Substitution of bridge carbons for sulphur in calix[4]arene-bis-α-hydroxymethylphosphonic acid transformed mobile carrier into ionic channel accompanied with evoked muscle contraction and impaired neurotransmission powered by membrane action of resulting thiocalix[4]arene-bis-α-hydroxymethylphosphonic acid.

The action of calix[4]arenes C-424, C-425 and C-1193 has been investigated on suspended cholesterol/egg phosphatidylcholine lipid bilayer in a voltage-clamp mode. Comparative analysis with the membrane action by calix[4]arene-bis-α-hydroxymethylphosphonic acid (C-99) has shown that the substitution of bridge carbons for sulphur and addition of another methyl group to two alkyl tales in the lower rim of former dipropoxycalix[4]arene C-99 transformed mobile carrier that C-99 created in lipid bilayer (Shatursky et al., 2014) into a transmembrane pore as exposure of the bilayer membrane to sulphur-containing derivative dibutoxythiocalix[4]arene C-1193 resulted in microscopic transmembrane current patterns indicative of a channel-like mode of facilitated diffusion. Within all calix[4]arenes tested a net steady-state voltage-dependent transmembrane current was readily achieved only after addition of calix[4]-arene C-1193. In comparison with the membrane action of C-99 the current induced by calix[4]-arene C-1193 exhibited a much weakened anion selectivity passing slightly more current at positive potentials applied from the side of bilayer membrane to which the calix[4]-arene was added. Testing C-1193 for the membrane action against smooth muscle cells of rat uterus or swine myometrium and synaptosomes of rat brain nerve terminals revealed an increase in intracellular concentration of Ca2+ with reduction of the effective hydrodynamic diameter of the smooth muscle cells and enhanced basal extracellular level of neurotransmitters (glutamate and γ-aminobutyric acid) after C-1193-induced depolarization of the nerve terminals.

Multipollutant reciprocal neurological hazard from smoke particulate matter and heavy metals cadmium and lead in brain nerve terminals.

Heavy metals, Cd2+ and Pb2+, and carbonaceous air pollution particulate matter are hazardous neurotoxicants. Here, a capability of water-suspended smoke particulate matter preparations obtained from poplar wood (WPs) and polypropylene fibers (medical facemasks) (MPs) to influence Cd2+/Pb2+-induced neurotoxicity, and vice versa, was monitored using biological system, i.e. isolated presynaptic rat cortex nerve terminals. Combined application of Pb2+ and WPs/MPs to nerve terminals in an acute manner revealed that smoke preparations did not change a Pb2+-induced increase in the extracellular levels of excitatory neurotransmitter L-[14C]glutamate and inhibitory one [3H]GABA, thereby demonstrating additive result and no interference of neurotoxic effects of Pb2+ and particulate matter. Whereas, both smoke preparations decreased a Cd2+-induced increase in the extracellular level of L-[14C]glutamate and [3H]GABA in nerve terminals. In fluorimetric measurements, the metals and smoke preparations demonstrated additive effects on the membrane potential of nerve terminals causing membrane depolarisation. WPs/MPs-induced reduction of spontaneous ROS generation was mitigated by Cd2+ and Pb2+. Therefore, a potential variety of multipollutant heavy metal-/airborne particulate-induced effects on key presynaptic processes was revealed. Multipollutant reciprocal neurological hazard through disturbance of the excitation-inhibition balance, membrane potential and ROS generation was evidenced. This multipollutant approach and data contribute to up-to-date environmental quality/health risk estimation.

Mercury-induced excitotoxicity in presynaptic brain nerve terminals: modulatory effects of carbonaceous airborne particulate simulants.

