IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals.

The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON-NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca2+]i which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.

Disruption of Functional Activity of Mitochondria during MTT Assay of Viability of Cultured Neurons.

The MTT assay based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium in the cell cytoplasm to a strongly light absorbing formazan is among the most commonly used methods for determination of cell viability and activity of NAD-dependent oxidoreductases. In the present study, the effects of MTT (0.1 mg/ml) on mitochondrial potential (ΔΨm), intracellular NADH, and respiration of cultured rat cerebellum neurons and isolated rat liver mitochondria were investigated. MTT caused rapid quenching of NADH autofluorescence, fluorescence of MitoTracker Green (MTG) and ΔΨm-sensitive probes Rh123 (rhodamine 123) and TMRM (tetramethylrhodamine methyl ester). The Rh123 signal, unlike that of NADH, MTG, and TMRM, increased in the nucleoplasm after 5-10 min, and this was accompanied by the formation of opaque aggregates of formazan in the cytoplasm and neurites. Increase in the Rh123 signal indicated diffusion of the probe from mitochondria to cytosol and nucleus due to ΔΨm decrease. Inhibition of complex I of the respiratory chain decreased the rate of formazan formation, while inhibition of complex IV increased it. Inhibition of complex III and ATP-synthase affected only insignificantly the rate of formazan formation. Inhibition of glycolysis by 2-deoxy-D-glucose blocked the MTT reduction, whereas pyruvate increased the rate of formazan formation in a concentration-dependent manner. MTT reduced the rate of oxygen consumption by cultured neurons to the value observed when respiratory chain complexes I and III were simultaneously blocked, and it suppressed respiration of isolated mitochondria if substrates oxidized by NAD-dependent dehydrogenases were used. These results demonstrate that formazan formation in cultured rat cerebellum neurons occurs primarily in mitochondria. The initial rate of formazan formation may serve as an indicator of complex I activity and pyruvate transport rate.

[BRAIN FOCAL ISCHEMIA-REPERFUSION CAUSES A DECREASED RESISTANCE OF ERYTHROCYTES FROM VENOUS BLOOD TO ACID HEMOLYSIS, WHICH IS PREVENTED BY ECDYSTERONE].

We investigated the resistance of erythrocytes from rat brain venous blood to acid hemolysis in the dynamics of brain ischemic period (15, 30, 45 and 60 min), as well as in the early (5 min) and distant (24h) period of brain reperfusion. Brain ischemia-reperfusion was made in rats that received ecdysterone (standartized extract of Serratula coronata) within 18 days (per os, 1 mg/kg). Analysis of the kinetic curves of acid hemolysis showed a pronounced (60 times, from 1.45 to 85.85% at 60 min of brain ischemia and at 5 min of brain reperfusion, respectively) increase of unstable erythrocytes that hemolyzed easily (< 2.5 min). In the preconditioned rats, this increase was only 8-fold. During the period of brain ischemia, with a maximum at 15th minute, in the venous blood from brain the diene conjugates (DK) pools increased from 2.40 to 9.48 ng/mg protein and LTC4 pools increased from 1.49 to 5.98 pmol/mg protein. Even more pools of DC and LTC4 were increased at 5th min of brain reperfusion. In animals received ecdysterone, during ischemia and early reperfusion period, both pools of DC and LTC4 in venous blood were lower than that in the controls. The latter implies a possible antiradical mechanism of the protective effect of ecdysterone.

[Induction of oxidative stress in heart mitochondria in brain focal ischemia-reperfusion and protective effect of ecdysterone].

