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The concentration dependenies of the chronotropic response and changes in blood supply to the isolated heart of 7-day-old newborn rats induced by application of α2-adrenergic receptor agonist clonidine hydrochloride in concentrations of 10-9-10-6 M were revealed. The minimum concentration of α2-adrenergic receptor agonist caused tachycardia, while higher concentrations led to bradycardia. The maximum effect manifesting in a decrease in coronary flow was recorded at the minimum concentration of the agonist, while the highest concentration had no effect on the coronary flow. When comparing these results with those obtained in control adult rats, we found that the most pronounced differences in the chronotropic effects were observed after addition of the minimum concentration of the α2-adrenergic receptor agonist: bradycardia in adult rats and tachycardia in newborns. The maximum differences in coronary flow parameters were observed after addition of α2-adrenergic receptor agonist in the maximum concentration that induced a two-phase response in adult rats and had no effect on the blood supply in newborns.

Intracellular protons play a special role in the regulation of presynaptic processes, since the functioning of synaptic vesicles and endosomes depends on their acidification by the H+-pump. Furthermore, transient acidification of the intraterminal space occurs during synaptic activity. Using microelectrode recording of postsynaptic responses (an indicator of neurotransmitter release) and exo-endocytic marker FM1-43, we studied the effects of intracellular acidification with propionate on the presynaptic events underlying neurotransmitter release. Cytoplasmic acidification led to a marked decrease in neurotransmitter release during the first minute of a 20-Hz stimulation in the neuromuscular junctions of mouse diaphragm and frog cutaneous pectoris muscle. This was accompanied by a reduction in the FM1-43 loss during synaptic vesicle exocytosis in response to the stimulation. Estimation of the endocytic uptake of FM1-43 showed no disruption in synaptic vesicle endocytosis. Acidification completely prevented the action of the cell-membrane permeable compound 24-hydroxycholesterol, which can enhance synaptic vesicle mobilization. Thus, the obtained results suggest that an increase in [H+]in negatively regulates neurotransmission due to the suppression of synaptic vesicle delivery to the sites of exocytosis at high activity. This mechanism can be a part of the negative feedback loop in regulating neurotransmitter release.

Amyotrophic lateral sclerosis (ALS) is a progressive incurable neurodegenerative disease, which is characterized by selective death of the motoneurons of the spinal cord and brain. Currently, ALS is considered as a disease with a wide range of etiological causes, which trigger the cellular and molecular mechanisms leading to the development of similar clinical symptoms associated with degeneration of motoneurons. At the same time, there is no single hypothesis combining various pathogenetic mechanisms, which makes it difficult to develop an effective neuroprotective therapy. This review is devoted to the analysis of the early pathogenetic mechanisms of ALS and their possible role in the initiation of disease on the basis of the published data and our own research. The data on participation of various elements of the motoneuron–skeletal muscle system and membrane disorders in the initiation and development of ALS in humans and in animal models are presented. The analysis of scientific results of the last decades allows us to conclude that a key role in the initiation of pathogenetic mechanisms of ALS is played by dysfunction/destruction of neuromuscular synapses, distal axonopathy, and changes in skeletal muscle metabolism; the methods for correction of these disorders are promising for the development of therapeutic strategies in ALS.

The study examined the effects of α1A-adrenoceptor stimulation on chronotropic function of Langendorff-perfused isolated heart ex vivo and on cardiac chronotropy in vivo in 7-day-old rats. α1A-Adrenergic receptor agonist A-61603 reduced heart chronotropy only in the whole organism. No chronotropic effects of selective stimulation of α1A-adrenergic receptors on isolated hearts were observed in ex vivo experiments. These findings suggest that α1A-adrenergic receptors are not implicated in HR regulation in newborn rats. Bradycardia induced by activation of these receptors in vivo is most likely associated with reflex influences on the heart and changes in the vascular tone in the whole organism.

Homocysteine is a sulfur-containing amino acid, which at high concentrations has neurotoxic effects and induces impairments to the development of the nervous system. Homocysteine is rapidly oxidized in the plasma, forming disulfide bonds with proteins and other low molecular weight thiols; it also undergoes transformation into the into homocysteine thiolactone. On chronic exposure, the neurotoxicity of homocysteine is therefore mediated mainly by its derivatives. The aim of the present work was to investigate the effects of homocysteine and its derivatives – homocystine and homocysteine thiolactone – on spontaneous network activity in the hippocampus of rats in the first week after birth. Giant depolarizing potentials (GDP) and multiple action potentials (MAP) were recorded using an extracellular electrode in hippocampal field CA3. All three study compounds were found to induce increases in the frequency of GDP and MAP at concentrations of 100 and 500 μM, homocystine producing the most significant increase in neuron network activity. The effects of homocysteine, homocystine, and homocysteine thiolactone on the spontaneous network activity of neurons were completely eliminated on blockade of NMDA and AMPA receptors. Thus, homocysteine and its derivatives lead to increased spontaneous network activity of hippocampal neurons in neonatal rats, which can induce impairments to the formation of the neural networks of the hippocampus in conditions of chronic hyperhomocysteinemia, and could also induce hyperexcitability and the risk of developing epilepsy in the postnatal period.