Multipollutant approach is a breakthrough in up-to-date environmental quality and health risk estimation. Both mercury and carbonaceous air particulate are hazardous neurotoxicants. Here, the ability of carbonaceous air particulate simulants, i.e. carbon dots obtained by heating of organics, and nanodiamonds, to influence Hg2+-induced neurotoxicity was monitored using biological system, i.e. presynaptic rat cortex nerve terminals. Using HgCl2 and classical reducing/chelating agents, an adequate synaptic parameter, i.e. the extracellular level of key excitatory neurotransmitter L-[14C]glutamate, was selected for further analysis. HgCl2 starting from 5 µM caused an acute and concentration-dependent increase in the extracellular L-[14C]glutamate level in nerve terminals. Combined application of Hg2+ and carbon dots from heating of citric acid/urea showed that this simulant was able to mitigate in an acute manner excitotoxic Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals by 37%. These carbon dots and Hg2+ acted as a complex in nerve terminals that was confirmed with fluorimetric data on Hg2+-induced changes in their spectroscopic features. Nanodiamonds and carbon dots from ß-alanine were not able to mitigate a Hg2+-induced increase in the extracellular L-[14C]glutamate level in nerve terminals. Developed approach can be applicable for monitoring capability of different particles/compounds to have Hg2+-chelating signs in the biological systems. Therefore, among testing simulants, the only carbon dots from citric acid/urea were able to mitigate acute Hg2+-induced neurotoxicity in nerve terminals, thereby showing a variety of effects of carbonaceous airborne particulate in situ and its potential to interfere and modulate Hg2+-associated health hazard.

Disposable facemask waste combustion emits neuroactive smoke particulate matter.

Tremendous deposits of disposable medical facemask waste after the COVID-19 pandemic require improvement of waste management practice according to WHO report 2022, moreover facemasks are still in use around the world to protect against numerous airborne infections. Here, water-suspended smoke preparations from the combustion of disposable medical facemasks (polypropylene fibers) were collected; size, zeta potential, surface groups of smoke particulate matter were determined by dynamic light scattering, FTIR and Raman spectroscopy, and their optical properties were characterized. Neurochemical study using nerve terminals isolated from rat cortex revealed a significant decrease in the initial rate of the uptake/accumulation of excitatory and inhibitory neurotransmitters, L-[14C]glutamate and [3H]GABA, and exocytotic release, and also an increase in the extracellular level of these neurotransmitters. Fluorescent measurements revealed that ROS generation induced by hydrogen peroxide and glutamate receptor agonist kainate decreased in nerve terminals. A decrease in the membrane potential of nerve terminals and isolated neurons, the mitochondrial potential and synaptic vesicle acidification was also shown. Therefore, accidental or intentional utilization of disposable medical facemask waste by combustion results in the release of neuroactive ultrafine particulate matter to the environment, thereby contributing to plastic-associated pollution of air and water resources and neuropathology development and expansion.

A comparative multi-level toxicity assessment of carbon-based Gd-free dots and Gd-doped nanohybrids from coffee waste: hematology, biochemistry, histopathology and neurobiology study.

Here, a comparative toxicity assessment of precursor carbon dots from coffee waste (cofCDs) obtained using green chemistry principles and Gd-doped nanohybrids (cofNHs) was performed using hematological, biochemical, histopathological assays in vivo (CD1 mice, intraperitoneal administration, 14 days), and neurochemical approach in vitro (rat cortex nerve terminals, synaptosomes). Serum biochemistry data revealed similar changes in cofCDs and cofNHs-treated groups, i.e. no changes in liver enzymes' activities and creatinine, but decreased urea and total protein values. Hematology data demonstrated increased lymphocytes and concomitantly decreased granulocytes in both groups, which could evidence inflammatory processes in the organism and was confirmed by liver histopathology; decreased red blood cell-associated parameters and platelet count, and increased mean platelet volume, which might indicate concerns with platelet maturation and was confirmed by spleen histopathology. So, relative safety of both cofCDs and cofNHs for kidney, liver and spleen was shown, whereas there were concerns about platelet maturation and erythropoiesis. In acute neurotoxicity study, cofCDs and cofNHs (0.01 mg/ml) did not affect the extracellular level of L-[14C]glutamate and [3H]GABA in nerve terminal preparations. Therefore, cofNHs demonstrated minimal changes in serum biochemistry and hematology assays, had no acute neurotoxicity signs, and can be considered as perspective biocompatible non-toxic theragnostic agent.

