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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.

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.

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.

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.

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 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.

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.

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.

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.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.

Cholesterol oxidation products frequently have a high biological activity. In the present study, we have used microelectrode recording of end plate currents and FM-based optical detection of synaptic vesicle exo-endocytosis to investigate the effects of two structurally similar oxysterols, olesoxime (cholest-4-en-3-one, oxime) and 5ɑ-cholestan-3-one (5ɑCh3), on neurotransmission at the frog neuromuscular junction. Olesoxime is an exogenous, potentially neuroprotective, substance and 5ɑCh3 is an intermediate product in cholesterol metabolism, which is elevated in the case of cerebrotendinous xanthomatosis. We found that olesoxime slightly increased evoked neurotransmitter release in response to a single stimulus and significantly reduced synaptic depression during high frequency activity. The last effect was due to an increase in both the number of synaptic vesicles involved in exo-endocytosis and the rate of synaptic vesicle recycling. In contrast, 5ɑCh3 reduced evoked neurotransmitter release during the low- and high frequency synaptic activities. The depressant action of 5ɑCh3 was associated with a reduction in the number of synaptic vesicles participating in exo- and endocytosis during high frequency stimulation, without a change in rate of the synaptic vesicle recycling. Of note, olesoxime increased the staining of synaptic membranes with the B-subunit of cholera toxin and the formation of fluorescent ganglioside GM1 clusters, and decreased the fluorescence of 22-NBD-cholesterol, while 5ɑCh3 had the opposite effects, suggesting that the two oxysterols have different effects on lipid raft stability. Taken together, these data show that these two structurally similar oxysterols induce marked different changes in neuromuscular transmission which are related with the alteration in synaptic vesicle cycle.

We investigated the role of the vesicular acetylcholine transporter in the mechanism of non-quantal (non-vesicular) secretion of neurotransmitter in the neuromuscular synapse of the rat diaphragm muscle. Non-quantal secretion was estimated electrophysiologically by the amplitude of end-plate hyperpolarization after inhibition of cholinesterase and nicotinic receptors (H-effect) or measured by the optical detection of acetylcholine in the bathing solution. It was shown that 1 mM methyl-β-cyclodextrin (MCD) reduced both endocytosis and, to much lesser extent, exocytosis of synaptic vesicles (SV) thereby increasing non-quantal secretion of acetylcholine with a concurrent decrease in axoplasm pH. During high-frequency stimulation of the motor nerve, that substantially increases vesicles exocytosis, the non-quantal secretion was further enhanced if the endocytosis of SV was blocked by MCD. In contrast, non-quantal secretion of acetylcholine did not increase when the MCD-treated neuromuscular preparations were superfused with either vesamicol, an inhibitor of vesicular transporter of acetylcholine, or sodium propionate, which decreases intracellular pH. These results suggest that the proton-dependent, vesamicol-sensitive vesicular transporters of acetylcholine, which become inserted into the presynaptic membrane during SV exocytosis and removed during endocytotic recycling of SV, play the major role in the process of non-quantal secretion of neurotransmitter.

The effects of sodium hydrosulfide NaHS, a donor of hydrogen sulfide H2S, on the force of muscle contraction were examined on isolated myocardial strips from frog ventricles. NaHS decreased the amplitude of muscle contractions in a dose-dependent manner under normal conditions and during inhibition of Ca channels with nifedipine. In contrast, under conditions of blockade of ATP-dependent potassium channels with glibenclamide, NaHS exerted a positive inotropic effect from the first minute of application. Neither blockade, nor activation of ATP-dependent K-channels with glibenclamide modulated the negative inotropic effect of NaHS. Inhibition of K-channels with tetraethylammonium (TEA) (3, 5, 10 mM) or 4-aminopyridine increased the amplitude of myocardial contractions. Preliminary application of 4-aminopyridine or TEA (3 mM) did not eliminate NaHS-induced negative inotropic effect, although higher TEA concentrations (5 or 10 mM) prevented it. The data indicate that the targets of H(2)S in frog myocardium are ATP-dependent, Ca-activated, and voltage-dependent K-channels.