Based on the fact that the acute phase of ischemic stroke is accompanied by the development of heart damage, manifestations of which are oxidative stress, morphological changes in the myocardium, in the model of brain focal ischemia-reperfusion, we investigated the oxidative stress in rat heart mitochondria and possible mechanisms of cardioprotective effect of ecdysterone. Under the conditions of brain focal ischemia-reperfusion, there is an increase rate of the generation of reactive oxygen species: superoxide (*O2-) and hydroxyl radicals (*OH), pools of stable hydrogen peroxide (H2O2), accumulate products of lipid peroxidation (diene conjugates and malonic dialdehyde), as a result of activation xanthine oxidase (marker uric acid), lipooxygenase (marker leukotriene C4) and cyclooxygenase (marker tromboksane B2) ways of *O2-(generating). In animals that received ecdysterone for 18 days, under conditions of brain focal ischemia-reperfusion, the rate of reactive oxygen species generation and the pools of lipid peroxidation products were decreased, and the survival of animals was increased. The obtained results support the development of oxidative stress in heart mitochondria of rats, powerful antiradical properties ofecdysterone, its cardioprotective effect, in conditions of brain focal ischemia-reperfusion.

Study on ATP concentration changes in cytosol of individual cultured neurons during glutamate-induced deregulation of calcium homeostasis.

For the first time, simultaneous monitoring of changes in the concentration of cytosolic ATP ([ATP]c), pH (pHc), and intracellular free Ca2+ concentration ([Ca2+]i) of the individual neurons challenged with toxic glutamate (Glu) concentrations was performed. To this end, the ATP-sensor AT1.03, which binds to ATP and therefore enhances the efficiency of resonance energy transfer between blue fluorescent protein (energy donor) and yellow-green fluorescent protein (energy acceptor), was expressed in cultured hippocampal neurons isolated from 1-2-day-old rat pups. Excitation of fluorescence in the acceptor protein allowed monitoring changes in pHc. Cells were loaded with fluorescent low-affinity Ca2+ indicators Fura-FF or X-rhod-FF to register [Ca2+]i. It was shown that Glu (20 µM, glycine 10 µM, Mg2+-free) produced a rapid acidification of the cytosol and decrease in [ATP]c. An approximately linear relationship (r(2) = 0.56) between the rate of [ATP]c decline and latency of glutamate-induced delayed calcium deregulation (DCD) was observed: higher rate of [ATP]c decrease corresponded to shorter DCD latency period. DCD began with a decrease in [ATP]c of as much as 15.9%. In the phase of high [Ca2+]i, the plateau of [ATP]c dropped to 10.4% compared to [ATP]c in resting neurons (100%). In the presence of the Na+/K+-ATPase inhibitor ouabain (0.5 mM), glutamate-induced reduction in [ATP]c in the phase of the high [Ca2+]i plateau was only 36.6%. Changes in [ATP]c, [Ca2+]i, mitochondrial potential, and pHc in calcium-free or sodium-free buffers, as well as in the presence of the inhibitor of Na+/K+-ATPase ouabain (0.5 mM), led us to suggest that in addition to increase in proton conductivity and decline in [ATP]c, one of the triggering factors of DCD might be a reversion of the neuronal plasma membrane Na+/Ca2+ exchange.

[Induction of nitrosative stress in mitochondria of rats hearts in experimental ischemia-reperfusion of the brain and its correction by ecdysterone].

On the model of focal ischemia-reperfusion of the brain investigated the induction of nitrosative stress in mitochondria of rats hearts and possible mechanisms of protective action of ecdysterone. It is shown that focal ischemia-reperfusion of the brain induced in myocardial mitochondria the activation of constitutive and inducible de novo synthesis of NO by oxidation of L-arginine and not oxidative synthesis of NO through the recovery of oxidized stable metabolites of NO. Strong evidence of induction of nitrosative stress in heart mitochondria by focal ischemia-reperfusion of the brain, was a significant increase in mitochondrial pool of nitrate- and nitrite-anions and pools of nitrosothiols, that is proof of the formation and decay of peroxynitrite--a key marker of nitrosative stress. Also was observed increase in heart mitochondria by focal ischemia-reperfusion of the brain, content key regulator of de novo synthesis of NO-hydrogen sulfide and activity of inducible arginase II and, as a result, the pool of carbamide, which is also a regulator of the synthesis of NO. Previous introduction for animals herbal extract Serratsula coronata, enriched ecdysterone, reduces induction nitrosative stress in mitochondria of rats hearts under conditions of focal ischemia-reperfusion of the brain.