Similar in vitro response of rat brain nerve terminals, colon preparations and COLO 205 cells to smoke particulate matter from different types of wood.

Major source of carbon-containing air born particular matter that significantly pollutes environment and provokes development of neuropathology is forest fires and wood combustion. Here, water-suspended smoke particulate matter preparations (SPs) were synthesized from birch, pine, poplar wood, and also birch bark and pine needles. Taking into account importance of the gut-brain communication system, SP properties were compared regarding their capability to modulate functioning of nerve terminals and gut cells/preparations. In cortex nerve terminals, poplar wood SP was more effective in decreasing uptake and increasing the extracellular levels of excitatory and inhibitory neurotransmitters L-[14C]glutamate and [3H]GABA, respectively. Spontaneous and H2O2-stimulated ROS generation in nerve terminals decreased by SPs, the most efficient one was from poplar wood. SPs from birch, pine and poplar wood caused membrane depolarization, poplar wood SP effect was 5-fold higher vs. birch and pine wood ones. Functional characteristics of gut cells/preparations, which tightly related to nerve terminal experiments, were assessed. SPs increased paracellular permeability of proximal colon mucosal-submucosal preparations monitored in Ussing chamber system (FITC-dextran, 4 kDa), where the most prominent effect had poplar wood SP. The latter demonstrated more considerable influence on COLO 205 cell causing 30 % loss of cell viability. PM emitted to the environment during combustion of various wood caused similar unidirectional harmful effects on brain and gut cell functioning, thereby triggering development of pathologies in gut and brain and gut-brain communication system.

Neuromodulation by selective angiotensin-converting enzyme 2 inhibitors.

Here, neuromodulatory effects of selective angiotensin-converting enzyme 2 (ACE2) inhibitors were investigated. Two different types of small molecule ligands for ACE2 inhibition were selected using chemical genetic approach, they were synthesized using developed chemical method and tested using presynaptic rat brain nerve terminals (synaptosomes). EBC-36032 (1 µM) increased in a dose-dependent manner spontaneous and stimulated ROS generation in nerve terminals that was of non-mitochondrial origin. Another inhibitor EBC-36033 (MLN-4760) was inert regarding modulation of ROS generation. EBC-36032 and EBC-36033 (100 µM) did not modulate the exocytotic release of L-[14C]glutamate, whereas both inhibitors decreased the initial rate of uptake, but not accumulation (10 min) of L-[14C]glutamate by nerve terminals. EBC-36032 (100 µM) decreased the exocytotic release as well as the initial rate and accumulation of [3H]GABA by nerve terminals. EBC-36032 and EBC-36033 did not change the extracellular levels and transporter-mediated release of [3H]GABA and L-[14C]glutamate, and tonic leakage of [3H]GABA from nerve terminals. Therefore, synthesized selective ACE2 inhibitors decreased uptake of glutamate and GABA as well as exocytosis of GABA at the presynaptic level. The initial rate of glutamate uptake was the only parameter that was mitigated by both ACE2 inhibitors despite stereochemistry issues. In terms of ACE2-targeted antiviral/anti-SARS-CoV-2 and other therapies, novel ACE2 inhibitors should be checked on the subject of possible renin-angiotensin system (RAS)-independent neurological side effects.

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission.

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.

A comparative study of wood sawdust and plastic smoke particulate matter with a focus on spectroscopic, fluorescent, oxidative, and neuroactive properties.