Cognitive dysfunction following surgery is a common complication, which increases the incidence of other co-morbid conditions, hospital and health-care costs. The reported rate of the occurrence of post-operative cognitive decline varies with different studies, depending on population profile, type of surgery, definition of cognitive disorder and detection methods, design of study, etc. It remains unclear whether these psychiatric signs and symptoms are direct results of the effects of surgery or general anesthesia. Nonetheless they are more frequent after cardiac surgery and are likely to be multi-factorial, but the patho-mechanisms are not yet fully characterized. This communication provides a synopsis of proteomics tools and delineates novel SELDI-TOF results to evaluate biomarkers in this regard. Presented for the first time is a classification of the clinically relevant forms of post-operative cognitive decline with the advent of a novel subclass.

We carried out a detailed analysis of rat model of esophageal achalasia previously developed by us. Manifest morphological and functional disorders were observed in experimental achalasia: hyperplasia of the squamous epithelium, reduced number of nerve fibers, excessive growth of fibrous connective tissue in the esophageal wall, high contractile activity of the lower esophageal sphincter, and reduced motility of the longitudinal muscle layer. Changes in rat esophagus observed in experimental achalasia largely correlate with those in esophageal achalasia in humans. Hence, our experimental model can be used for the development of new methods of disease treatment.

Calcium/calmodulin-dependent protein-kinase II (CaMKII) is a ubiquitous intracellular enzyme, which is implicated in learning and memory mechanisms in the central nervous system, however its contribution to peripheral cholinergic neurotransmission is not well characterized. This study evaluated the impact of CaMKII on the function of frog neuromuscular synapse using electrophysiological recordings. Application of the selective CaMKII inhibitor KN-93 (5 microM) did not significantly alter the parameters of evoked and spontaneous quantal acetylcholine release under low-frequency stimulation (0.03 Hz). KN-93, on the other hand, produced pronounced changes in short-term synaptic plasticity: particularly, KN-93 inhibits the second component of paired-pulse facilitation (interpulse intervals of 100 ms and longer) and strengthens the depression of synaptic transmission under high-frequency stimulation (50 Hz). These results imply that CaMKII plays an important role in presynaptic functions at the frog neuromuscular junction, and potentiates quantal acetylcholine release under high-frequency activity.

Experiments on frog neuromuscular synapses using extracellular microelectrode recording of endplate currents (EPC) and nerve ending (NE) responses were performed to study the mechanisms of facilitation of quantum secretion of acetylcholine on replacement of extracellular Ca ions with Sr ions. Solutions with a Ca ion concentration of 0.5 mM (calcium solutions) or a Sr ion concentration of 1 mM (strontium solutions) were used; the basal levels of neurotransmitter secretion (in conditions of low-frequency stimulation) were essentially identical. In calcium solutions, the drop in EPC facilitation on paired-pulse stimulation as the interimpulse interval was increased from 5 to 500 msec was described by the sum of three exponential components - the early, the first, and the second. In strontium solutions, facilitation was decreased as compared with the level in calcium solutions predominantly because of decreases in the early and first components. At the same time, EPC facilitation in conditions of rhythmic stimulation (10 or 50 impulses/sec) in strontium solution was significantly increased as compared with the level in calcium solutions. In strontium solutions in conditions of high-frequency stimulation at 50 impulses/sec, there was also a marked decrease in the amplitude of the third phase of the NE response, reflecting NE potassium currents. These data lead to the conclusion that the facilitation sites underlying the first and early components had lower affinities for Sr ions than for Ca ions. Increases in facilitation in strontium solutions in conditions of high-frequency rhythmic activity resulted from two mechanisms: more marked widening of the NE action potential and an increase in the divalent cation influx current due to weak activation of the Ca2+-dependent potassium current in the presence of Sr ions, as well as the slow dynamics of the removal of Sr ions from the NE axoplasm as compared with that in the presence of Ca ions.

Experiments on the mouse diaphragm muscle using intracellular microelectrode recordings and fluorescence microscopy were performed to study the dynamics of transmitter secretion and synaptic vesicle recycling processes (the exocytosis-endocytosis cycle) in motor nerve endings (NE) during prolonged rhythmic stimulation (20 impulses/sec). During stimulation, there were triphasic changes in the amplitude of endplate potentials (EPP): an initial rapid reduction, followed by prolonged (1-2 min) stabilization of amplitude, i.e., a plateau, and then a further slow decrease. Restoration of EPP amplitude after stimulation for 3 min occurred over a period of several seconds. Loading of synaptic vesicles with the fluorescent endocytic stain FM1-43 showed that rhythmic stimulation led to a gradual (over 5-6 min) decrease in NE fluorescence, demonstrating exocytosis of synaptic vesicles. Quantum analysis of the electrophysiological data and comparison of these data with results from fluorescence studies suggested that mouse NE have a high rate of endocytosis and reutilization of synaptic vesicles (the mean recycling time was about 50 sec), which may support the maintenance of reliable synaptic transmission during prolonged high-frequency activity. The sizes of the release-ready and recycling pools of synaptic vesicles were determined quantitatively. It is suggested that vesicle recycling in mouse NE occurs via a short, rapid pathway with incorporation into the recycling pool. Vesicles of the reserve pool are not used for transmitter secretion in the stimulation conditions used here.

