Uridine Administration Promotes Normalization of Heart Mitochondrial Function in Dystrophin-Deficient Mice and Decreases Tissue Fibrosis.

The work shows the effect of the metabolic modulator uridine on the functioning and ultrastructure of heart mitochondria in dystrophin-deficient mdx mice. Intraperitoneal administration of uridine (30 mg/kg/day for 28 days) improved K+ transport and increased its content in the heart mitochondria of mdx mice to the level of wild-type animals. This was accompanied by a significant decrease in the level of malondialdehyde and an increase in the number of mitochondria in the heart of mdx mice. At the same time, uridine did not affect the hyperfunctionality of mitochondria in mdx mice, which manifested in an increase in the calcium retention capacity. Nevertheless, we noted that uridine causes a significant decrease in the level of fibrosis in the heart of mdx mice, which attested to a positive effect of therapy.

Autophagy in Neurons of the Prefrontal Cortex and Hippocampus of Rats after Trimethyltin Chloride Intoxication.

Ultrastructural studies of the hippocampus and the prefrontal cortex of rats were performed 7, 30, and 50 days after their damage by neurotoxicant trimethyltin chloride (TMT). Significant damage to neurons was observed in both brain structures. In the hippocampus, a large number of autophagosomes (0.9±0.1 per µm2) appeared in the soma of neurons, dendrites, and axons in 7 days after intoxication. In addition, we observed the appearance of hyperchromic neurons with abnormal structure of mitochondria. In the prefrontal cortex, damaged neurons also contained autophagosomes, but their number was significantly lower (0.3±0.1 per µm2). The number of autophagosomes decreased with increasing the time after TMT administration: 30 days after injection, the content of autophagosomes in the hippocampus was 0.10±0.01 per µm2, while in the prefrontal cortex, autophagosomes were no longer found. We hypothesized that autophagy in the hippocampus was not effective enough to prevent neuronal death caused by the neurotoxicant.

Effect of Alisporivir on Calcium Ion Transport and Mitophagy in Skeletal Muscle and Heart Mitochondria in Dystrophin-Deficient Mice.

We studied the effect of the mitochondrial calcium-dependent pore (MPT pore) inhibitor alisporivir (5 mg/kg per day for 4 weeks) on the parameters of calcium ion transport and the intensity of mitophagy in mitochondria of the heart and skeletal muscles of dystrophin-deficient C57BL/10ScSn-mdx mice. Alisporivir increased the rate of calcium uptake by skeletal muscle mitochondria of mdx mice, which was accompanied by changes in the level of the MCU and MCUb subunits of the calcium uniporter. At the same time, the intensity of calcium uniport in the heart mitochondria did not change. Alisporivir was found to reduce the expression of Pink1 and Parkin genes regulating the intensity of mitophagy in skeletal muscles of mdx mice, but did not affect the expression of these genes in the heart. This effect of alisporivir was accompanied by fragmentation and a decrease in the mean size of organelles. Possible mitochondrion-related mechanisms of the protective effect of alisporivir on the skeletal muscle and heart cells are discussed.

Kainate-Induced Degeneration of Hippocampal Neurons. Protective Effect of Activation of the Endocannabinoid System.

We studied the prolonged action of kainic acid on glutamatergic neurons in the dorsal hippocampus and the endocannabinoid-dependent protection against neurodegeneration. The pyramidal neurons of the CA3 field of the hippocampus, as well as granular and mossy cells of the dentate gyrus were examined. Light and electron microscopy revealed substantial damage to the components of the protein-synthesizing (rough endoplasmic reticulum, Golgi apparatus, and polyribosomes) and catabolic (lysosomes, autophagosomes, multivesicular structures, and lipofuscin formations) systems in all cells. Pyramidal and mossy neurons die mainly by the necrotic pathway. The death of granular cells occurred through both apoptosis and necrosis. The most vulnerable cells are mossy neurons located in the hilus. Activation of the endocannabinoid system induced by intracerebral injection of URB597, an inhibitor of degradation of endocannabinoid anandamide, protected the normal structure of the hippocampus and prevented neuronal damage and death induced by KA.

Does the Operation of Mitochondrial ATP-Dependent Potassium Channels Affect the Structural Component of Mitochondrial and Endothelial Dysfunctions in Experimental Parkinsonism?

We have previously demonstrated that the development of oxidative stress in some pathologies can be prevented by activation of the mitochondrial ATP-dependent potassium channel (mitoKATP). Here we studied the effect of modulation of mitoKATP on the development of mitochondrial and endothelial dysfunction in the medulla oblongata and myocardium of rats with experimental parkinsonism. It is known that uridine-5'-diphosphate, activator of mitoKATP, does not penetrate the plasma membrane, but it can be synthesized in cells from exogenous uridine that is delivered into cells by special transport systems. Our results suggest that mitoKATP is involved in the development of mitochondrial and endothelial dysfunction in experimental parkinsonism and prove the cardio- and neuroprotective effects of uridine.