Contemporary research has been enriched by the new directions in which the light plays a key role as a tool for modulation of cellular activity and invasive monitoring of intracellular ions and other components. The main advantages of these approaches are the possibilities to precisely control the intensity, spectral characteristics and durations of light signals in space and time. This review summarizes the key areas, optogenetics and photopharmacology, - directions that allow to control cellular activity with light. Optogenetics is the use of light-sensitive transmembrane proteins capable of exciting or inhibiting cellular activity under illumination by different wavelengths. In 2003 a light-sensitive protein canalo-rodopsine was isolated and cloned which is capable of inducing ion currents and changing cellular rest potential with its excitation under the blue light when embedded into the neurons or other cell types. Inhibition of cellular activity is caused by expression of other lightsensitive proteins - chloride or hydrogen pumps, or anion-selective ion channels. These principles turns out to be efficacious for the study of the functions of solitary cells and neural nets as well as for the control of living organisms behavior but their use in medicine is complicated because of necessary genetic manipulations. Photopharmacology is based on creating and using of chemical compounds changing conformations and/or activity under the light. Photochromic compounds with the use of photosensitive switches are capable of selective activation or inhibition of the activity of functionally important proteins - receptors, ion channels, enzymes, etc. The principles and the potential use of optogenetics and photopharmacology in the analysis of the neuronal functions and the perspectives for new approaches to treat some diseases of the nervous system are discussed.

The effects of neuropeptide Y (10—9-10—6 M) on electrical activity of right atrial cardiomyocytes of rats aging 7, 21, and 100 days were examined in vitro. Neuropeptide Y affected the amplitude-temporal parameters of the action potential in these cells. It decreased the duration of repolarization phase in 7-day-old rats in concentrations of 10—8 and 10—7 M, in 21-day-old rats at 10—8 and 10—6 M, and in 100-day-old at 10—6 M. The data indicate elevation of total membrane potassium current under the action of neuropeptide Y.

Amyotrophic lateral sclerosis (ALS) is a progressive incurable neurodegenerative disease with selective loss of lower and upper motoneurons. Dysfunction and destruction of neuromuscular synapses leading to skeletal muscle denervation is one of the early and major events in ALS pathogenesis. Despite of the presence of studies devoted to investigation of neuromuscular transmission in ALS mouse models, no detailed information about molecular mechanisms underlying synaptic dysfunction in ALS and their temporal dynamics during ALS progression is provided. The goal of present work was to study the processes of neurotransmitter release and presynaptic vesicle recycling in neuromuscular synapses of mutated SOD1 (mSOD1) transgenic mice at different clinical stages of disease. Utilizing combination of electrophysiological recording and FM 1–43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide) fluorescent imaging, we found that mSOD1 mice at symptomatic and terminal stages of disease showed decreased baseline quantal content of end-plate potentials and prolonged synaptic vesicle recycling time comparing to wild-type mice. Despite the decrease of end-plate potential (EPP) quantal content, studied mSOD1 mice groups showed unchanged dynamics of EPP relative amplitude comparing to WT mice. We also found an increase of miniature end-plate potential amplitude in mSOD1 mice at symptomatic stage, which may reflect compensatory mechanism that alleviates reduction of EPP amplitude. Thus, we provided one of the first detailed characteristics of presynaptic dysfunction at neuromuscular junction in ALS model. Obtained data expand our understanding of the ALS pathogenesis and contribute to stage- and localization-specific description of ALS pathogenetic mechanisms.

Septins are GTP-binding proteins recognized as a component of the cytoskeleton. Despite the fact that septins are highly expressed by neurons and can interact with the proteins that participate in synaptic vesicle exocytosis and endocytosis, the role of septins in synaptic transmission and the synaptic vesicle recycling mechanisms is poorly understood. In this study, neurotransmitter release and synaptic vesicle exocytosis and endocytosis were investigated by microelectrode intracellular recording of end-plate potentials and fluorescent confocal microscopy in mouse diaphragm motor nerve endings during septin polymerization induced by forchlorfenuron application. It was shown that forchlorfenuron application reduces neurotransmission during prolonged high-frequency (20 and 50 pulses/s) stimulation. Application of pairs of short high-frequency stimulation trains showed that forchlorfenuron slows the replenishment of the readily releasable pool. Forchlorfenuron enhanced FM 1-43 fluorescent dye loading by synaptic vesicle endocytosis but decreased dye unloading from the preliminarily stained nerve endings by synaptic vesicle exocytosis. It was concluded that the septin polymerization induced by forchlorfenuron application slows the rate of synaptic vesicle recycling in motor nerve endings due to the impairment of synaptic vesicle transport.

Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions, (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.