Numerous findings obtained over the last decades suggest that accumulation of beta-amyloid peptide (betaAP) plays the central role in the pathogenesis of Alzheimer's disease. It is well established that betaAP has wide range of toxic effects on neurons in vitro and in vivo, however the influence of betaAP in the periphery and on various other types of excitable tissues, eg. skeletal muscle cells, is almost unknown despite the many non-cognitive and other extra-neuronal symptoms associated with Alzheimer's dementia. Here we utilized conventional electrophysiological technique to investigate the effects and mechanisms of betaAP action on the resting membrane potential of frog skeletal muscle fibers. betaAP in the range of concentrations from 10(-6) to 10(-8)M produced slow, significant, reversible depolarization of muscle fiber membranes. The impact developed and was washed out faster at higher concentrations of betaAP (10(-6)-0(-7)M). The effect of betaAP was completely absent when applied in Na+-free Tris+ solutions. betaAP-mediated depolarization was also prevented by tetrodotoxin (10(-5)M) pre-treatment and rescued by tetrodotoxin after-treatment. These findings suggest that betaAP-induced depolarization of skeletal muscle plasma membranes can significantly disturb the functioning of skeletal muscles and therefore contribute to motor dysfunction observed in Alzheimer's disease and other disorders associated with betaAP accumulation.

Carbon monoxide (CO) is an endogenous gaseous messenger, which regulates numerous physiological functions in a wide variety of tissues. Using extracellular microelectrode recording from frog neuro-muscular preparation the mechanisms of exogenous and endogenous CO action on evoked quantal acetyl-choline (Ach) release were studied. It was shown that CO application increases Ach-release in dose-dependent manner without changes in pre-synaptic Na+ and K+ currents. The effect of exogenous CO on Ach-release was decreased by prior application of guanylate cyclase inhibitor ODQ and prevented by application of a cyclic guanylate monophosphate (cGMP) analog 8Br-cGMP. Pre-treatment of the preparation with adenylate cyclase inhibitor MDL-12330A has completely abolished the effect of CO, whereas elevation of intracellular level of cyclic adenosine monophosphate (cAMP) mimicked and eliminated CO action. Application of cGMP-activated phosphodiesterase-2 inhibitor EHNA did not prevent CO action, whereas inhibition of cGMP-inhibited phosphodiesterase-3 by quazinone has partially blocked the effect of CO. Utilizing immuno-histochemical methods CO-producing enzyme heme-oxygenase-2 (HO-2) was shown to be expressed in skeletal muscle fibers, mostly in sub-sarcolemmal region, karyolemma and sarcoplasmic reticulum. Zn-protoporphirin-IX, the selective HO-2 blocker, has depressed Ach-release, suggesting the tonic activating effect of endogenous CO on pre-synaptic function. These results suggest that facilitatory effect of CO on Ach-release is mediated by elevation of intracellular cAMP level due to activation of adenylate cyclase and decrease of cAMP breakdown. As such, endogenous skeletal muscle-derived CO mediates tonic retrograde up-regulation of neuro-transmitter release at the frog neuro-muscular junction.