Here, water-suspended smoke aerosol preparation was synthesized from biomass-based fuel, i.e., a widespread product for residential heating, wood sawdust (WP) (pine, poplar, and birch mixture), and its properties were compared in parallel experiments with the smoke preparation from plastics (PP). Molecular groups in the PM preparations were analyzed using Raman and Fourier-transform infrared spectroscopy. WP was assessed in neurotoxicity studies using rat cortex nerve terminals (synaptosomes). Generation of spontaneous and H2O2-evoked reactive oxygen species (ROS) detected using fluorescent dye 2',7'-dichlorofluorescein in nerve terminals was decreased by WP. In comparison with PP, WP demonstrated more pronounced reduction of spontaneous and H2O2-evoked ROS production. WP completely inhibited glutamate receptor agonist kainate-induced ROS production, thereby affecting the glutamate receptor-mediated signaling pathways. WP decreased the synaptosomal membrane potential in fluorimetric experiments and the synaptosomal transporter-mediated uptake of excitatory and inhibitory neurotransmitters, L-[14C]glutamate and [3H] γ-aminobutyric acid (GABA), respectively. PP decreased the ambient synaptosomal level of [3H]GABA, whereas it did not change that of L-[14C]glutamate. Principal difference between WP and PP was found in their ability to influence the ambient synaptosomal level of [3H]GABA (an increase and decrease, respectively), thereby showing riskiness in mitigation of synaptic inhibition by PP and triggering development of neuropathology.

Unique features of brain metastases-targeted AGuIX nanoparticles vs their constituents: A focus on glutamate-/GABA-ergic neurotransmission in cortex nerve terminals.

Gadolinium-based radiosensitizing AGuIX nanoparticles (AGuIX) currently tested two phase 2 clinical trials in association with radiotherapy for the treatment of brain metastases. Here, excitatory/inhibitory neurotransmission was assessed in rat cortex nerve terminals in the presence of AGuIX and their constituents (DOTAGA and DOTAGA/Gd3+) at concentrations used for medical treatment, and those 5-24 times higher. The ambient level, transporter-mediated, tonic and exocytotic release of L-[14C]glutamate and [3H]GABA, the membrane potential of nerve terminals were not changed in the presence of AGuIX at concentrations used for medical treatment ([Gd3+] = 0.25 mM, corresponding to 0.25 g.L-1), and DOTAGA (0.25 mM) and DOTAGA/Gd3+ (0.25 mM/0.01 mM). Difference between AGuIX and the precursors was uncovered, when their concentrations were increased. AGuIX (1.25-6 mM) did not change any transport characteristics of L-[14C]glutamate and [3H]GABA, whereas, DOTAGA (1.25-6 mM) affected the membrane potential, ambient level, and exocytotic release of L-[14C]glutamate and [3H]GABA. Gd3+ did not mask, but even enhanced above effects of DOTAGA. Therefore, AGuIX did not influence glutamate- and GABA-ergic neurotransmission at the presynaptic site. In contrast, DOTAGA and mixture DOTAGA/Gd3+ significantly affected synaptic neurotransmission at high concentrations. AGuIX own structure that overcomes neurotoxic features of their constituents.

Transient coating of γ-Fe2O3 nanoparticles with glutamate for its delivery to and removal from brain nerve terminals.

Glutamate is the main excitatory neurotransmitter in the central nervous system and excessive extracellular glutamate concentration is a characteristic feature of stroke, brain trauma, and epilepsy. Also, glutamate is a potential tumor growth factor. Using radiolabeled ʟ-[14C]glutamate and magnetic fields, we developed an approach for monitoring the biomolecular coating (biocoating) with glutamate of the surface of maghemite (γ-Fe2O3) nanoparticles. The nanoparticles decreased the initial rate of ʟ-[14C]glutamate uptake, and increased the ambient level of ʟ-[14C]glutamate in isolated cortex nerve terminals (synaptosomes). The nanoparticles exhibit a high capability to adsorb glutamate/ʟ-[14C]glutamate in water. Some components of the incubation medium of nerve terminals, that is, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and NaH2PO4, decreased the ability of γ-Fe2O3 nanoparticles to form a glutamate biocoating by about 50% and 90%, respectively. Only 15% of the amount of glutamate biocoating obtained in water was obtained in blood plasma. Albumin did not prevent the formation of a glutamate biocoating. It was shown that the glutamate biocoating is a temporal dynamic structure at the surface of γ-Fe2O3 nanoparticles. Also, components of the nerve terminal incubation medium and physiological fluids responsible for the desorption of glutamate were identified. Glutamate-coated γ-Fe2O3 nanoparticles can be used for glutamate delivery to the nervous system or for glutamate adsorption (but with lower effectiveness) in stroke, brain trauma, epilepsy, and cancer treatment following by its subsequent removal using a magnetic field. γ-Fe2O3 nanoparticles with transient glutamate biocoating can be useful for multifunctional theranostics.

