Tatiana Borisova

Cholesterol depletion attenuates tonic release but increases the ambient level of glutamate in rat brain synaptosomes.

The low level of ambient glutamate is important for the brains spontaneous activity and proper synaptic transmission. Cholesterol deficiency has been implicated in the pathogenesis of several neurodegenerative disorders. It was examined whether membrane cholesterol modulated the extracellular glutamate level in nerve terminals and the processes responsible for its maintenance. The ambient L-[(14)C]glutamate level, being an equilibrium between Na(+)-dependent uptake and tonic release, was increased from 0.193+/-0.013 nmol/mg protein to 0.282+/-0.013 (extracellular endogenous glutamate-from 6.9+/-2.0 to 16.6+/-2.0, respectively) in rat brain synaptosomes treated with a cholesterol acceptor methyl-beta-cyclodextrin (MbetaCD). This alteration was not due to the change in the activity of glutamine synthetase that was shown with the specific blocker L-methionine sulfoximine. In the presence of DL-threo-beta-benzyloxyaspartate, which significantly reduced the contribution of glutamate transporters, net tonic release of L-[(14)C]glutamate was decreased by 38% and release in low-Na(+) medium was attenuated by 41% after cholesterol extraction. Also, cholesterol-deficient synaptosomes showed a reduced content of cytosolic L-[(14)C]glutamate and a lower initial velocity of L-[(14)C]glutamate uptake. We suggested that cholesterol deficiency altered the intra-to-extracellular glutamate ratio by the reduction of the cytosolic level of the neurotransmitter and the augmentation of the ambient glutamate level, thereby favoring a decrease in tonic glutamate release. Thus, increased extracellular glutamate in cholesterol-deficient nerve terminals was not a result of the changes in tonic release and/or glutamine synthetase activity, but was set by lack function of glutamate transporters.

Presynaptic malfunction: the neurotoxic effects of cadmium and lead on the proton gradient of synaptic vesicles and glutamate transport.

Exposure to Cd(2+) and Pb(2+) has neurotoxic consequences for human health and may cause neurodegeneration. The study focused on the analysis of the presynaptic mechanisms underlying the neurotoxic effects of non-essential heavy metals Cd(2+) and Pb(2+). It was shown that the preincubation of rat brain nerve terminals with Cd(2+) (200 μM) or Pb(2+) (200 μM) resulted in the attenuation of synaptic vesicles acidification, which was assessed by the steady state level of the fluorescence of pH-sensitive dye acridine orange. A decrease in L-[(14)C]glutamate accumulation in digitonin-permeabilized synaptosomes after the addition of the metals, which reflected lowered L-[(14)C]glutamate accumulation by synaptic vesicles inside of synaptosomes, may be considered in the support of the above data. Using isolated rat brain synaptic vesicles, it was found that 50 μM Cd(2+) or Pb(2+) caused dissipation of their proton gradient, whereas the application of essential heavy metal Mn(2+) did not do it within the range of the concentration of 50-500 μM. Thus, synaptic malfunction associated with the influence of Cd(2+) and Pb(2+) may result from partial dissipation of the synaptic vesicle proton gradient that leads to: (1) a decrease in stimulated exocytosis, which is associated not only with the blockage of voltage-gated Ca(2+) channels, but also with incomplete filling of synaptic vesicles; (2) an attenuation of Na(+)-dependent glutamate uptake.

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 (40-800 μg/ml) in dose-dependent manner: (1) decreased exocytotic release of [(3)H]GABA and L-[(14)C]glutamate; (2) reduced acidification of synaptic vesicles; (3) attenuated the initial velocity of Na(+)-dependent transporter-mediated uptake of [(3)H]GABA and L-[(14)C]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 800 μg/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.

Cholesterol Depletion from the Plasma Membrane Impairs Proton and Glutamate Storage in Synaptic Vesicles of Nerve Terminals

We report that cholesterol depletion with methyl-β-cyclodextrin (MβCD) acutely applied to rat brain synaptosomes is accompanied by an immediate increase in transporter-mediated glutamate release and decrease in exocytotic release. To clarify the possible mechanisms underlying these phenomena, we investigated the influence of MβCD on synaptic vesicle acidification and exo/endocytotic process in nerve terminals. As shown by acridine orange fluorescence measurements, the application of MβCD to synaptosomes, as well as to isolated synaptic vesicles, led to the gradual leakage of the protons from the vesicles, whereas the application of MβCD complexed with cholesterol stimulated additional vesicle acidification and an increase in Ca2+-dependent exocytotic response. It was found that the treatment of nerve terminals with MβCD did not block Ca2+-triggered vesicle recycling. We suggest that cholesterol depletion of the plasma membrane with MβCD induces the removal of cholesterol from the membrane of synaptic vesicles resulting in immediate dissipation of synaptic vesicle proton gradient and redistribution of the neurotransmitter between the vesicular and cytosolic pools. The latter appears to be the main cause of a dramatic decrease in exocytotic and considerable increase in transporter-mediated release of l-[14C]glutamate.