Hydrogen sulfide (H2S) is a widespread gasotransmitter also known as a powerful neuroprotective agent in the central nervous system. However, the action of H2S in peripheral synapses is much less studied. In the current project we studied the modulatory effects of the H2S donor sodium hydrosulfide (NaHS) on synaptic transmission in the mouse neuromuscular junction using microelectrode technique. Using focal recordings of presynaptic response and evoked transmitter release we have shown that NaHS (300 μM) increased evoked end-plate currents (EPCs) without changes of presynaptic waveforms which indicated the absence of NaHS effects on sodium and potassium currents of motor nerve endings. Using intracellular recordings it was shown that NaHS increased the frequency of miniature end-plate potentials (MEPPs) without changing their amplitudes indicating a pure presynaptic effect. Furthermore, NaHS increased the amplitude of end-plate potentials (EPPs) without influencing the resting membrane potential of muscle fibers. L-cysteine, a substrate of H2S synthesis induced, similar to NaHS, an increase of EPC amplitudes whereas inhibitors of H2S synthesis (β-cyano-L-alanine and aminooxyacetic acid) had the opposite effect. Inhibition of adenylate cyclase using MDL 12,330A hydrochloride (MDL 12,330A) or elevation of cAMP level with 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (pCPT-cAMP) completely prevented the facilitatory action of NaHS indicating involvement of the cAMP signaling cascade. The facilitatory effect of NaHS was significantly diminished when intracellular calcium (Ca(2+)) was buffered by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester (BAPTA-AM) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Activation of ryanodine receptors by caffeine or ryanodine increased acetylcholine release and prevented further action of NaHS on transmitter release, likely due to an occlusion effect. Inhibition of ryanodine receptors by ryanodine or dantrolene also reduced the action of NaHS on EPC amplitudes. Our results indicate that in mammalian neuromuscular synapses endogenously produced H2S increases spontaneously and evoked quantal transmitter release from motor nerve endings without changing the response of nerve endings. The presynaptic effect of H2S appears mediated by intracellular Ca(2+) and cAMP signaling and involves presynaptic ryanodine receptors.

Selective blockade of α2C-adrenoceptors had different effects on the cardiovascular system in rats of various age groups. Blockade of α2C-adrenoceptors in adult rats and 3-week-old animals produced the positive and negative chronotropic effects, respectively. HR in 1-week-old and 6-week-old rats did not change during α2C-adrenoceptor blockade. Selective blockade of α2C-adrenoceptors in adult rats and 3-week-old animals was followed by the increase in BP. BP in 6-week-old rats was shown to decrease under these conditions.

In the present study we combined FM 1-43 imaging and electrophysiological recording of miniature end-plate currents (MEPCs) to determine the role of extracellular calcium in synaptic vesicle exo- and endocytosis at the frog motor nerve terminals. We replaced extracellular Ca2+ ions with other bivalent cations (Sr2+, Ba2+, Cd2+, Mg2+) or used a calcium-free solution and monitored fluorescent staining of the nerve terminals in the presence of caffeine, which promotes the release of Ca2+ from intracellular stores. Caffeine has induced FM1-43 internalization only in the presence of bivalent cations in the external solution. The exposure of the neuromuscular junction to caffeine in a calcium-free solution caused a reversible failure of FM 1-43 loading and an increase in the nerve terminal width. This effect of a calcium-free solution was not due to a decrease in exocytosis, because caffeine-induced FM1-43 unloading from the previously loaded nerve terminals, as well as a degree of the MEPCs frequency increase, was unchanged. We conclude that the presence of Ca2+ or other bivalent cations in extracellular space is necessary for endocytosis but not for exocytosis of synaptic vesicles, while transmitter release is promoted by efflux of Ca2+ from intracellular stores. The effect of extracellular Ca2+ on endocytosis might be driven by the non-specific interactions with membrane lipids.

High-frequency synaptic activity can cause facilitation of transmitter release due to accumulation of "residual Ca(2+)" at the nerve terminal. However, the mechanism of this phenomenon is still under debate. Here we show that, using extracellular recording from frog cutaneous pectoris muscle, paired-pulse facilitation (PPF) at the frog neuro-muscular junction decays in two or three-exponential manner depending upon the extracellular Ca(2+) concentration ([Ca(2+)](e)). First, second and "early" PPF components are analyzed and described in this study. Considering the dependence of PPF on [Ca(2+)](e), existence of several specific high-affinity intra-terminal Ca(2+)-binding sites that underlie the facilitation of transmitter release at the frog neuro-muscular junction is proposed.

Assessment of calcium-dependent inactivation of calcium current in nerve terminals is not feasible due to technical reasons. Perineural measurement of calcium-flow, however, might be utilized as indirect means to evaluate synaptic currents. Using perineural recording from frog neuromuscular junction, supra-threshold stimuli applied to motor nerve in paired-pulse manner with varying inter-pulse intervals (5-50 ms) are demonstrated in this study to cause paired-pulse depression (PPD) of Ca(2+)-current. PPD of Ca(2+)-flow was reduced at lower extracellular Ca(2+) concentrations, in BAPTA-AM and EGTA-AM treated preparations and after replacing extracellular Ca(2+) with Sr(2+). Using perineural measurement of calcium current as an indirect model to investigate synaptic ionic activity, our findings demonstrate that PPD may be attributed to calcium-dependent inactivation of Ca(2+)-current, which may serve as negative feedback in response to massive Ca(2+) entry to motor nerve terminals. A putative sensor of Ca(2+)-current is also proposed in this study.