Dopamine Improves Resistance of Dendrites of Mauthner Neurons to Destruction Induced by Sensory Stimulation and Application of Β-Amyloid.

Mauthner neurons in goldfish fry were studied by the methods of light and electron microscopy. The structure and volume of individual dendrites as well as the structure of axodendritic synapses were examined using virtual images of neurons formed from serial 3-µ sections. In short-time (5 h) experiments with application of dopamine, ß-amyloid fragment (25-35), and long-term sensory stimulation affecting afferent inputs to Mauthner neurons, the dendrites were larger than the same dendrites under the same conditions without dopamine application. Application of dopamine induced no pathological changes in the structure of axodendritic chemical and electric synapses containing desmosome-like contacts.

Ultrastructural Alterations in Granular Neurons of the Dentate Fascia Caused by Intrahippocampal Injection of Beta-Amyloid 1-42.

The deposition of beta-amyloid (Aß) in the brain is detected in Alzheimer's disease and during ageing. Until now, ultrastructural studies of changes caused by Aß in the dentate gyrus are very scarce. The effects of Aß 1-42 injection into the CA1 field of rat hippocampus were studied by electron microscopy. In 2 weeks after injection of aggregated Aß in low concentrations, destructive changes were seen in the structure of dentate gyrus cells, which consisted in a decrease in the number of dentate gyrus neurons and axo-dendritic synapses. These changes were accompanied by enlargement of the endoplasmic reticulum cisterns and widening of the active zones of synapses. Thus, injection of aggregated Aß 1-42 into the hippocampus led to irreversible (a decrease in the number of neurons and axo-dendritic synapses, agglutination of synthetic vesicles) and adaptive changes (an increase in the sizes of endoplasmic reticulum cisterns and active zones of synapses) in dentate gyrus neurons aimed at the maintenance of functional activity of the nervous system.

Effect of Interleukin-10 on Localization of AMPA Receptors in Synapses during Long-Term Posttetanic Potentiation in Cultured Hippocampal Slices.

The effect of the anti-inflammatory cytokine IL-10 on the ultrastructural distribution of AMPA receptor GluR1 subunit in CA1 field of cultured hippocampal slices was studied by using immunohistochemical technique. It was found that long-term posttetanic potentiation increased the content of GluR1 in the postsynaptic density of the axo-spinous synapse. Addition of IL-10 in concentrations of 1 and 10 ng/ml to the medium facilitated long-term posttetanic potentiation thereby changing the distribution of GluR1 in the spine: the number of receptors increased in the cytoplasm and decreased in the postsynaptic density. It is assumed that activation of neuronal IL-10 receptors affects the distribution of AMPA receptors in axo-spinous synapses of hippocampal field CA1 through interplay of intracellular signaling pathways, thereby participating in the mechanisms of synaptic plasticity under normal conditions.

Effect of TGF-beta1 on long-term synaptic plasticity and distribution of AMPA receptors in the CA1 field of the hippocampus.

Using the methods of electrophysiology and immunohistochemistry, the effect of the transforming factor beta-1(TGF-ß1), an anti-inflammatory cytokine, on the long-term post-tetanic potentiation (LTP) in CA1 field hippocampal slices and the distribution of the GluR1 subunit of the AMPA receptor has been studied. It was shown that TGF-ß1 at a concentration of 10 ng/ml did not significantly affect the initial stage of LTP and substantially changed the distribution of synaptic AMPA receptors in response to tetanic stimulation. Twenty five minutes after the tetanization, the main pool of AMPA receptors (90%) was due to the postsynaptic density (PSD). By contrast, LTP in the presence of TGF-ß1 was accompanied by less pronounced changes in the distribution of AMPA receptors. Their localization in both pre- and postsynaptic regions remained nearly the same as that in the control. It may be suggested that the normal distribution of AMPA receptors in spinous synapses promotes the stabilization of potentiated synapses, thereby retaining LTP for longer terms.

Expression of mGlu Receptor Genes in the Hippocampus After Intoxication with Trimethyltin.

