomes) using cholesterol-depleting agents decreases the glutamate uptake and increases the extracellular level of glutamate in nerve terminals. Extraction of cholesterol from the plasma membrane and its further removal from the synaptosomes by external magnetic field can be achieved by means of magnetic nanoparticles with immobilized cholesterol-depleting agent such as O-methyl-β-cyclodextrin (MCD). A simple approach is developed for preparation of maghemite (γ-Fe2O3) nanoparticles containing chemically bonded MCD. The method is based on preparation of a silanization agent containing MCD. It is synthesized by the reaction of triethoxy(3-isocyanatopropyl)silane with MCD. Base-catalyzed silanization of superparamagnetic γ-Fe2O3 provides a relatively stable colloid product containing 48μmol of MCDg−1. MCD-modified γ-Fe2O3 nanoparticles decrease the initial rate of the uptake and accumulation of l-[14C]glutamate and increase the extracellular l-[14C]glutamate level in the preparation of nerve terminals. The effect of MCD-immobilized nanoparticles is the same as that of MCD solution; moreover, magnetic manipulation of the nanoparticles enables removal of bonded cholesterol.

Ethanol-Induced Motor Impairment Mediated by Inhibition of α7 Nicotinic Receptors.

Nicotine and ethanol (EtOH) are among the most widely co-abused substances, and nicotinic acetylcholine receptors (nAChRs) contribute to the behavioral effects of both drugs. Along with their role in addiction, nAChRs also contribute to motor control circuitry. The α7 nAChR subtype is highly expressed in the laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic center that contributes to motor performance through its projections to thalamic motor relay centers, including the mediodorsal thalamus. We demonstrate that EtOH concentrations just above the legal limits for intoxication in humans can inhibit α7 nAChRs in LDTg neurons from rats. This EtOH-induced inhibition is mediated by a decrease in cAMP/PKA signaling. The α7 nAChR-positive allosteric modulator PNU120596 [N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl)-urea], which interferes with receptor desensitization, completely eliminated EtOH modulation of these receptors. These data suggest that EtOH inhibits α7 responses through a PKA-dependent enhancement of receptor desensitization. EtOH also inhibited the effects of nicotine at presynaptic α7 nAChRs on glutamate terminals in the mediodorsal thalamus. In vivo administration of PNU120596 either into the cerebral ventricles or directly into the mediodorsal thalamus attenuated EtOH-induced motor impairment. Thus, α7 nAChRs are likely important mediators of the motor impairing effects of moderate EtOH consumption. The motor-impairing effects of ethanol contribute to intoxication-related injury and death. Here we explore the cellular and neural circuit mechanisms underlying ethanol-induced motor impairment. Physiologically relevant concentrations of ethanol inhibit activity of a nicotinic receptor subtype that is expressed in brain areas associated with motor control. That receptor inhibition is mediated by decreased receptor phosphorylation, suggesting an indirect modulation of cell signaling pathways to achieve the physiological effects.

Trypanosoma brucei DHFR-TS Revisited: Characterisation of a Bifunctional and Highly Unstable Recombinant Dihydrofolate Reductase-Thymidylate Synthase

Bifunctional dihydrofolate reductase–thymidylate synthase (DHFR-TS) is a chemically and genetically validated target in African trypanosomes, causative agents of sleeping sickness in humans and nagana in cattle. Here we report the kinetic properties and sensitivity of recombinant enzyme to a range of lipophilic and classical antifolate drugs. The purified recombinant enzyme, expressed as a fusion protein with elongation factor Ts (Tsf) in ThyA- Escherichia coli, retains DHFR activity, but lacks any TS activity. TS activity was found to be extremely unstable (half-life of 28 s) following desalting of clarified bacterial lysates to remove small molecules. Stability could be improved 700-fold by inclusion of dUMP, but not by other pyrimidine or purine (deoxy)-nucleosides or nucleotides. Inclusion of dUMP during purification proved insufficient to prevent inactivation during the purification procedure. Methotrexate and trimetrexate were the most potent inhibitors of DHFR (Ki 0.1 and 0.6 nM, respectively) and FdUMP and nolatrexed of TS (Ki 14 and 39 nM, respectively). All inhibitors showed a marked drop-off in potency of 100- to 1,000-fold against trypanosomes grown in low folate medium lacking thymidine. The most potent inhibitors possessed a terminal glutamate moiety suggesting that transport or subsequent retention by polyglutamylation was important for biological activity. Supplementation of culture medium with folate markedly antagonised the potency of these folate-like inhibitors, as did thymidine in the case of the TS inhibitors raltitrexed and pemetrexed.