The molecular mechanisms that underlie neuromuscular junction plasticity are complex and remain to be fully elucidated. Experimental models of various forms of impaired motor activity may be promising for their study. The dysferlin protein plays a key role in the multimolecular complex, which maintains sarcolemma integrity and the functioning of skeletal muscle cells. We studied the structural and functional characteristics of the diaphragm muscle motor end plates of dysferlin-deficient Bla/J mice (a model of dysferlinopathy), dystrophin-deficient mdx mice (a model of Duchenne muscular dystrophy), and control C57Bl/6 mice. Increased end plate fragmentation and a decrease in the area of individual fragments were observed in mdx mice and absent in Bla/J mice, which indicates a difference in these models of myodystrophy from these characteristics. However, end plates of both mice lines were characterized by a decrease in the density of distribution of nicotinic acetylcholine receptors, as well as by membrane depolarization, presumably, due to altered functional interaction between the α2 isoform of Na,K-ATPase and the nicotinic acetylcholine receptors.

Hydrogen sulfide (H2S) is an endogenously synthesized gaseous transmitter that participates in the regulation of the cardiovascular system and has a cardioprotective effect under ischemia–reperfusion conditions. Here, we studied possible mechanisms of the interaction of H2S and muscarinic acetylcholine receptors in the regulation of mice atrium contractility in the isometric conditions. We show that sodium hydrosulfide (NaHS), an exogenous donor of H2S, caused dose-dependent and reversible depression of the contractile force in the concentration range from 1 μM to 5 mM. The negative inotropic effect of NaHS did not change after the activation of muscarinic acetylcholine receptors by carbachol. However, we observed that the negative inotropic effect of carbachol increased after preliminary application of NaHS. The application of the reducing agent dithiothreitol did not change the effects of carbachol, which indicated that the effects of NaHS was not related to a direct action on the disulfide bonds of the receptor’s protein subunits. The increased effects of carbachol after NaHS application were not prevented by the inhibition of intracellular signaling pathway that mediated activation of M-cholinergic receptors, including adenylate cyclase, guanylate cyclase, and NO-synthase. However, an increase in the carbachol negative inotropic effect was not observed when ATP-dependent potassium channels were inhibited by glibenclamide. In its turn, activation of ATPdependent potassium channels by diazoxide resulted in an increase in carbachol negative inotropic action in the atrial myocardium of mice similar to the effect of NaHS. Our data indicate that the enhanced negative inotropic effect of carbachol under the action of H2S in the mouse atrium was mediated by the activation of ATP-dependent potassium channels.

The effect of sodium hydrosulfide (NaHS), a hydrogen sulfide (H2S) donor, on spontaneous contractile activity of rat gastric smooth muscle cells was analyzed. Experiments were conducted on gastric stripes under conditions of isometric contraction. It was shown that NaHS has a biphasic effect on spontaneous contractile activity, increasing tonic tension and the amplitude of phasic contractions within the first minutes since application. This initial phase is followed by a decrease in amplitude, basal tone, and frequency of spontaneous contractions. The inhibitory effect of NaHS was dose-dependent at concentrations from 10 to 600 μM. Preliminary application of tetraethylammonium and 4-aminopirydine, inhibitors of voltage-gated and calciumactivated potassium channels, prevented a NaHS-induced initial increase in basal tone and phasic contraction amplitude. Activation of ATP-dependent potassium channels (KATP-channels) by diazoxide prevented in part a NaHS-induced decrease in basal tone and amplitude of spontaneous contractions. Glibenclamide, an inhibitor of KATP-channels, decreased the inhibitory effect of NaHS on amplitude, basal tone and frequency of spontaneous contractions. It was concluded that in rat gastric smooth muscles the excitatory effect of H2S is mediated by the inhibition of voltagegated and calcium-activated potassium channels, while its inhibitory effect involves the activation of KATP-channels.

We studied the effect of α2-adrenoreceptor agonist clonidine hydrochloride in concentrations of 10—9-10—6 M on inotropy, chronotropy, and coronary flow in Langendorff-isolated heart of adult rats. It was found that α2-adrenoreceptor agonist changed all studied parameters. Left ventricular myocardium contraction force decreased after application of all tested concentrations, the maximum effect was observed at a concentration of 10—6 M. Stimulation of α2-adrenergic receptors in concentrations of 10—8, 10—7, and 10—6 M produced a two-phase effect (initial increase and a subsequent decrease) on the coronary flow. Clonidine hydrochloride in the maximum concentration (10—6 M) caused a decrease in HR in one group and an increase in the other.

The study examined the effect of clonidine hydrochloride (10-9-10-5 М) on electrical activity of working cardiomyocytes in rat right atrium. Stimulation of α2-adrenergic receptor with clonidine changed electrical activity of the heart. All tested concentrations of the agonist lengthened the action potential and decreased the firing rate of cardiomyocytes in a dose-dependent manner. The maximum effects of clonidine were observed at concentration of 10-5 М.

