The multifunctional promyelocytic leukemia protein (PML) is involved in the regulation of various cellular processes in both physiological and pathological conditions. Specifically, PML is one of the inositol-1,4,5-trisphosphate receptors (IP3Rs) activity regulators and can influence Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria. In this work, the effects of PML knockout on calcium homeostasis in the cytosol, ER, and mitochondria of HeLa cells were studied upon stimulation with histamine, which induces Ca2+ mobilization from the ER via IP3Rs. We utilized calcium indicators with different subcellular localizations, including synthetic dyes Fura-2 (cytosolic), Xrhod-5F (mitochondrial), and protein sensor R-CEPIAer (ER), as well as mitochondrial potential-sensitive probes Rh123 and TMRM. Our results show that PML knockout induced changes in HeLa cell and mitochondrial morphology, slightly decreased basal and integral Ca2+ levels, enhanced mitochondrial Ca2+ uptake from the cytoplasm, and maintained residual mitochondrial potential after depolarization. Additionally, it reduced the Ca2+ pool in ER membranes not associated with histamine receptor activation and, consequently, IP3Rs. These findings suggest that changes in calcium ion transport due to PML knockout in HeLa cells affect mitochondrial activity.

Pannexins are capable of forming in the cell membrane anion channels with relatively low conductivity, as well as channels with high nonspecific conductivity that can transport from the cytoplasm into the extracellular space not only inorganic ions but also low molecular weight metabolites with a molecular weight below 1 kDa, in particular ATP, glutamate, glutathione and others. Due to substantial differences in specificity and conductivity, pannexins are involved both in the normal physiological regulation of body functions and in a variety of pathological processes. The review examines the role of pannexin 1 (Panx1) in the occurrence and progression of pathologies of the central nervous system such as ischemia, Parkinson’s and Alzheimer’s diseases, and neuropathic pain. Blockade of Panx1 diminishes the negative consequences of pathological processes, indicating that Panx1 channels may be a promising therapeutic target for the pharmacological correction of brain disorders.

Hydrogen sulfide (H2S), which under physiological conditions exists in cells mainly in the form of the HS– anion, is considered as a gaseous transmitter of inter- and intracellular signals along with nitrogen monoxide and carbon monoxide. Analysis of the dynamics of H2S content in living cells is impossible without the creation of sensitive and specific probes. The group of K.H. Ahn synthesized several acedan-based compounds, which in the presence of H2S attached a sulfhydryl group, forming fluorescent carbocyclic compounds. According to the spectral characteristics and reaction rate with H2S, the optimal substance was P3, which forms the carbocyclic compound csP3 with the same large Stokes shift as P3 (approx. 130 nm) and has a brighter fluorescence. In this work, we tested the suitability of csP3 for recording changes in H2S in solutions simulating the minimum salt composition of the intracellular medium, as well as in cells of primary neuronal culture from the rat cerebral cortex. It was found that the fluorescence intensity of csP3, which was formed when Na2S (H2S donor, 100 and 300 µM) was added to the P3 solution, differed for solutions corresponding in salt composition to the extracellular medium and cytosol. In both cases, fluorescence increased in the presence of bicarbonate (NaHCO3, 10 mM). A decrease in the polarity of solutions due to the addition of dimethyl sulfoxide (30