A variety of localization and signaling properties of eight subtypes of metabotropic glutamate receptors (mGluRs) in the brain provide glutamate an important regulatory role in many processes, including neurodegeneration and repair of neuronal damage. To identify specific subtypes of mGluRs, which are involved in neurodegeneration process, we assessed expression levels of their genes under pathophysiological conditions. Using quantitative real-time RT-PCR analysis, we studied transcription levels of mGlu2-5 and mGlu7 genes in the hippocampus after its damage by neurotoxicant trimethyltin chloride (TMT) in Wistar rats. This organotin compound is known to cause neurodegeneration in the brain, especially in the hippocampus. Morphological studies confirmed neuronal damage in CA3-CA4 subfields of the hippocampus 6 weeks after the treatment with TMT. Step-through passive avoidance test revealed memory deterioration in rat-treated TMT. Interestingly, 3 and 6 weeks after the treatment with TMT, expression levels of the mGlu2 and mGlu7 genes were not changed in comparison to the control values while expression level of mGlu4 genes was upregulated throughout the whole studied period of TMT action. The dynamics of mGlu3 gene expression revealed the existence of neuroinflammation 3 weeks after the treatment with TMT, which was further confirmed by the upregulation of cyclooxygenase-2 gene expression. The expression level of mGlu5 receptors was downregulated 6 weeks after the treatment with TMT. Our results revealed a significant role of mGlu4, mGlu5, and mGlu3 receptors in the neurodegenerative/reparative processes in the hippocampus after the treatment with TMT. Ligands of these receptor subtypes can be, therefore, considered potential therapeutic targets for prevention or reduction of neurodegeneration.

Structure of Interneuronal Contacts in the Neuropil of the Oculomotor Nuclei in Mouse Brain under Conditions of Long-Term Microgravity.

Ultrastructure of the neuropil of the brain oculomotor nuclei was studied in mice after 30-day exposure to microgravity on Bion-M1 biosatellite and after 13-h exposure to Earth gravity. The number of axo-dendritic synapses in the neuropil of the oculomotor nucleus significantly decreased after the flight. Degenerated axon terminals containing conglomerates of presynaptic vesicles appeared. The number of synapses with high functional activity increased and the length of active zones of the axo-dendritic synapses significantly increased. The observed ultrastructural changes of the neuropil of the oculomotor nuclei of mice exposed to microgravity reflect the development of long-term deafferentation of the analyzed brain structures. These changes in the neuropil ultrastructure can determine the disturbances in the oculomotor system, e.g. development of atypical nystagmus under conditions of microgravity.

Specific Features of Immediate Ultrastructural Changes in Brain Cortex Mitochondria of Rats with Different Tolerance to Hypoxia under Various Modes of Hypoxic Exposures.

We performed ultrastructural study of cerebral cortex mitochondria in rats with different tolerance to oxygen deficiency (low resistant and highly resistant specimens). Low resistant rats were characterized by the prevalence of mitochondria with lightened matrix due to the nondense packing of cristae. By contrast, mitochondria of highly resistant animals had the dense packing of cristae. The structure of mitochondria underwent adaptive changes at 14-10% O2 in the inspired air. Under these conditions, structural characteristics of the cerebral cortex in hypoxia-sensitive rats resembled those in resistant animals. The decrease in O2 concentration to 8% was accompanied by ultrastructural signs of mitochondrial damage, which correlated with de-energization of the cell and dysfunction of adaptive signaling systems. Ultrastructural features of cerebral cortex mitochondria in animals with low and high tolerance to acute oxygen deficiency confirm the hypothesis that they are associated with two different "functionaland-metabolic portraits".

[Ultrastructure of afferent synapses on the ventral dendrite of mauthner neurons after goldfish adaptation to optokinetic stimulation].

Using the morphometric techniques, the ultrastructural changes of the afferent synapses on the ventral dendrite of the Mauthner neurons (MNs) were studied after the adaptation of goldfish to long-term fatiguing sensory (visual) stimulation, characterized by the growth of MN resistance. It was shown that after the adaptation, the length of active zones (AZs) in the synapses located on the MN ventral dendrite was significantly reduced by 23%. At the same time, the length the AZs of the excitatory visual synapses was reduced by 29% in comparison with the control, while the length of desmosome-like contacts (DLCs) bordering AZs was increased by 71%. It was also found that the length of AZs in the inhibitory synapses was decreased by 19% after the adaptation, which is consistent with the important role of inhibitory processes in the sensory pathways during the memory formation. Taking into account the actin nature of the DLCs, the basis of the adaptation to the visual stimulation is suggested to be in the presynaptic mechanism of neurotransmitter secretion regulation by actin.

[Serotoninergic synapses on the ventral dendrite of the mauthner neuron (ultrastructural study with immunogold labeling)].

Using immunogold labeling, excitatory serotoninergic synapses of both chemical and mixed types, were found on the ventral dendrite (VD) of goldfish Mauthner neuron (MN).They are characterized by the presence of several mitochondria in the bouton and by an obligatory desmosome-like contact (DLC) besides the active zone (AZ). Their AZs were commonly found to make contact with the unlabeled chemical crested synapses, which, in turn, directly interacted with VD. These synapses were practically devoid of mitochondria and had no DLCs, thus allowing to identify them as the inhibitory ones. This "two-level" organization of excitatory serotoninergic and inhibitory synapses appears to be related to the reciprocal mechanism of the regulation of MN functional activity by visual input.