Neuroactivity of detonation nanodiamonds: dose-dependent changes in transporter-mediated uptake and ambient level of excitatory/inhibitory neurotransmitters in brain nerve terminals.

BackgroundNanodiamonds are one of the most perspective nano-sized particles with superb physical and chemical properties, which are mainly composed of carbon sp3 structures in the core with sp2 and disorder/defect carbons on the surface. The research team recently demonstrated neuromodulatory properties of carbon nanodots with other than nanodiamonds hybridization types, i.e., sp2 hybridized graphene islands and diamond-like sp3 hybridized elements.ResultsIn this study, neuroactive properties of uncoated nanodiamonds produced by detonation synthesis were assessed basing on their effects on transporter-mediated uptake and the ambient level of excitatory and inhibitory neurotransmitters, glutamate and γ-aminobutyric acid (GABA), in isolated rat brain nerve terminals. It was shown that nanodiamonds in a dose-dependent manner attenuated the initial velocity of Na+-dependent transporter-mediated uptake and accumulation of l-[14C]glutamate and [3H]GABA by nerve terminals and increased the ambient level of these neurotransmitters. Also, nanodiamonds caused a weak reduction in acidification of synaptic vesicles and depolarization of the plasma membrane of nerve terminals.ConclusionsTherefore, despite different types of hybridization in nanodiamonds and carbon dots, they exhibit very similar effects on glutamate and GABA transport in nerve terminals and this common feature of both nanoparticles is presumably associated with their nanoscale size. Observed neuroactive properties of pure nanodiamonds can be used in neurotheranostics for simultaneous labeling/visualization of nerve terminals and modulation of key processes of glutamate- and GABAergic neurotransmission. In comparison with carbon dots, wider medical application involving hypo/hyperthermia, external magnetic fields, and radiolabel techniques can be perspective for nanodiamonds.

Putative duality of presynaptic events

The main structure in the brain responsible not only for nerve signal transmission but also for its simultaneous regulation is chemical synapse, where presynaptic nerve terminals are of considerable importance providing release of neurotransmitters. Analyzing transport of glutamate, the major excitatory neurotransmitter in the mammalian CNS, the authors suggest that there are two main relatively independent mechanisms at the presynaptic level that can influence the extracellular glutamate concentration, and so signaling, and its regulation. The first one is well-known precisely regulated compound exocytosis of synaptic vesicles containing neurotransmitters stimulated by membrane depolarization, which increases significantly glutamate concentration in the synaptic cleft and initiates glutamate signaling through postsynaptic glutamate receptors. The second one is permanent glutamate turnover across the plasma membrane that occurs without stimulation and is determined by simultaneous non-pathological transporter-mediated release of glutamate thermodynamically synchronized with uptake. Permanent glutamate turnover is responsible for maintenance of dynamic glutamatein/glutamateout gradient resulting in the establishment of a flexible extracellular level of glutamate, which can be unique for each synapse because of dependence on individual presynaptic parameters. These two mechanisms, i.e. exocytosis and transporter-mediated glutamate turnover, are both precisely regulated but do not directly interfere with each other, because they have different intracellular sources of glutamate in nerve terminals for release purposes, i.e. glutamate pool of synaptic vesicles and the cytoplasm, respectively. This duality can set up a presynaptic base for memory consolidation and storage, maintenance of neural circuits, long-term potentiation, and plasticity. Arguments against this suggestion are also considered.

Glutaminase Increases in Rat Dorsal Root Ganglion Neurons after Unilateral Adjuvant-Induced Hind Paw Inflammation.