Актуальность. При исследованиях внутриклеточного сигналинга и межнейрональной передачи сигнала в мозге в норме и при патологии все большее применение находят трансгенные животные, экспрессирующие в нейронах флуоресцентный Са2+-сенсор. Вместе с тем, пока недостаточно исследовано, насколько изменения кальциевого гомеостаза в интактном мозге соотносятся с гораздо более подробно изученными изменениями этого важнейшего параметра в нейроглиальных культурах, служащих модельными системами живого мозга. Целью работы было на модели ишемического инсульта выяснить в какой степени изменения внутриклеточной концентрации Са2+ ([Ca2+]) в мозге трансгенной мыши, измеренные с помощью флуоресцентного белкового сенсора GCaMP6f, соотносятся с изменениями [Ca2+] в первичных нейроглиальных культурах из кортекса этих животных. Методы. Методом широкопольной оптической нейровизуализации (ШОН) измерены изменения концентрации свободного Са2+ в цитозоле нейронов ([Ca2+]c) головного мозга мышей. Измерения проводили перед и после фотоиндуцированного инсульта в сенсомоторной зоне коры. Изменения [Ca2+]c отслеживали по флуоресценции GCaMP6f, экспрессируемого в нейронах кортекса. На первичных нейроглиальных культурах из коры головного мозга мышей той же линии проверено влияние эксайтотоксических доз глутамата (Glu) на [Ca2+]c и на изменения средней концентрации свободного Са2+ в цито- и нуклеоплазме ([Ca2+]i). Измерения [Ca2+]i выполнены методом флуоресцентной микроскопии с использованием синтетических Са2+- индикаторов Fura-2 и Fura-FF. В культивируемых нейронах дополнительно к измерениям кальциевого гомеостаза выполнены измерения внутриклеточного рН (pHi), митохондриального потенциала (ΔΨm) и эндогенной флуоресценции NADH. Результаты. Фотоиндуцированная ишемия вызывает сильный рост [Ca2+]c в зоне облучения ~1,1 мм2 (n=9). В течение сутокобласть высокой [Ca2+]c расширяется до ~6 мм2, но к 7-м сут практически возвращается к размерам необратимого повреждения. В нейроглиальных культурах из коры головного мозга мышей этой же линии кинетика изменений [Ca2+]c, индуцированных Glu, напоминает кинетику [Ca2+]i, однако [Ca2+]c имеет значительно меньшую амплитуду при развитии отсроченной кальциевой дисрегуляции (ОКД). Сопоставление изменений [Ca2+] и pHi показывает, что различия могут быть обусловлены тушением флуоресценции GCaMP6f при закислении цитозоля в результате эксайтотоксического действия Glu. Заключение. Сопоставление сигналов экспрессируемого нейронами Са2+-сенсора GCaMP6f в мозге и синтетических Са2+-индикаторов в первичных нейроглиальных культурах, полученных из животных той же линии, показывает, что феномен ОКД, впервые обнаруженный в культурах, вероятно, реализуется и в нейронах целого мозга при инсульте. Вместе с тем, необходимо учитывать, что относительные изменения [Ca2+]c на разных стадиях развития ишемического повреждения и последующего восстановления мозга после фотоиндуцированного инсульта, могут быть искажены за счет влияния pHi на флуоресценцию белкового сенсора. Relevance. In studies of brain intracellular and intercellular signaling in normal and pathological conditions, transgenic animals expressing a fluorescent Ca2+ sensor in neurons are increasingly used. Calcium homeostasis was studied in detail in primary neuroglial cultures, which serve as model systems of the living brain. But how change in calcium homeostasis during ischemic conditions in the intact brain correlate with experiments on cell cultures has been poorly studied so far. The aim of this work was to compare ischemia-driven changes in the intracellular concentration of Ca2+ ([Ca2+]) in vivo and in vitro: in the brain of transgenic mice, using the GCaMP6f fluorescent protein sensor, and in the neuroglial cell culture, obtained from the cortex of these animals. Methods. Changes in the cytosolic concentration of free Ca2+ ([Ca2+]c) in the neurons was measured by wide-field optical imaging (WFOI). Measurements were taken before and after photothrombotic stroke performed in the sensorimotor cortex. Changes in [Ca2+]c were monitored by the fluorescence of GCaMP6f expressed in cortical neurons. In primary neuroglial cultures from the cerebral cortex glutamate (Glu) in excitotoxic doses was used to model ischemic injury. Measurements of averaged Ca2+ concentration in the cyto- and nucleoplasm ([Ca2+]i) were carried out by fluorescence microscopy using synthetic Ca2+ indicators Fura-2 and Fura-FF. In addition, measurements of intracellular pH (pHi) and mitochondrial potential (ΔΨm) were carried out in vitro. Results. Photothrombotic stroke caused a strong increase in [Ca2+]c in the illuminated zone ~1.1 mm2 (n=9). During the 24 hours, the area with high [Ca2+]c expands to ~6 mm2, but by the 7th day it almost returns to the size of the primary damage. In neuroglial cultures from the cerebral cortex of the same mice strain, the [Ca2+]c kinetics measured by GCaMP6f resembles the [Ca2+]i kinetics, but [Ca2+]c has a significantly lower amplitude during the development of delayed calcium deregulation (DCD). Comparison of changes in [Ca2+]i and pHi shows that the differences may be due to the quenching of GCaMP6f fluorescence due to cytosol acidification as a result of the excitotoxic Glu action. Conclusion. Comparison of measurements by the genetically-encoded GCaMP6f Ca2+ sensor in vivo and by the synthetic Ca2+ indicators in primary neuroglial cultures shows that the DCD phenomenon, first discovered in cultures, is probably realized in intact brain neurons in stroke. It should be taken into account that the relative changes in [Ca2+]c can be distorted due to the effect of pHi on the fluorescence of the protein sensor at different stages of ischemia development and subsequent brain recovery after a photothrombotic stroke.