Permanent dynamic transporter-mediated turnover of glutamate across the plasma membrane of presynaptic nerve terminals: arguments in favor and against

Abstract Mechanisms for maintenance of the extracellular level of glutamate in brain tissue and its regulation still remain almost unclear, and criticism of the current paradigm of glutamate transport and homeostasis has recently appeared. The main premise for this study is the existence of a definite and non-negligible concentration of ambient glutamate between the episodes of exocytotic release in our experiments with rat brain nerve terminals (synaptosomes), despite the existence of a very potent Na+-dependent glutamate uptake. Glutamate transporter reversal is considered as the main mechanisms of glutamate release under special conditions of energy deprivation, hypoxia, hypoglycemia, brain trauma, and stroke, underlying an increase in the ambient glutamate concentration and development of excitotoxicity. In the present study, a new vision on transporter-mediated release of glutamate as one of the main mechanisms involved in the maintenance of definite concentration of ambient glutamate under normal energetical status of nerve terminals is forwarded. It has been suggested that glutamate transporters act effectively in outward direction in a non-pathological manner, and this process is thermodynamically synchronized with uptake and provides effective outward glutamate current, thereby establishing and maintaining permanent and dynamic glutamatein/glutamateout gradient and turnover across the plasma membrane. In this context, non-transporter tonic glutamate release by diffusion, spontaneous exocytosis, cystine-glutamate exchanger, and leakage through anion channels can be considered as a permanently added ‘new’ exogenous substrate using two-substrate kinetic model calculations. Permanent glutamate turnover is of value for tonic activation of post/presynaptic glutamate receptors, long-term potentiation, memory formation, etc. Counterarguments against this mechanism are also considered.

Putative duality of presynaptic events

Abstract 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.

Perinatal hypoxia: different effects of the inhibitors of GABA transporters GAT1 and GAT3 on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals

Aim To analyze the effects of highly selective blocker GAT1, NO-711, and substrate inhibitor GAT3, β-alanine, on the initial velocity of [3H]GABA uptake by cortical, hippocampal, and thalamic nerve terminals (synaptosomes) after perinatal hypoxia. Methods Animals were divided into two groups: control (n = 17) and hypoxia (n = 12). Rats in the hypoxia group underwent hypoxia and seizures (airtight chamber, 4% O2 and 96% N2) at the age of 10-12 postnatal days and were used in the experiments 8-9 weeks after hypoxia. Results In cortical synaptosomes, the effects of NO-711 (30 μΜ) and β-alanine (100 μΜ) on [3H]GABA uptake were similar in control and hypoxia groups. In hippocampal synaptosomes, NO-711 inhibited 84.3% of the initial velocity of [3H]GABA uptake in normal conditions and 80.1% after hypoxia, whereas the effect of β-alanine was increased after hypoxia from 14.4% to 22.1%. In thalamic synaptosomes, the effect of NO-711 was decreased by 79.6% in controls and by 70.9% in hypoxia group, whereas the effect of β-alanine was increased after hypoxia from 20.2% to 30.2%. Conclusions The effectiveness of β-alanine to influence GABA uptake was increased in hippocampal and thalamic nerve terminals as a result of perinatal hypoxia and the effectiveness of NO-711 in thalamic nerve terminals was decreased. These results may indicate changes in the ratio of active GAT1/GAT3 expressed in the plasma membrane of nerve terminals after perinatal hypoxia. We showed a possibility to modulate non-GAT1 GABA transporter activity in different brain regions by exogenous and endogenous β-alanine.

The proton gradient of secretory granules and glutamate transport in blood platelets during cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin.