Glutamate is a neurotransmitter used at both the peripheral and central terminals of nociceptive primary sensory neurons, yet little is known concerning regulation of glutamate metabolism during peripheral inflammation. Glutaminase (GLS) is an enzyme of the glutamate-glutamine cycle that converts glutamine into glutamate for neurotransmission and is implicated in producing elevated levels of glutamate in central and peripheral terminals. A potential mechanism for increased levels of glutamate is an elevation in GLS expression. We assessed GLS expression after unilateral hind paw inflammation by measuring GLS immunoreactivity (ir) with quantitative image analysis of L4 dorsal root ganglion (DRG) neurons after one, two, four, and eight days of adjuvant-induced arthritis (AIA) compared to saline injected controls. No significant elevation in GLS-ir occurred in the DRG ipsilateral to the inflamed hind paw after one or two days of AIA. After four days AIA, GLS-ir was elevated significantly in all sizes of DRG neurons. After eight days AIA, GLS-ir remained elevated in small (<400 µm2), presumably nociceptive neurons. Western blot analysis of the L4 DRG at day four AIA confirmed the elevated GLS-ir. The present study indicates that GLS expression is increased in the chronic stage of inflammation and may be a target for chronic pain therapy.

Region-specific changes in presynaptic agmatine and glutamate levels in the aged rat brain

During the normal aging process, the brain undergoes a range of biochemical and structural alterations, which may contribute to deterioration of sensory and cognitive functions. Age-related deficits are associated with altered efficacy of synaptic neurotransmission. Emerging evidence indicates that levels of agmatine, a putative neurotransmitter in the mammalian brain, are altered in a region-specific manner during the aging process. The gross tissue content of agmatine in the prefrontal cortex (PFC) of aged rat brains is decreased whereas levels in the temporal cortex (TE) are increased. However, it is not known whether these changes in gross tissue levels are also mirrored by changes in agmatine levels at synapses and thus could potentially contribute to altered synaptic function with age. In the present study, agmatine levels in presynaptic terminals in the PFC and TE regions (300 terminals/region) of young (3month; n=3) and aged (24month; n=3) brains of male Sprague–Dawley rats were compared using quantitative post-embedding immunogold electron-microscopy. Presynaptic levels of agmatine were significantly increased in the TE region (60%; p<0.001) of aged rats compared to young rats, however no significant differences were detected in synaptic levels in the PFC region. Double immunogold labeling indicated that agmatine and glutamate were co-localized in the same synaptic terminals, and quantitative analyses revealed significantly reduced glutamate levels in agmatine-immunopositive synaptic terminals in both regions in aged rats compared to young animals. This study, for the first time, demonstrates differential effects of aging on agmatine and glutamate in the presynaptic terminals of PFC and TE. Future research is required to understand the functional significance of these changes and the underlying mechanisms.

Incubation of cocaine-craving relates to glutamate over-flow within ventromedial prefrontal cortex

Craving elicited by drug-associated cues intensifies across protracted drug abstinence – a phenomenon termed “incubation of craving” – and drug-craving in human addicts correlates with frontal cortical hyperactivity. Herein, we employed a rat model of cue-elicited cocaine-craving to test the hypothesis that the time-dependent incubation of cue-elicited cocaine-craving is associated with adaptations in dopamine and glutamate neurotransmission within the ventromedial prefrontal cortex (vmPFC). Rats were trained to self-administer intravenous cocaine (6 h/day × 10 days) and underwent in vivo microdialysis procedures during 2 h-tests for cue-elicited cocaine-craving at either 3 or 30 days withdrawal. Controls rats were trained to either self-administer sucrose pellets or received no primary reinforcer. Cocaine-seeking rats exhibited a withdrawal-dependent increase and decrease, respectively, in cue-elicited glutamate and dopamine release. These patterns of neurochemical change were not observed in either control condition. Thus, cue-hypersensitivity of vmPFC glutamate terminals is a biochemical correlate of incubated cocaine-craving that may stem from dopamine dysregulation in this region.

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.