Boris Izrailevich Khudy-Khodorov was a biophysicist-electrophysiologist and neurophysiologist, professor, author of a number of monographs and dozens of articles in leading scientific journals, and the founder of a scientific school, who educated a Pleiad of neurophysiologists and neurobiologists who head laboratories or work in research, both in domestic and in foreign scientific institutes and universities. Boris Khodorov passed away in 2014. This year, students and former colleagues of Boris Khodorov celebrated his 100th birthday, and in honor of this event, a conference was organized at the Institute of General Pathology and Pathophysiology, where he worked until his final days. This work was written by disciples of Boris Khodorov. It briefly outlines his biography, and also provides an overview of the scientific areas in which Boris Khodorov was engaged throughout his productive scientific activity: from the study of reflexes, to the analysis of the mechanisms of action of anesthetics and functioning of channels, and ending with studies on the mechanisms of regulation of intracellular calcium levels during toxic glutamate exposure.

The effect of high concentrations of glutamate (Glu) on primary cultures of neurons from the rat brain led to a strong depolarization of mitochondria, which developed synchronously with a secondary increase in the intracellular free Ca 2+ concentration (delayed calcium deregulation, DCD). Simultaneously with measurements of the intracellular free Ca 2+ concentration ([Ca 2+ ] i ), pH was measured in the mitochondrial matrix (pH m ) and cytosol (pH c ) of neurons when exposed to a toxic dose of Glu (100 µM). For this purpose, pH-sensitive green fluorescent protein mtYFP in mitochondria and pH-sensitive red fluorescent protein mKate in cytosol were expressed in primary cultures from the hippocampus of newborn rats. The resulting neuronal culture was loaded with the Ca 2+ indicator Fura-FF; [Ca 2+ ] i , pH m and pH c were simultaneously measured in those neurons that expressed both mtYFP and mKate. It was found that during the first phase of the [Ca 2+ ] i response to Glu, when partial depolarization of mitochondria was observed, there was an increase in the pH gradient between the mitochondrial matrix and the cytosol (ΔpH), which compensated for the decrease in the electrical component of the mitochondrial potential (∆Ψ m ), thereby maintaining the constancy of the electrochemical potential of mitochondria. The development of DCD led to an abrupt decrease in ∆Ψ m and ΔpH in the soma of neurons; however, a complete collapse of ΔpH was not observed. This may mean that DCD was not caused by a nonspecific megapore in the inner mitochondrial membrane (mPTP), as is commonly believed. Alternatively, part of the mitochondria in the soma of neurons could retain the barrier properties of the inner membrane and did not form mPTP even with the development of DCD and reaching a high [Ca 2+ ] i plateau.

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.

Glutamate (Glu) excitotoxicity, which accompanies brain ischemia or traumatic brain injury, is the leading mechanism of neuronal death. In the present work, we studied the effects of the peptides HFRWPGP (ACTH6–9PGP), KKRRPG, and PyrRP on the survival of cultured cortical neurons on the background of excitotoxic effect of Glu (100 µM). Biochemical (MTT/WST) and morphometric analyzes showed that, depending on the dose, ACTH6–9PGP and KKRRPGP protect neurons from the cells death, while PyrRP, conversely, enhances it. The neuroprotective effect of ACTH6–9PGP is accompanied by a slowdown in the development of delayed calcium dysregulation and synchronous mitochondrial depolarization. Among the studied peptides, only ACTH6–9PGP significantly increased the number of neurons that restored Ca2+ homeostasis after Glu was abolished. The influence of KKRRPGP was less pronounced, whereas PyrRP, on the contrary, reduced the number of neurons with low [Ca2+]i. Thus, this study revealed the high therapeutic significance of ACTH6–9PGP and allowed assessing the prospects for its possible clinical use.

Over the last decade, a number of hydrogels attracted great attention in the area of brain tissue engineering. The hydrogels are composed of hydrophilic polymers forming 3D network in water. Their function is promoting structural and functional restoration of damaged brain tissues by providing mechanical support and navigating cell fate. This paper reports on the neurocompatibility of chitosan-g-oligo(L,L-lactide) copolymer hydrogel with primary rat cortical neuron culture. The hydrogel was produced by a molding technique on the base of photocurable composition consisting of chitosan-g-oligo(L,L-lactide) copolymer, poly(ethylene glycol) diacrylate and photosensitizer Irgacure 2959. The influence of the hydrogel on cell viability, phenotype and calcium homeostasis, mitochondrial potential and oxygen consumption rate in glutamate excitotoxicity was analyzed using primary neuron cultures obtained from a neonatal rat cortex. This study revealed that the hydrogel is non-cytotoxic. Dissociated neonatal rat cortical cells were actively attaching to the hydrogel surface and exhibited the phenotype, calcium homeostasis and mitochondrial function in both standard conditions and glutamate excitotoxicity (100 μM) similar to the control cells cultured without the hydrogel. To conclude, in this study we assessed the feasibility of the application of chitosan-g-oligo(L,L-lactide) copolymer hydrogel for tissue engineering therapy of brain injury in an in vitro model. The results support that the hydrogel is able to sustain realization of the functional metabolic activity of neonatal rat cortical cells in response to glutamate excitotoxicity.

