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.

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

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

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.

Inhibition of sigma-1 receptors substantially modulates GABA and glutamate transport in presynaptic nerve terminals.

Sigma-1 receptors (Sig-1Rs) have been implicated in many neurological and psychiatric disorders and are a novel target for the treatment of such disorders. Sig-1R expression/activity deficits are linked to neurodegeneration, whereas the mechanisms mediated by Sig-1R are still unclear. Here, presynaptic [3H]GABA and L-[14C]glutamate transport was analysed in rat brain nerve terminals (synaptosomes) in the presence of the Sig-1R antagonist NE-100. NE-100 at doses of 1 and 10 µM increased the initial rate of synaptosomal [3H]GABA uptake, whereas 50 and 100 µM NE-100 decreased this rate, exerting a biphasic mode of action.Antagonists of GABAA and GABAB receptors, flumazenil and saclofen, respectively, prevented an increase in [3H]GABA uptake caused by 10 µM NE-100. L-[14C]glutamate uptake was decreased by 10-100 µM NE-100. A decrease in the uptake of both neurotransmitters mediated by NE-100 (50-100 µM) may have resulted from simultaneous antagonist-induced membrane depolarization, which was measured using the potential-sensitive fluorescent dye rhodamine 6G. The extracellular level of [3H]GABA was decreased by 1-10 µM NE-100, but that of L-[14C]glutamate remained unchanged. The tonic release of [3H]GABA measured in the presence of NO-711 was not changed by the antagonist, suggesting that NE-100 did not disrupt membrane integrity. The KCl- and FCCP-induced transporter-mediated release of L-[14C]glutamate was decreased by the antagonist; this may underlie the neuroprotective action of the antagonist in hypoxia/ischaemia. NE-100 (10-100 µM) decreased the KCl-evoked exocytotic release of [3H]GABA and L-[14C]glutamate, whereas the induction of the release of both neurotransmitters by the Ca2+ ionophore ionomycin was not affected by the antagonist; therefore, the mitigation of KCl-evoked exocytosis was associated with the NE-100-induced dysfunction of potential-dependent Ca2+ channels. Therefore, the Sig-1R antagonist can specifically act in an acute manner at the presynaptic level through the modulation of GABA and glutamate uptake, transporter-mediated release and exocytosis.

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.

Age-Dependency of Levetiracetam Effects on Exocytotic GABA Release from Nerve Terminals in the Hippocampus and Cortex in Norm and After Perinatal Hypoxia.

Perinatal hypoxia can lead to multiple chronic neurological deficits, e.g., mental retardation, behavioral abnormalities, and epilepsy. Levetiracetam (LEV), 2S-(2-oxo-1-pyrrolidiny1) butanamide, is an anticonvulsant drug with proven efficiency in treating patients with focal and generalized seizures. Rats were underwent hypoxia and seizures at the age of 10-12 postnatal days (pd). The ambient level and depolarization-induced exocytotic release of [3H]GABA (γ-aminobutyric acid) were analyzed in nerve terminals in the hippocampus and cortex during development at the age of pd 17-19 and pd 24-26 (infantile stage), pd 38-40 (puberty) and pd 66-73 (young adults) in norm and after perinatal hypoxia. LEV had no effects on the ambient [3H]GABA level. The latter increased during development and was further elevated after perinatal hypoxia in nerve terminals in the hippocampus during the whole period and in the cortex in young adults. Exocytotic [3H]GABA release from nerve terminals increased after perinatal hypoxia during development in the hippocampus and cortex, however this effect was preserved at all ages during blockage of GABA transporters by NO-711 in the hippocampus only. LEV realized its anticonvulsant effects at the presynaptic site through an increase in exocytotic release of GABA. LEV exerted more significant effect after perinatal hypoxia than in norm. Action of LEV was strongly age-dependent and can be registered in puberty and young adults, but the drug was inert at the infantile stage.

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.

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.

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.

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, ß-alanine, on the initial velocity of [(3)H]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 ß-alanine (100 µΜ) on [(3)H]GABA uptake were similar in control and hypoxia groups. In hippocampal synaptosomes, NO-711 inhibited 84.3% of the initial velocity of [(3)H]GABA uptake in normal conditions and 80.1% after hypoxia, whereas the effect of ß-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 ß-alanine was increased after hypoxia from 20.2% to 30.2%. CONCLUSIONS. The effectiveness of ß-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 ß-alanine.