Maintaining the presynaptic glutamate supply for excitatory neurotransmission

Glutamate released from synapses during excitatory neurotransmission must be rapidly recycled to maintain neuronal communication. This review evaluates data from physiological experiments at hippocampal CA3 to CA1 synapses and the calyx of Held synapse in the brainstem to analyze quantitatively the rates of release and resupply of glutamate required to sustain neurotransmission. We calculate that, without efficient recycling, the presynaptic glutamate supply will be exhausted within about a minute of normal synaptic activity. We also discuss replenishment of the presynaptic pool by diffusion from the soma, direct uptake of glutamate back into the presynaptic terminal, and uptake of glutamate precursor molecules. Diffusion of glutamate from the soma is calculated to be fast enough to resupply presynaptic glutamate in the hippocampus but not at the calyx of Held. However, because the somatic cytoplasm will also quickly run out of glutamate and synapses can function continually even if the presynaptic axon is severed, mechanisms other than diffusion must be present to resupply glutamate for release. Direct presynaptic uptake of glutamate is not present at the calyx of Held but may play a role in glutamate recycling in the hippocampus. Alternatively, glutamine or tricarboxylic acid cycle intermediates released from glia can serve as a precursor for glutamate in synaptic terminals, and we calculate that the magnitude of presynaptic glutamine uptake is sufficient to supply enough glutamate to sustain neurotransmission. The nature of these mechanisms, their relative abundance, and the co-ordination between them remain areas of intensive investigation.

Protective effects of bupivacaine against kainic acid-induced seizure and neuronal cell death in the rat hippocampus.

The excessive release of glutamate is a critical element in the neuropathology of epilepsy, and bupivacaine, a local anesthetic agent, has been shown to inhibit the release of glutamate in rat cerebrocortical nerve terminals. This study investigated whether bupivacaine produces antiseizure and antiexcitotoxic effects using a kainic acid (KA) rat model, an animal model used for temporal lobe epilepsy, and excitotoxic neurodegeneration experiments. The results showed that administering bupivacaine (0.4 mg/kg or 2 mg/kg) intraperitoneally to rats 30 min before intraperitoneal injection of KA (15 mg/kg) increased seizure latency and reduced the seizure score. In addition, bupivacaine attenuated KA-induced hippocampal neuronal cell death, and this protective effect was accompanied by the inhibition of microglial activation and production of proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in the hippocampus. Moreover, bupivacaine shortened the latency of escaping onto the platform in the Morris water maze learning performance test. Collectively, these data suggest that bupivacaine has therapeutic potential for treating epilepsy.

Signaling through Homomeric and Heteromeric Dopamine D2 and Cannabinoid Cb1 Receptors

G protein-coupled receptors (GPCRs) play important roles in signal transduction and represent about half of the drug targets. Accumulating evidence suggests that GPCRs exist and function not only as homomers but as heteromers as well. Coimmunoprecipitation and fluorescence resonance energy transfer (FRET) results have provided evidence for heteromerization of D2-CB1 receptors in co-transfected cell lines. Besides, co-localization of CB1 and D2 receptors in the somata and dendrites of striato-pallidal GABA neurons and cortical-striatal glutamate terminals has been demonstrated. Studies have shown that homomeric D2 and CB1 receptors are Gi/o coupled able to inhibit upon stimulation the forskolin-induced adenylyl-cyclase activation leading to a decrease in cAMP levels. However, stimulation of the CB1 receptor upon the co-expression of D2-CB1 receptors in cell lines increased cAMP levels presumably because CB1 receptors switch their specificity to becoming Gs-coupled when complexed with the D2 receptor. In this study, we utilized Xenopus laevis oocytes as a heterologous expression system in which G protein-sensitive inwardly rectifying K+ (GIRK) channels are co-expressed with GPCRs. Stimulation of GPCRs with specific ligands activates GIRK channels via the betagamma subunits of the G proteins (Gbg). The GIRK channel current is monitored by two-electrode voltage clamp (TEVC) to access receptor activity. We have found that stimulation of homomeric D2 and CB1 receptors increases the GIRK currents. This increase could be blocked by pertussis toxin (PTX) that specifically ADP-ribosylates Gi/o. Overexpression of Gs in Xenopus oocytes biases signaling to couple Gs coupled receptors to GIRK channels via Gbg. Using this approach, we demonstrated that neither the CB1 nor the D2 homomeric receptors can couple to Gs. We are in the process of testing whether the channel response of CB1 receptor ligands through the heteromeric D2-CB1 receptors proceeds via Gs.