[Ultrastructure of Mauthner neurons after optokinetic stimulation and eye enucleation].

It was previously shown that the contralateral (relative to preferred side of turns) optokinetic stimulation and ipsilateral eye enucleation cause a significant, 2- to 4-fold reduction of the ventral dendrite (VD) volume in one of two goldfish Mauthner neurons (MN) that becomes more active functionally. In this study, we investigated the MN ultrastructure after mentioned unilateral visual effects. In both cases, devastation of the afferent synapses was detected along the full length of the reduced VD, with simultaneous compaction of its cytoskeleton, in contrast to those of VD of the contralateral MN and of lateral dendrites and cell bodies of both neurons. It is suggested that the depleted synapses belong to the excitatory visual afferent input, and both cytoskeletal and synaptic mechanisms are involved in the regulation of MN functional activity through VD.

[Localization in a cell of a protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane. An ultrastructural study].

The localization in the cell of the protein forming the ATP-dependent potassium-selective channels in the bilayer lipid membrane has been studied. The electron microscope investigation of rat liver and heart tissue sections after their incubation with Abs against this protein and the visualization of the protein with secondary Abs conjugated with colloid gold were carried out. Colloid gold particles were observed both in mitochondrial membranes and in membranes of endoplasmic and sarcoplasmic reticulum. In heart mitochondria, these particles were significantly greater than in liver mitochondria. The localization of the channel protein both in mitochondria and reticulum, as well as the structural similarity between the mitochondrial channel and the precursor of calreticulin suggest that the channel protein belongs to the family of calreticulins. The possible function of the protein as a channel subunit of the mitochondrial ATP-dependent potassium channel is discussed.

[Cytoskeletal regulation of the cellular function by dopamine].

The interaction of dopamine with model membranes, isolated G-actin, and living cells, such as Mauthner neurons and fibroblast-like BHK-21 cells has been studied. It was found that in vitro dopamine passes through the phospholipid membrane and directly polymerizes G-actin due to incorporation into threads as their integral part. In in vivo conditions, it penetrates inside the cell and induces the appearance of a network of actin filaments in loci rich in globular actin. The data suggest that there exists a mechanism of dopamine interaction with living cells, which is based on direct polymerization of cytosolic G-actin as its cellular target. The reorganization of the actin cytoskeleton leads to changes in the morphofunctional status of cells.

The structure of mixed synapses in Mauthner neurons during exposure to substances altering gap junction conductivity.

The aim of the present work was to study the effects of dopamine, ecdysone, and chlorpromazine, substances which alter the conductivity of gap junctions (GJ), on the ultrastructure of mixed synapses in goldfish Mauthner neurons. These studies showed that dopamine, which increased the electrical conductivity of mixed synapses, appeared to target desmosome-like contacts (DLC). Hypertrophy of DLC, along with increases in the numbers of bridges within their clefts, showed that the mechanism by which dopamine increased electrical conductivity involved neuronal actin. This was indicated by the transformation of isolated monomeric muscle actin into polymerized actin in the presence of dopamine. Conversely, GJ were degraded by dopamine. Ecdysone, which also increased GJ conductivity, altered GJ structure, increasing the numbers of GJ at the attachment zone and decreasing the sectional length. but had virtually no effect on DLC structure. Ecdysone also showed no interaction with DLC in in vitro conditions. The mechanism of action of ecdysone is thus associated primarily with GJ function. Chlorpromazine, which decreased GJ conductivity, partially or completely degraded the fibrillar juxtamembrane material of DLC, preventing actin polymerization, with corresponding in vitro effects, but produced no changes in GJ. The mechanism of its action therefore appears to be based on changes in the state of neuronal actin.

Morphofunctional changes in incubated Mauthner neurons in goldfish treated with peptides from scorpion venom.

Electron microscopy with negative contrast showed that direct interaction of one of the peptide fractions of scorpion venom with monomeric chromatographically pure actin led to polymerization of actin, transforming it from the globular form to the fibrillar form. The effects of prolonged orthodromic stimulation on the evoked electrical activity and ultrastructure of Mauthner neurons (MN) were studied in incubated slices of goldfish medulla oblongata in the presence of this actin-polymerizing venom fraction. Peptides in this fraction were found to stabilize the amplitude of the electrical response of MN to exhaustion and to protect the ultrastructure of afferent chemical synapses and the neurons themselves from damage induced by stimulation. Enhancements in morphofunctional resistance were accompanied by stabilization of actin-containing specialized synaptic structures--desmosome-like contacts. The data obtained here provide evidence that peptides of this fraction of scorpion venom have direct actions on the actin component of the MN cytoskeleton and demonstrate potential for its use as a pharmacological tool able to penetrate living cells with value for studying the role of actin in the mechanisms of adaptation and memory.