Glutamate transport in blood platelets resembles that in brain nerve terminals because platelets contain neuronal Na(+)-dependent glutamate transporters, glutamate receptors in the plasma membrane, vesicular glutamate transporters in secretory granules, which use the proton gradient as a driving force, and can release glutamate during aggregation/activation. The acidification of secretory granules and glutamate transport were assessed during acute treatment of isolated platelets with cholesterol-depleting agent methyl-β-cyclodextrin (MβCD). Confocal imaging with the cholesterol-sensitive fluorescent dye filipin showed a quick reduction of cholesterol level in platelets. Using pH-sensitive fluorescent dye acridine orange, we demonstrated that the acidification of secretory granules of human and rabbit platelets was decreased by ∼15% and 51% after the addition of 5 and 15mM MβCD, respectively. The enrichment of platelet plasma membrane with cholesterol by the application of complex MβCD-cholesterol (1:0.2) led to the additional accumulation of acridine orange in secretory granules indicating an increase in the proton pumping activity of vesicular H(+)-ATPase. MβCD did not evoke release of glutamate from platelets that was measured with glutamate dehydrogenase assay. Flow cytometric analysis did not reveal alterations in platelet size and granularity in the presence of MβCD. These data showed that the dissipation of the proton gradient of secretory granules rather than their exocytosis caused MβCD-evoked decrease in platelet acidification. Thus, the depletion of plasma membrane cholesterol in the presence of MβCD changed the functional state of platelets affecting storage capacity of secretory granules but did not evoke glutamate release from platelets.

Impaired Na+-dependent glutamate uptake in platelets during depolarization of their plasma membrane

Blood platelets contain neuronal Na(+)-dependent glutamate transporters and are able to accomplish glutamate uptake. Applying high-KCl, we have demonstrated dose-dependent depolarization of the plasma membrane of rabbit platelets that was registered as an increase in the fluorescence of the potential-sensitive fluorescent dye rhodamine 6G. The initial velocity of L-[(14)C]glutamate uptake (10 microM) in platelets was decreased by 20% during 35 mM KCl-evoked depolarization and consisted of 1.2+/-0.09 pmol x min(-1) x mg(-1) protein in control and 0.96+/-0.08 pmol x min(-1) x mg(-1) protein during depolarization. Confocal laser scanning microscopy and flow cytometry revealed that these changes in glutamate uptake were not a result of platelet aggregation/activation. Also, addition of high-KCl did not change acidification of platelet secretory granules that was found with pH-sensitive fluorescent dye acridine orange, thereby showing that changes in their proton gradient could not cause glutamate uptake alteration. This malfunction of glutamate transporters has to take place under: (i) the conditions of pseudohyperkalemia or hyperkalemia, i.e. activation and clotting of platelets, haemolysis, leucocytosis, acute renal failure, hypofunction of adrenal cortex, lack of aldosterone, stroke, trauma and (ii) depolarization of the plasma membrane of platelets during their activation by ADP, thrombin, platelet-activating factor. Weak glutamate uptake might have considerable consequences for platelets per se (and thus for haemostatic system) and glutamate homeostasis in the CNS.

Personalized approach in brain protection by hypothermia: individual changes in non-pathological and ischemia-related glutamate transport in brain nerve terminals

BackgroundBoth deep and profound hypothermia are effectively applied in cardiac surgery of the aortic arch, when the reduction of cerebral circulation facilitates operations, and for the prevention of ischemic stroke consequences. Neurochemical discrimination of the effects of deep and profound hypothermia (27 and 17 °C, respectively) on non-pathological and pathological ischemia-related mechanisms of presynaptic glutamate transport with its potential contribution to predictive, preventive and personalized medicine (PPPM) was performed.MethodsExperiments were conducted using nerve terminals isolated from rat cortex (synaptosomes). Glutamate transport in synaptosomes was analyzed using radiolabel l-[14C]glutamate. Diameter of synaptosomes was assessed by dynamic light scattering.ResultsSynaptosomal transporter-mediated uptake and tonic release of l-[14C]glutamate (oppositely directed processes, dynamic balance of which determines the physiological extracellular level of the neurotransmitter) decreased in a different range in deep/profound hypothermia. As a result, hypothermia-induced changes in extracellular l-[14C]glutamate are not evident (in one half of animals it increased, and in other it decreased). A progressive decrease from deep to profound hypothermia was shown for pathological mechanisms of presynaptic glutamate transport, that is, transporter-mediated l-[14C]glutamate release (*) stimulated by depolarization of the plasma membrane and (**) during dissipation of the proton gradient of synaptic vesicles by the protonophore FCCP.ConclusionsTherefore, the direction of hypothermia-induced changes in extracellular glutamate is unpredictable in “healthy” nerve terminals and depends on hypothermia sensitivity of uptake vs. tonic release. In affected nerve terminals (e.g., in brain regions suffering from a reduction of blood circulation during cardiac surgery, and core and penumbra zones of the insult), pathological transporter-mediated glutamate release from nerve terminals decreases with progressive significance from deep to profound hypothermia, thereby underlying its potent neuroprotective action. So, alterations in extracellular glutamate during hypothermia can be unique for each patient. An extent of a decrease in pathological glutamate transporter reversal depends on the size of damaged brain zone in each incident. Therefore, test parameters and clinical criteria of neuromonitoring for the evaluation of individual hypothermia-induced effects should be developed and delivered in practice in PPPM.