Alzheimer’s disease is a progressive incurable neurodegenerative disease manifested by dementia and other cognitive disorders. Gene-cell therapy is one of the most promising trends in the development of treatment for Alzheimer’s disease. The study was aimed to evaluate the therapeutic potential of intravenous transplantation of human umbilical cord blood mononuclear cells (UCBMCs) transduced with adenoviral vectors overexpressing nerve growth factor (NGF) for the treatment of Alzheimer’s disease in an APP/PS1 transgenic mice model. The transplantation of NGF-expressing UCBMCs was found to improve spatial memory and decrease anxiety in APP/PS1 mice. Grafted cells and their expression of NGF were detected in the cortex and hippocampus of transgenic mice in the period up to 90 days after transplantation. Thus, gene-cell therapy based on the use of NGF-overexpressing UCBMCs is a promising approach for the development of Alzheimer’s disease treatments.

Diabetes mellitus (DM) is a systemic disease characterized by changes in many organs and tissues, including the motor system. The processes of exo- and endocytosis in the motor nerve ending of the mouse diaphragm muscle during high-frequency activity in experimental alloxan model of DM were studied. Endplate potentials (EPPs) were recorded using intracellular microelectrodes during single and high-frequency (50 Hz, 1 min) stimulation. In mice with the experimental DM, the amplitude-time parameters of EPPs did not differ from those of the control; however, an increase in EPPs depression and a slower recovery were observed during high-frequency stimulation. Using an endocytosis marker FM 1-43, it was shown that in animals with experimental DM fluorescence intensity of the nerve terminals loaded with the dye by high-frequency stimulation increased that was prevented by 1-azakenpaullone (2 μM), an inhibitor of slow dynamin-1-mediated endocytosis. In addition, in the model animals, the destaining of the pre-loaded nerve terminals during high-frequency (50 Hz) stimulation slowed down. The obtained data indicate that in the experimental first type DM recycling of synaptic vesicles via long path becomes more pronounced and the mechanisms of the vesicular transport are impaired, which was confirmed by methods of mathematical modeling.

In our previous review, we described brain cholesterol metabolism in control conditions and in the case of some rare neurological pathologies linked to defects in the genes which are directly involved in the synthesis and/or traffic of cholesterol. Here, we have analyzed disruptions in cholesterol homeostasis in widespread neurodegenerative diseases (Alzheimers and Parkinsons diseases) and autism spectrum disorders. We particularly focused on the synaptic dysfunctions that could arise from changes in both membrane cholesterol availability and oxysterol production. Notably, alterations in the brain cholesterol metabolism and neurotransmission occur in the early stages of these pathologies and the polymorphism of the genes associated with cholesterol homeostasis and synaptic communication affects the risk of onset and severity of these diseases. In addition, pharmacological and genetic manipulations of brain cholesterol homeostasis in animal models frequently have marked effects on the progression of neurodegenerative diseases. Thus, the development of Alzheimers, Parkinsons and autism spectrum disorders may be partially associated with an imbalance of cholesterol homeostasis that leads to changes in the membrane cholesterol and oxysterol levels that, in turn, modulates key steps in the synaptic transmission.

Background/objective Alzheimer's disease (AD) is a progressive incurable neurodegenerative disorder. Glial cell line-derived neurotrophic factor (GDNF) is a prominent regulator of brain tissue and has an impressive potential for use in AD therapy. While its metabolism is still not fully understood, delivering neuropeptides such as GDNF via umbilical cord blood mononuclear cells (UCBMCs) to the sites of neurodegeneration is a promising approach in the development of innovative therapeutic avenues. Methods UCBMCs were transduced with adenoviral vectors expressing GDNF and injected into AD transgenic mice. Various parameters including homing and survival of transplanted cells, expression of GDNF and synaptic proteins, as well as spatial memory were evaluated. Results UCBMCs were observed in the hippocampus and cortex several weeks after transplantation, and their long-term presence was associated with improved spatial memory. Post-synaptic density protein 95 (PSD-95) and synaptophysin levels in the hippocampus were also effectively restored following the procedure in AD mice. Conclusions Our data indicate that gene-cell therapy with GDNF-overexpressing UCBMCs may produce long-lasting neuroprotection and stimulation of synaptogenesis. Such adenoviral constructs could potentially possess a high therapeutic potential for the treatment of AD.

Dysferlin protein plays a key role in the multimolecular complex responsible for the maintenance of sarcolemma integrity and skeletal muscle cell functioning. We studied the membrane distribution of nicotinic acetylcholine receptors and α2 isoform of Na,K-ATPase in motor endplates of m. soleus in dysferlin-deficient Bla/J mice (a dysferlinopathy model). Endplates of Bla/J mice were characterized by increased area (without changes in fragmentation degree) and reduced density of the membrane distribution of nicotinic acetylcholine receptors in comparison with the corresponding parameters in control С57Bl/6 mice. The density of the membrane distribution of α2 isoform of Na,K-ATPase was also reduced, but the level of the corresponding mRNA remained unchanged. It can be hypothesized that abnormal membrane localization of α2 isoform of Na,K-ATPase results from adaptive skeletal muscle remodeling under conditions of chronic motor dysfunction.