Experimental myocardial infarction (coronary ligation without drug infusion) reduced heart rate, stroke volume, cardiac output, and blood pressure. Under conditions of blocked NO synthesis, experimental myocardial infarction was accompanied by a transient increase in heart rate, stroke volume, cardiac output, and blood pressure, followed by their decrease throughout the experiment. Experimental myocardial infarction against the background of L-arginine treatment increased all these parameters with their subsequent stabilization at the attained level. Animal survival after administration of L-arginine was 80% vs. 33 in the control, and 22% after administration of L-NAME. It can be hypothesized that L-arginine possesses an intrinsic NO-independent cardioprotective effect.

We studied the role of ryanodine receptors in the effects of hydrogen sulfide on transmitter release from frog motor nerve ending. Sodium hydrosulfide (300 μM), a donor of hydrogen sulfide, reversibly increased the frequency of miniature endplate current without changes in its amplitude-time parameters. These effects were associated with reversible increase in endplate current amplitude, which was abolished by activation of ryanodine receptors of intracellular Ca(2+)stores with caffeine (3 mM) and ryanodine (0.5 μM). Under conditions of ryanodine receptors blockade with ryanodine (10 μM), sodium hydrosulfide had no effect on induced transmitter release, but its effects remained unchanged during ryanodine receptors blockade with dantrolene (25 μM). We concluded that an enhanced acetylcholine release induced by hydrogen sulfide is related to an increase of intracellular Ca(2+)concentration due to activation of ryanodine receptors for intracellular Ca(2+)-pool.

Rhythmic stimulation of nerve-muscle preparation of frog sternal muscle bathed in low-Ca(2+) saline increased the release of neurotransmitter (facilitation) and modified the shape of extracellular response of nerve terminal (decreased phase III amplitude). Iberiotoxin and 4-aminopyridine modified the dynamics these processes. We conclude that inactivation of potential-dependent K(+)-channels and activation of calcium-dependent K(+)-channels in frog motor nerve terminals during rhythmic activity modulate Ca(2+) influx into nerve terminals and contribute into facilitation of neurotransmitter secretion. The degree of these mechanisms depends on the rate of synaptic rhythmic activity.

Electrophysiological and optical methods were used to study exo- and endocytosis of synaptic vesicles underlying secretion of the neurotransmitter from motor nerve terminals in frog sternocutaneous muscle. Increase in extracellular concentration of K+ or sucrose produced similar increase in the frequency of miniature endplate currents recorded by extracellular microelectrode. Fluorescent microscopy revealed bright spots in nerve terminal during stimulation of secretion with high-potassium solutions in the presence of endocytosis marker FM1-43. These spots corresponded to clusters of synaptic vesicles that passed through the cycles of exo- and endocytosis. Subsequent high-potassium stimulation of exocytosis in normal Ringer solution led to disappearance of marker spots, while in hyperosmotic saline the spots were preserved. No spots were seen after stimulation of neurotransmitter secretion with sucrose in the presence of FM1-43. It is concluded that quantal secretion of the neurotransmitter in frog motor nerve endings can be realized via both complete exocytosis of synaptic vesicles with subsequent endocytosis and kiss-and-run mechanism with the formation of a temporary pore.

Using electrophysiological and optical techniques, we studied the mechanisms by which cholesterol depletion stimulates spontaneous transmitter release by exocytosis at the frog neuromuscular junction. We found that methyl-β-cyclodextrin (MCD, 10 mM)-mediated exhaustion of cholesterol resulted in the enhancement of reactive oxygen species (ROS) production, which was prevented by the antioxidant N-acetyl cysteine (NAC) and the NADPH oxidase inhibitor apocynin. An increase in ROS levels occurred both extra- and intracellularly, and it was associated with lipid peroxidation in synaptic regions. Cholesterol depletion provoked a rise in the intracellular Ca(2+) concentration, which was diminished by NAC and transient receptor potential vanilloid (TRPV) channel blockers (ruthenium red and capsazepine). By contrast, the MCD-induced rise in [Ca(2+)]i remained unaffected if Ca(2+) release from endoplasmic stores was blocked by TMB8 (8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride). The effects of cholesterol depletion on spontaneous release and exocytosis were significantly reduced by the antioxidant, intracellular Ca(2+) chelation with BAPTA-AM and blockers of TRPV channels. Bath application of the calcineurin antagonist cyclosporine A blocked MCD-induced enhancement of spontaneous release/exocytosis, whereas okadaic acid, an inhibitor of phosphatases PP1 and PP2A, had no effect. Thus, our findings indicate that enhancement of spontaneous exocytosis induced by cholesterol depletion may depend on ROS generation, leading to an influx of Ca(2+) via TRPV channels and, subsequently, activation of calcineurin.