Determination of the structural-functional significance of astrocytes in the physiology and pathology of the CNS is an actual problem of modern neuroscience and clinical neurology. Astrocytes are glial cells of the brain, constitute the substance of the brain, support neurons and separate them with their bodies into compartments. They participate in the immune response of the brain, they are able to maintain the chronic inflammation and progressive neurodegeneration due to overexpression of cytokines, growth factors, and chemokines. This review discusses the key features of astrogliosis as complex of molecular, cellular and functional changes of astrocytes in the response to various brain injuries. Reactive astrogliosis is critical for regeneration and remodeling of neural networks after the injury and ischemia and can have both positive and negative impact. The overexpression of S100b protein is an index of the astrocyte activation, which is characteristic for glial cells as this protein is located mainly in astrocytes. In cerebral ischemia, traumatic brain injury or neurodegenerative diseases there is the modulation of astrogliosis, aimed at the provision of repair mechanisms of the damaged parts of the brain that determines search capabilities of the new means of pharmacological correction of activated astrocytes and other glial components for the treatment of neurological diseases.

Актуальность. Моделирование in vitro травматического повреждения мозга помогает выяснить патологические механизмы, ответственные за гибель клеток или их последующую дисфункцию в деталях, труднодостижимых in vivo. Цель. Определить изменения внутриклеточной концентрации свободного Са2+ ([Ca2+]i) и митохондриального потенциала (m) в первичной нейроглиальной культуре непосредственно в момент нанесения механической травмы. Методы и материалы. Методом флуоресцентной микроскопии отслеживали изменения [Ca2+]i) и m в первичной нейроглиальной культуре из коры головного мозга 1-2-дневных крыс. Возраст культуры в момент измерений 11-14 дней. Результаты. Обнаружено, что нейротравма вызывает скачок [Ca2+]i и совпадающее с ним по времени резкое падение m. Эти изменения затрагивали клетки, расположенные не далее 100мкм от границы травмы. Блокирование ионотропных глутаматных рецепторов NMDA-типа с помощью МК-801 снижало в 8,5 раз долю нейронов, имевших высокий подъем [Ca2+]i. Выводы. Поступления Са2+ в клетки при механическом повреждении первичной нейроглиальной культуры происходит преимущественно по NMDA-каналам и отчасти, вероятно, по АТФ-активируемым каналам. Background. In vitro modeling of traumatic brain injury helps clarifying pathological mechanisms responsible for cell death or their subsequent dysfunction in detail, which is difficult to accomplish in vivo. Aim. To determine changes in intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial potential (m) in a primary neuroglial culture during infliction of a mechanical injury (scratch). Methods and materials. Changes in [Ca2+]i and m in the primary neuroglial culture from the cerebral cortex of 1-2 day old rats were monitored using a fluorescence microscopy technique. Measurements were performed in 11-14-day old cultures. Results. Neurotrauma resulted in a sharp increase in [Ca2+]i and a synchronous profound drop of m. These changes affected cells located not farther than 100 µm from the boundary of the injury. Inhibition of NMDA-type ionotropic glutamate receptors with MK-801 reduced by approximately 8.5 times the proportion of neurons, which indicated a high [Ca2+]i rise. Conclusion. Са2+ influx into cells during mechanical injury of the primary neuroglial culture occurs predominantly through NMDA-channels and perhaps partially through ATP-activated channels.

Exposure of cultured neurons to high concentrations of Glu leads to a strong depolarization of mitochondria, which develops synchronously with the secondary rise in the intracellular Ca2+ concentration (delayed calcium deregulation, DCD). In this study, using the primary culture of rat cerebellar neurons, we investigated the mechanism of neuronal sensitization, which manifests itself in the reduction of latent periods of DCD during repeated exposures to Glu. It was shown that the most likely cause of sensitization is the inability of mitochondria to maintain a high transmembrane potential (ΔΨm) as a result of an increase in the proton conductivity of the internal mitochondrial membrane, but not the opening of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Mitochondrial dysfunction reduces the production of ATP, leading to the inability of neurons to quickly restore the concentration of Na+, ATP, and NADH in the intervals between successive Glu administrations. One of the reasons that aggravate the dysfunction of mitochondria and contribute to the sensitization of neurons to the repeated action of Glu is Ca2+ accumulated in the mitochondria during the first glutamate impact.