Monitoring of the velocity of high-affinity glutamate uptake by isolated brain nerve terminals using amperometric glutamate biosensor

Glutamate is the major excitatory neurotransmitter in the central nervous system, which is involved in the main aspects of normal brain functioning. High-affinity Na+-dependent glutamate transporters is key proteins, which transport extracellular glutamate to the cytoplasm of nerve cells, thereby preventing continuous activation of glutamate receptors, and thus the development of neurotoxicity. Disturbance in glutamate uptake is involved in the pathogenesis of major neurological disorders. Amperometric biosensors are the most promising and successful among electrochemical biosensors. In this study, we developed (1) amperometric glutamate biosensor, (2) methodological approach for the analysis of glutamate uptake in liquid samples of isolated rat brain nerve terminals (synaptosomes). The basal level of glutamate, the initial velocity of glutamate uptake and time-dependent accumulation of glutamate by synaptosomes were determined using developed glutamate biosensor. Comparative analysis of the data with those obtained by radioactive analysis, spectrofluorimetry and ion exchange chromatography was performed. Therefore, the methodological approach for monitoring of the velocity of glutamate uptake, which takes into consideration the definite level of endogenous glutamate in nerve terminals, was developed using glutamate biosensor.

Relationship between alexithymia and dopamine D2-type receptor availability: Methamphetamine-dependent subjects differ from healthy controls

Aims: Repeated cocaine administration produces perturbations in dopamine and glutamate neurotransmission that contribute to the reinstatement of extinguished cocaine seeking. Postsynaptic adenosine A2A receptors co-localize with dopamine D2 receptors in nucleus accumbens neurons, while presynaptic adenosine A2A receptors co-localize with adenosine A1 receptors on glutamate terminals. The synaptic localizationof adenosineA2A receptors represent targets for altering the dopamine and glutamate systems to effect the expression of behaviors associatedwith relapse. The goal of these studieswas to determine the effects of presynaptic or postsynaptic adenosine A2A receptor antagonism on the reinstatement of cocaine seeking. Methods:Male Sprague-Dawley rats self-administered cocaine in 10 daily self-administration sessions on a fixed-ratio 1 schedule. Lever pressing was extinguished in 6 daily extinction sessions. We first tested whether presynaptic and postsynaptic A2A receptor antagonism (SCH 442416 and KW 6002, respectively) was sufficient to reinstate cocaine seeking. We next tested the effects of SCH442416 and KW 6002 on reinstatement to cocaine seeking induced by cocaine. Results: Administration of the postsynaptic adenosine A2A receptor antagonist dose-dependently reinstated cocaine seeking and facilitated reinstatement induced by 5mg/kg cocaine, a dose that was alone insufficient to reinstate cocaine seeking. Administration of the presynaptic adenosine A2A receptor antagonist did not induce cocaine seekingwhen administered alone, but impaired cocaine-induced reinstatement induced by 15mg/kg cocaine. Conclusions: These findings highlight the importance of synaptic locations of adenosine A2A receptors in regulating cocaine seeking. Thus, antagonism of postsynaptic A2A receptors facilitates cocaine seeking, perhaps by enabling D2 receptor signaling. Antagonism of presynaptic A2A receptors, on the other hand, impairs cocaine seeking, perhaps by enabling adenosine A1 receptor activation and inhibition of glutamate transmission. Financial support: DA033358, DA029420, and CU Innovative Seed Grant.

Neuromodulatory properties of fluorescent carbon dots: Effect on exocytotic release, uptake and ambient level of glutamate and GABA in brain nerve terminals

Carbon dots (C-dots), a recently discovered class of fluorescent nano-sized particles with pure carbon core, have great bioanalytical potential. Neuroactive properties of fluorescent C-dots obtained from β-alanine by microwave heating were assessed based on the analysis of their effects on the key characteristics of GABA- and glutamatergic neurotransmission in isolated rat brain nerve terminals. It was found that C-dots (40–800μg/ml) in dose-dependent manner: (1) decreased exocytotic release of [3H]GABA and l-[14C]glutamate; (2) reduced acidification of synaptic vesicles; (3) attenuated the initial velocity of Na+-dependent transporter-mediated uptake of [3H]GABA and l-[14C]glutamate; (4) increased the ambient level of the neurotransmitters, nevertheless (5) did not change significantly the potential of the plasma membrane of nerve terminals. Almost complete suppression of exocytotic release of the neurotransmitters was caused by C-dots at a concentration of 800μg/ml. Fluorescent and neuromodulatory features combined in C-dots create base for their potential usage for labeling and visualization of key processes in nerve terminals, and also in theranostics. In addition, natural presence of carbon-containing nanoparticles in the human food chain and in the air may provoke the development of neurologic consequences.