Plastic smoke aerosol: Nano-sized particle distribution, absorption/fluorescent properties, dysregulation of oxidative processes and synaptic transmission in rat brain nerve terminals.

Smoke from plastic waste incineration in an open air travels worldwide and is a major source of air pollution particulate matter (PM) that is very withstand to degradation and hazard to human health. Suspension of smoke aerosol components in water occurs during rains and fire extinguishing. Here, water-suspended plastic smoke aerosol (WPS) preparations suitable for biotesting were synthesized. It has been revealed using dynamic light scattering that WPS contained major nano-sized (∼30 nm) PM fraction, and this result was confirmed by electron microscopy. Optical absorption of WPS was in the UV region and an increase in λex led to a red-shift in fluorescence emission with a corresponding decrease in fluorescence intensity. WPS was analyzed in neurotoxicity studies in vitro using presynaptic rat cortex nerve terminals (synaptosomes). Generation of spontaneous reactive oxygen species (ROS) detected using fluorescent dye 2',7-dichlorofluorescein in nerve terminals was decreased by WPS (10-50 µg/ml) in a dose-dependent manner. WPS also reduced the H2O2-evoked ROS production in synaptosomes, thereby influencing cellular oxidative processes and this effect was similar to that for carbon nanodots. WPS (0.1 mg/ml) decreased the synaptosomal membrane potential and synaptic vesicle acidification in fluorimetric experiments. WPS (1.0 mg/ml) attenuated the synaptosomal transporter-mediated uptake of excitatory and inhibitory neurotransmitters, L-[14C]glutamate and [3H]GABA, respectively. This can lead to an excessive increase in the glutamate concentration in the synaptic cleft and neurotoxicity via over activation of ionotropic glutamate receptors. Therefore, WPS was neurotoxic and provoked presynaptic malfunction through changes of oxidative activity, reduction of the membrane potential, synaptic vesicle acidification, and transporter-mediated uptake of excitatory and inhibitory neurotransmitters in nerve terminals. In summary, synthesis and emission to the environment of ultrafine PM occur during combustion of plastics, thereby polluting air and water resources, and possibly triggering development of neuropathologies.

Membrane action of polyhexamethylene guanidine hydrochloride revealed on smooth muscle cells, nerve tissue and rat blood platelets: A biocide driven pore-formation in phospholipid bilayers.

A well-known cationic biocide of guanidine polymer family, polyhexamethylene guanidine hydrochloride (PHMG) has been tested against smooth muscle cells isolated from swine myometrium, synaptosomes of rat brain nerve terminals and rat blood platelets for the membrane action. It was established that PHMG blocked the activity of Na+,K+-ATPase of smooth muscle cells plasma membrane by 82.2 ±â€¯0.9% at a concentration of 7 ppm, whilst a dose-dependent depolarization of synaptosomes and platelets became appreciable at 100-500 ppm. Comparative studies by the methods of mass spectrometry (MALDI-TOF and PDMS-TOF), viscosimetry, dynamic light scattering and model phospholipid membranes revealed PHMG oligomers with various number of repeat units (8-16) that formed K+-selective potential-dependent pores in sterol-free phosphatidylethanolamine-containing phospholipid bilayers at a concentration of 1 ppm. Obtained results suggest that besides acidic lipids and membrane proteins phosphatidylethanolamine and cholesterol are the other major factors responsible for the differences between PHMG-induced plasma membrane depolarization of microbial and eukaryotic cells and thus, diverse modes of PHMG membrane action.

Vitamin D3 deficiency in puberty rats causes presynaptic malfunctioning through alterations in exocytotic release and uptake of glutamate/GABA and expression of EAAC-1/GAT-3 transporters.