Correction to the article “Peculiar Aspects in Influence of α1-Adrenoceptor Stimulation on Isolated Rat Heart,” by T. L. Zefirov, I. I. Khabibrakhmanov, N. I. Ziyatdinova, and A. L. Zefirov, Vol. 162, No. 1, pp. 4-6, November 2016

The effects of general anesthetics ketamine and midazolam, the drugs that cause neuroapoptosis at the early stages of CNS development, on electrical activity of the somatosensory cortex in newborn rats were studied using extracellular recording of local field potentials and action potentials of cortical neurons. Combined administration of ketamine (40 mg/kg) and midazolam (9 mg/kg) induced surgical coma and almost completely suppressed early oscillatory patterns and neuronal firing. These effects persisted over 3 h after injection of the anesthetics. We concluded that general anesthesia induced by combined administration of ketamine and midazolam profoundly suppressed cortical activity in newborn rats, which can trigger neuroapoptosis in the developing brain.

Cholesterol is an important constituent of cell membranes and plays a crucial role in the compartmentalization of the plasma membrane and signaling. Brain cholesterol accounts for a large proportion of the bodys total cholesterol, existing in two pools: the plasma membranes of neurons and glial cells and the myelin membranes . Cholesterol has been recently shown to be important for synaptic transmission, and a link between cholesterol metabolism defects and neurodegenerative disorders is now recognized. Many neurodegenerative diseases are characterized by impaired cholesterol turnover in the brain. However, at which stage the cholesterol biosynthetic pathway is perturbed and how this contributes to pathogenesis remains unknown. Cognitive deficits and neurodegeneration may be associated with impaired synaptic transduction. Defects in cholesterol biosynthesis can trigger dysfunction of synaptic transmission. In this review, an overview of cholesterol turnover under physiological and pathological conditions is presented (Huntingtons, Niemann-Pick type C diseases, Smith-Lemli-Opitz syndrome). We will discuss possible mechanisms by which cholesterol content in the plasma membrane influences synaptic processes. Changes in cholesterol metabolism in Alzheimers disease, Parkinsons disease, and autistic disorders are beyond the scope of this review and will be summarized in our next paper.

Location, location, location. The Na-K pump of skeletal muscle is regulated differently at neuromuscular junctions., The Na,K-ATPase is essential for the contractile function of skeletal muscle, which expresses the α1 and α2 subunit isoforms of Na,K-ATPase. The α2 isozyme is predominant in adult skeletal muscles and makes a greater contribution in working compared with noncontracting muscles. Hindlimb suspension (HS) is a widely used model of muscle disuse that leads to progressive atrophy of postural skeletal muscles. This study examines the consequences of acute (6–12 h) HS on the functioning of the Na,K-ATPase α1 and α2 isozymes in rat soleus (disused) and diaphragm (contracting) muscles. Acute disuse dynamically and isoform-specifically regulates the electrogenic activity, protein, and mRNA content of Na,K-ATPase α2 isozyme in rat soleus muscle. Earlier disuse-induced remodeling events also include phospholemman phosphorylation as well as its increased abundance and association with α2 Na,K-ATPase. The loss of α2 Na,K-ATPase activity results in reduced electrogenic pump transport and depolarized resting membrane potential. The decreased α2 Na,K-ATPase activity is caused by a decrease in enzyme activity rather than by altered protein and mRNA content, localization in the sarcolemma, or functional interaction with the nicotinic acetylcholine receptors. The loss of extrajunctional α2 Na,K-ATPase activity depends strongly on muscle use, and even the increased protein and mRNA content as well as enhanced α2 Na,K-ATPase abundance at this membrane region after 12 h of HS cannot counteract this sustained inhibition. In contrast, additional factors may regulate the subset of junctional α2 Na,K-ATPase pool that is able to recover during HS. Notably, acute, low-intensity muscle workload restores functioning of both α2 Na,K-ATPase pools. These results demonstrate that the α2 Na,K-ATPase in rat skeletal muscle is dynamically and acutely regulated by muscle use and provide the first evidence that the junctional and extrajunctional pools of the α2 Na,K-ATPase are regulated differently.

The study examined the dose-dependent effects of selective antagonists of α2A/D-, α2B-, and α2C- adrenoceptors applied in concentrations of 10—9-10—5 M on atrial and ventricular contractility of rat myocardium in vitro. Selective blockade of each α2-adrenoceptor subtype affected the contractile force of the atrial and ventricular strips. Various concentrations of α2A/D- and α2C-adrenoceptor antagonists produced positive inotropic effect on ventricular strips and negative effect on atrial strips. α2B-Adrenoceptor blocker in the majority of the tested concentrations produced a positive inotropic effect in both atria and ventricles.