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.

Excitotoxic potential of exogenous ferritin and apoferritin: Changes in ambient level of glutamate and synaptic vesicle acidification in brain nerve terminals

Ferritin, an iron storage protein, is present in the serum and cerebrospinal fluid, has receptors on the cell surface, able to penetrate the brain-blood barrier, can be secreted from the cells, and leaks from destroyed cell in insult and brain trauma. The effect of exogenous ferritin on the key characteristic of glutamatergic neurotransmission was assessed in rat brain nerve terminals (synaptosomes). Exogenous ferritin (80 μg/ml, iron content 0.7%) significantly increased the ambient level of L-[(14)C]glutamate (0.200±0.015 versus 0.368±0.016 nmol/mg of protein) and endogenous glutamate (fluorimetric glutamate dehydrogenase assay) in the nerve terminals. This increase was not a result of augmentation of tonic release because the velocity of tonic release of L-[(14)C]glutamate was not changed significantly in ferritin-treated synaptosomes as compared to the control. Ferritin caused a decrease in synaptic vesicle acidification that was shown using fluorescent dye acridine orange. Iron-dependence of the effects of ferritin was analyzed with apoferritin (0.0025% residual iron). Apoferritin weakly affected the proton electrochemical gradient of synaptic vesicles but increased the ambient level and decreased the initial velocity of uptake of L-[(14)C]glutamate by synaptosomes, nevertheless these effects were ~30% lesser than those caused by ferritin. Exogenous ferritin can provoke the development of excitotoxicity increasing the ambient level of glutamate and lowering synaptic vesicle acidification and glutamate uptake in the nerve terminals, however these effects are not completely iron-dependent. Thus, in the CNS exogenous ferritin can act as a modulator of glutamate homeostasis in iron-dependent and iron-independent manner.

Neurotoxic Potential of Lunar and Martian Dust: Influence on Em, Proton Gradient, Active Transport, and Binding of Glutamate in Rat Brain Nerve Terminals

The harmful effects of lunar dust (LD) on directly exposed tissues are documented in the literature, whereas researchers are only recently beginning to consider its effects on indirectly exposed tissues. During inhalation, nano-/microsized particles are efficiently deposited in nasal, tracheobronchial, and alveolar regions and transported to the central nervous system. The neurotoxic potential of LD and martian dust (MD) has not yet been assessed. Glutamate is the main excitatory neurotransmitter involved in most aspects of normal brain function, whereas disturbances in glutamate homeostasis contribute to the pathogenesis of major neurological disorders. The research was focused on the analysis of the effects of LD/MD simulants (JSC-1a/JSC, derived from volcanic ash) on the key characteristics of glutamatergic neurotransmission. The average size of LD and MD particles (even minor fractions) before and after sonication was determined by dynamic light scattering. With the use of radiolabeled l-[(14)C]glutamate, it was shown that there is an increase in l-[(14)C]glutamate binding to isolated rat brain nerve terminals (synaptosomes) in low [Na(+)] media and at low temperature in the presence of LD. MD caused significantly lesser changes under the same conditions, whereas nanoparticles of magnetite had no effect at all. Fluorimetric experiments with potential-sensitive dye rhodamine 6G and pH-sensitive dye acridine orange showed that the potential of the plasma membrane of the nerve terminals and acidification of synaptic vesicles were not altered by LD/MD (and nanoparticles of magnetite). Thus, the unique effect of LD to increase glutamate binding to the nerve terminals was shown. This can have deleterious effects on extracellular glutamate homeostasis in the central nervous system and cause alterations in the ambient level of glutamate, which is extremely important for proper synaptic transmission. During a long-term mission, a combination of constant irritation due to dust particles, inflammation, stress, low gravity and microgravity, radiation, UV, and so on may consequently change the effects of the dust and aggravate neurological consequences.