Recent experimental and epidemiologic investigations have revealed that the central nervous system is a target for vitamin D3 action and also linked vitamin D3 deficiency to Alzheimer's and Parkinson's disease, autism and dementia. Abnormal homeostasis of glutamate and GABA and signaling disbalance are implicated in the pathogenesis of major neurological diseases. Here, key transport characteristics of glutamate and GABA were analysed in presynaptic nerve terminals (synaptosomes) isolated from the cortex of vitamin D3 deficient (VDD) rats. Puberty rats were kept at the VDD diet up to adulthood. VDD caused: (i) a decrease in the initial rates of L-[14C]glutamate and [3H]GABA uptake by plasma membrane transporters of nerve terminals; (ii) a decrease in exocytotic release of L-[14C]glutamate and [3H]GABA; (iii) changes in expression of glutamate (EAAC-1) and GABA (GAT-3) transporters. Whereas, the synaptosomal ambient levels and Ca2+-independent transporter-mediated release of L-[14C]glutamate and [3H]GABA were not significantly altered in VDD. Vitamin D3 is a potent neurosteroid and its nutritional deficiency can provoke development of neurological consequences changing glutamate/GABA transporter expressions and excitation/inhibition balance. Also, changes in glutamate transport can underlie lower resistance to hypoxia/ischemia, larger infarct volumes and worsened outcomes in ischemic stroke patients with VDD.

Combined Application of Glutamate Transporter Inhibitors and Hypothermia Discriminates Principal Constituent Processes Involved in Glutamate Homo- and Heteroexchange in Brain Nerve Terminals.

Deep and profound hypothermia is successfully practiced in the prevention of ischemic stroke consequences and aortic arch cardiac surgery accompanied by reduction of cerebral circulation. Hypothermia is a current neuroprotection standard in hypoxic/ischemic encephalopathy. Drug-hypothermia administration is proposed as a new approach in pharmacotherapy for neonatal seizures. Also, hypothermia is useful as neuroprotective approach in long-term interplanetary space missions. We recently revealed gradual dynamics of hypothermia-induced decrease in transporter-mediated release and uptake of L-[14C]glutamate in presynaptic rat brain nerve terminals (synaptosomes), thereby confirming potent unspecific neuroprotective effect of hypothermia. Glutamate homo- and heteroexchange are significant mechanisms involved in the maintenance of the extracellular glutamate level in nerve terminals. We have analyzed whether glutamate homo- and heteroexchange in nerve terminals is temperature sensitive. In this study we showed that synaptosomal glutamate-induced L-[14C]glutamate release (homoexchange) and D-aspartate- and DL-threo-ß-hydroxyaspartate-induced L-[14C]glutamate release (heteroexchange) gradually decreased from deep (27°C) to profound (17°C) hypothermia with dynamics similar to that of glutamate transporter reversal. Interestingly, ambient L-[14C]glutamate concentration in the nerve terminal preparations remained unaltered during hypothermia administration. Therefore, we demonstrated that glutamate homo- and heteroexchange decreased from deep to profound hypothermia thereby preventing further elevation of extracellular glutamate. Hypothermia uncovered the principal processes contributing to glutamate homo- and heteroexchange in nerve terminals and the maintenance of definite ambient glutamate concentration. Additionally, we showed that glutamate transporter reversal can be nonpathological and occurs under physiological conditions at least as a part of homo- and heteroexchange mechanisms.

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.

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 ß-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 10 nm, 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 (0.5-1.0 mg/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 0.2 mg/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 0.1-0.5 mg/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 ß-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 ß-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.

Effect of O-methyl-ß-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-ß-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.

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

BACKGROUND: Both 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. METHODS: Experiments 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. RESULTS: Synaptosomal 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. CONCLUSIONS: Therefore, 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.

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

BACKGROUND: Nanodiamonds are one of the most perspective nano-sized particles with superb physical and chemical properties, which are mainly composed of carbon sp(3) structures in the core with sp(2) 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., sp(2) hybridized graphene islands and diamond-like sp(3) hybridized elements. RESULTS: In 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-[(14)C]glutamate and [(3)H]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. CONCLUSIONS: Therefore, 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.