The study examined the effect of α1-adrenoceptor stimulation with methoxamine on chronotropic function of isolated heart perfused ex vivo according to Langendorff and cardiac chronotropy in vivo. Stimulation of α1-adrenoceptors in isolated heart induced gradually developing bradycardia, which progressed during several minutes. Similar stimulation in vivo produced a short-term bradycardia probably terminated by the compensatory influences in the whole organism. Comparison of the data obtained in both experimental paradigms during α1-adrenoceptor stimulation revealed unidirectional changes in cardiac chronotropy characterized with time-related peculiarities.

The function of synaptic transmission and presynaptic vesicular cycle in the neuromuscular synapses of the diaphragm was studied in transgenic APP/PS1 mice (Alzheimer's disease model). The decrease in the quantal content of end-plate potential, intense depression of the amplitude of terminal plate potentials under conditions of lasting high frequency stimulation (50 Hz), a drastic prolongation of the synaptic vesicle recycling time in APP/PS1 mice in comparison with wild type mice were detected. Manifest dysfunction of the neuromuscular synapses, caused by disordered neurosecretion and recycling of the synaptic vesicles in the presynaptic nerve endings, was detected in the Alzheimer's disease model on transgenic APP/PS1 mice. The study supplemented the notions on the pathogenesis of Alzheimer's disease as a systemic disease, while the detected phenomena could just partially explain the development of motor disorders in this disease.

We studied the effect of neuropeptide Y in concentrations of 10-8-10-6 M on electrical activity of adult rat right atrial cardiomyocytes with preserved spontaneous activity. Neuropeptide Y was found to modulate the amplitude-time parameters of action potential: in concentrations of 10-7 and 10-6 M it reduced the membrane potential, increased the amplitude of action potential and duration of the repolarization phase, and reduced the frequency of action potential generation. In concentration of 10-6 M, neuropeptide Y produced stronger effect on the analyzed parameters, while in concentration of 10-8 M it produced no significant changes.

Transmitter release and synaptic vesicle exo- and endocytosis during high-frequency stimulation (20 pulses/s) in the extracellular presence of different bivalent cations (Ca2+, Sr2+ or Ba2+) were studied in frog cutaneous pectoris nerve-muscle preparations. It was shown in electrophysiological experiments that almost only synchronous transmitter release was registered in a Ca2+-containing solution; a high intensity of both synchronous and asynchronous transmitter release was registered in a Sr2+-containing solution, and asynchronous transmitter release almost only was observed in a Ba2+-containing solution. It was shown in experiments with a FM 1-43 fluorescent dye that the synaptic vesicles that undergo exocytosis-endocytosis during synchronous transmitter release (Ca-solutions) are able to participate in asynchronous exocytosis in Ba-solutions. The vesicles that had participated in the asynchronous transmitter release (Ba-solutions) could subsequently participate in a synchronous release (Ca-solutions). It was shown in experiments with isolated staining of recycling and reserve synaptic vesicle pools that both types of evoked transmitter release originate from the same synaptic vesicle pool.

Aim. Study is aimed to investigate contractility impairments and receptor mechanisms of adrenergic regulation of myocardium inotropic function in Alzheimer’s disease model on transgenic mice.Methods. Experiments were performed on isolated preparations of atria and ventricles myocardium of mice. Transgenic mice of B6C3-Tg(APP695)85Dbo Tg(PSENI)85Dbo genotype were used as animal model of Alzheimer’s disease. Contractile responses of myocardium were registered by conventional myographic technique in isometric conditions. To evaluate the expression of adrenergic receptors, immunofluorescence staining of myocardium with specific antibodies was performed.Results. Transgenic mice showed not only a decreased effect of norepinephrine on myocardium inotropic function but also the inversion of the effect of norepinephrine - the use of 10-5-10-4 M of norepinephrine decreased myocardium inotropic function. Immunofluorescent staining showed decrease of expression of β1- and especially β2-adrenergic receptors ventricular myocardium of transgenic mice comparing to wild type mice. Adrenergic deregulation was registered in ventricles, but not in atria. The features of adrenergic regulatory mechanisms of myocardial contractility in transgenic APP/PS1 mice aged 8-10 months are specific, although somewhat similar to wild type mice aged 8-10 months, and are evidently due to Alzheimer’s disease. The inversion of norepinephrine inotropic effect (from positive to negative) may be explained by switching the intracellular cascade pathway of β2-adrenergic receptors effects to another type of G-protein.Conclusion. The results indicate that peripheral adrenergic mechanisms of myocardial contractility regulation are impaired in studied transgenic mice model of Alzheimer’s disease. Obtained data widen our understanding of Alzheimer’s disease pathogenesis, as well as our conception of relations between cardiovascular diseases and neurodegeneration.

The effect of verapamil-induced blockade of L-type calcium ionic currents (ICa,L) on the action of non-selective adrenergic cardiac stimulation by norepinephrine was examined during different periods of early postnatal ontogeny. In 1-week-old rats, intravenous norepinephrine induced a short-term tachycardia both with and without preliminary injected verapamil. In 3-week-old rats, norepinephrine alone produced no chronotropic effect; in contrast, it induced a biphasic tachycardia in verapamil-treated rats. In 6- and 20-month-old rats, norepinephrine induced a short-term tachycardia, which could be prevented by verapamil. The age-related peculiarities of chronotropic action of non-selective adrenergic stimulation are indicative of the role of L-type calcium ionic channels in the development of sympathetic control over the heart.

Neurotransmitter release and exocytosis of synaptic vesicles in the motor nerve endings of the frog cutaneous-pectoris muscle were studied using electrophysiological and optical methods under the conditions of inhibition of the myosin light-chain kinase and non-muscle myosin by the specific inhibitors ML-7 (12 μM) and (-)-blebbistatin (100 μM). At high-frequency stimulation (20 pulses/s), these inhibitors strengthened suppression of transmitter release during the first 20-25 s and slowed down the release of the fluorescent dye FM 1-43. The obtained results indicate that myosin accelerates rapid synaptic vesicle recycling upon high-frequency stimulation.

We studied the effects of selective blockade of various subtypes of α2-adrenoceptors on cardiac chronotropy in newborn rats. This period in rats is characterized by the absence of adrenergic regulation of heart function. Blockade of α2A/D- and α2B-adrenoceptors in 1-weekold rats provoked tachycardia. In contrast, blockade of α2C-adrenoceptors in newborn rats had no effect on heart rate.

Marked loss of skeletal muscle mass occurs under various conditions of disuse, but the molecular and cellular mechanisms leading to atrophy are not completely understood. We investigate early molecular events that might play a role in skeletal muscle remodeling during mechanical unloading (disuse). The effects of acute (6–12 h) hindlimb suspension on the soleus muscles from adult rats were examined. The integrity of plasma membrane lipid rafts was tested utilizing cholera toxin B subunit or fluorescent sterols. In addition, resting intracellular Ca2+level was analyzed. Acute disuse disturbed the plasma membrane lipid-ordered phase throughout the sarcolemma and was more pronounced in junctional membrane regions. Ouabain (1 µM), which specifically inhibits the Na-K-ATPase α2 isozyme in rodent skeletal muscles, produced similar lipid raft changes in control muscles but was ineffective in suspended muscles, which showed an initial loss of α2 Na-K-ATPase activity. Lipid rafts were able to recover with cholesterol supplementation, suggesting that disturbance results from cholesterol loss. Repetitive nerve stimulation also restores lipid rafts, specifically in the junctional sarcolemma region. Disuse locally lowered the resting intracellular Ca2+concentration only near the neuromuscular junction of muscle fibers. Our results provide evidence to suggest that the ordering of lipid rafts strongly depends on motor nerve input and may involve interactions with the α2 Na-K-ATPase. Lipid raft disturbance, accompanied by intracellular Ca2+dysregulation, is among the earliest remodeling events induced by skeletal muscle disuse.

This study provides further insight into the molecular mechanisms that control neurotransmitter release. Experiments were performed on larval neuromuscular junctions of transgenicDrosophila melanogasterlines with different levels of human amyloid precursor protein (APP) production. To express human genes in motor neurons ofDrosophila, the UAS-GAL4 system was used. HumanAPPgene expression increased the number of synaptic boutons per neuromuscular junction. The total number of active zones, detected by Bruchpilot protein puncta distribution, remained unchanged; however, the average number of active zones per bouton decreased. These disturbances were accompanied by a decrease in frequency of miniature excitatory junction potentials without alteration in random nature of spontaneous quantal release. Similar structural and functional changes were observed with co-overexpression of humanAPPandβ-secretasegenes. InDrosophilaline with expression of human amyloid-β42 peptide itself, parameters analyzed did not differ from controls, suggesting the specificity of APP effects. These results confirm the involvement of APP in synaptogenesis and provide evidence to suggest that humanAPPoverexpression specifically disturbs the structural and functional organization of active zone and results in altered Bruchpilot distribution and lowered probability of spontaneous neurotransmitter release.

Experiments on neuromuscular preparations from the frog Rana ridibunda assessed the effects of oxidation of membrane cholesterol on the presynaptic vesicle cycle. Application of cholesterol oxidase (1 activity unit) for 30 min oxidized about 0.007 mg of cholesterol per g of preparation and decreased the stability of lipid rafts in nerve terminals. Electrophysiological studies demonstrated that oxidation of cholesterol decreased evoked transmitter release. Experiments using fluorescent FM dyes demonstrated suppression of synaptic vesicle exo- and endocytosis processes, along with dispersal of clusters of synaptic vesicles. Comparative analysis of electrophysiological and optical data, along with experiments using a dye quencher, demonstrated that transmitter could be released from some synaptic vesicles through transient fusion pores (the kiss-and-run mechanism). It is suggested that oxidation of cholesterol suppresses evoked exocytosis and delivery of synaptic vesicles of the reserve pool to the exocytosis site, thus interfering with their clustering. Vesicles of the recycling pool release transmitter by the “kiss-and-run” mechanism.

We studied the effect of sodium hydrosulfite (NaHS), a donor of hydrogen sulfide (H2S), on spontaneous contractive activity of isolated preparations of rat stomach and jejunum under isometric conditions. NaHS in concentrations of 10-200 μM reduced the amplitude, tonic tension, and frequency of contractions of the preparations. Blockade of K(+) channels with a non-specific antagonist tetraethylammonium (10 mM) increased contraction amplitude in the stomach strip and jejunum segment. The effects of NaHS on all parameters of contractile activity of the stomach and jejunum were fully preserved against the background of tetraethylammonium application. These data suggest that H2S in physiologically relevant concentrations inhibited spontaneous contractile activity of smooth muscle cells in rat stomach and jejunum by reducing the amplitude and frequency of contractions and decreased tonic tension without affecting the function of voltage- and calcium-dependent K(+) channels.

Alzheimer's disease (AD) is characterized by memory decline, but is often associated with non-cognitive symptoms, including muscular dysfunction. In the majority of cases these motor disturbances are seen when other neuro-degenerative disorders such as Parkinson's disease overlap dementia, however these can also be directly related to AD itself. Although the patho-mechanism remains largely unclear, β-amyloid peptide (βAP) is thought to be a key role-player in both the brain and periphery. Here we studied the electro-genesis of skeletal muscle fibers in a mouse transgenic AD model. Membrane potential was recorded by standard electro-physiological techniques. Compared to wild-type rodents, AD mice show severe disturbances in skeletal muscle electro-genesis manifested by significant depolarization of myo-fibers. These changes are not affected by short-term βAP treatment, the mark of a chronic degenerative process in the periphery directly related to AD whereby ion pumps on muscle membranes exhibit reduced activity. This phenomenon may explain ionic imbalance and cellular dysfunction both in the neuro-muscular system and in the brain. The observed motor disturbances might play a key role in impaired activities of daily living, and addressing the muscular patho-physiology could improve quality of life in AD.

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNF&alpha, receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.

The third author’s name should read: V. V. Matchkov.

The transmitter release and synaptic vesicle exo- and endocytosis induced by constant current depolarization of nerve terminals were studied by microelectode extracellular recording of miniature endplate currents and fluorescent microscopy (FM 1-43 styryl dye). Depolarization of the plasma membrane of nerve terminals in the control specimen was shown to significantly increase the MEPC frequency (quantal transmitter release) and exocytotic rate (FM 1-43 unloading from the synaptic vesicles preliminarily stained with the dye), which was caused by a rise in the intracellular Ca 2+ concentration due to opening of voltage-gated Ca channels. A slight increase in the MEPC frequency and in the rate of synaptic vesicle exocytosis was observed under depolarization in case of blockade of Ca channels and chelating of intracellular Ca 2+ ions (cooperative action of Cd 2+ and EGTA-AM). The processes of synaptic vesicle endocytosis (FM 1-43 loading) were proportional to the number of synaptic vesicles that had undergone exocytosis both in the control and in case of cooperative action of Cd 2+ and EGTA-AM. A hypothesis has been put forward that Ca-independent synaptic vesicle exo- and endocytosis that can be induced directly by depolarization of the membrane exists in the frog motor terminal in addition to the conventional Ca-dependent process.

Age-related development of behavioral disorders in transgenic mice with modeled Alzheimer's disease carrying V6S3-Tg(APP695)85Dbo Tg(PSENI)85Dbo) genotype was assessed at the age of 7.5, 10 and 20 months in the following tests: open-field, plus maze, T-maze, conditioned passive avoidance response, rotarod, conflict situation with water deprivation, behavioral despair, and arecoline tremor. The main behavioral disorder in transgenic mice at all observation terms was memory impairment in conditioning with positive (but not negative) reinforcement. At the age of 7.5 and 10 months, transgenic mice also showed signs of nonspecific excitement and anxiety, depression-like state, and symptoms of cholinergic deficit. Our results suggest that appropriate age for behavioral tests in studies of effects of potential anti-Alzheimer drugs in transgenic V6S3-Tg(APP695)85Dbo Tg(PSENI)85Dbo) mice is 7.5-10 months.

Selective blockade of various α2-adrenoceptors exerts various effects on the cardiovascular system in rats. Blockade of α2A/D-adrenoceptors in experimental animals decelerates and then accelerates HR. Blockade of α2B-adrenoceptors produces a negative chronotropic effect; blockade of α2C-adrenoceptors has a positive chronotropic effect. Administration of selective blockers of α2A/D- and α2B-adrenoceptors causes hypotension, while selective blockade of α2C-adrenoceptors increases BP.