We have investigated the effects of 5α-cholesten-3-one (5Ch3, 200 nM) on synaptic transmission in mouse diaphragm. 5Ch3 had no impact on the amplitude or frequency of miniature endplate currents (MEPCs, spontaneous secretion), but decreased the amplitude of EPCs (evoked secretion) triggered by single action potentials. Treatment with 5Ch3 increased the depression of EPC amplitude and slowed the unloading of the dye FM1-43 from synaptic vesicles (exocytosis rate) during high-frequency stimulation. The estimated recycling time of vesicles did not change, suggesting that the decline of synaptic efficiency was due to the reduction in the size of the population of vesicles involved in release. The effects of 5Ch3 on synaptic transmission may be related to changes in the phase properties of the membrane. We have found that 5Ch3 reduces the staining of synaptic regions with the B-subunit of cholera toxin (a marker of lipid rafts) and increases the fluorescence of 22-NBD-cholesterol, indicating a phase change within the membrane. Manipulations of membrane cholesterol (saturation or depletion) strongly reduced the influence of 5Ch3 on both FM1-43 dye unloading and staining with the B-subunit of cholera toxin. Thus, 5Ch3 reduces the number of vesicles which are actively recruited during synaptic transmission and alters membrane properties. These effects of 5Ch3 depend on membrane cholesterol.

Amyotrophic lateral sclerosis (ALS) is an incurable, chronic, fatal neuro-degenerative disease characterized by progressive loss of moto-neurons and paralysis of skeletal muscles. Reactivating dysfunctional areas is under earnest investigation utilizing various approaches. Here we present an innovative gene-cell construct aimed at reviving inert structure and function. Human umbilical cord blood cells (hUCBCs) transduced with adeno-viral vectors encoding human VEGF, GDNF and/or NCAM genes were transplanted into transgenic ALS mice models. Significant improvement in behavioral performance (open-field and grip-strength tests), as well as increased life-span was observed in rodents treated with NCAM-VEGF or NCAM-GDNF co-transfected cells. Active trans-gene expression was found in the spinal cord of ALS mice 10 weeks after delivering genetically modified hUCBCs, and cells were detectable even 5 months following transplantation. Our gene-cell therapy model yielded prominent symptomatic control and prolonged life-time in ALS. Incredible survivability of xeno-transpanted cells was also observed without any immune-suppression. These results suggest that engineered hUCBCs may offer effective gene-cell therapy in ALS.

Methyl-β-cyclodextrin (0.1 mM) reduced resting potential of muscle fibers and abolished local endplate membrane hyperpolarization in rat diaphragm. This effect was associated with selective reduction of electrogenic activity of α2-isoform of Na,K-ATPase without changes in the level of intracellular acetylcholine. Experiments with cholesterol marker filipin showed that methyl-β-cyclodextrin in this dose induced cholesterol translocation from lipid rafts to liquid phase of the membrane without its release into extracellular space. This modification of lipid rafts by methyl-β-cyclodextrin presumably impaired the mechanism maintaining electrogenesis in endplates mediated by modulation of Na,K-ATPase by non-quantum acetylcholine. Cholesterol can serve as a molecular component of this mechanism.

Some of the complexities of surgical interventions include neurological and psychiatric disturbances. Prompt identification and early treatment of these complications are pivotal in achieving excellent clinical results. Recognizing major adverse events such as stroke, seizure or delirium is usually straight-forward, however the discovery of less frequent or more subtle post-operative changes such as cognitive dysfunction might be delayed due to lack of appropriate diagnostic tools. This review summarizes biological markers that can be utilized as surrogates in evaluating surgery-related neuro-psychiatric disorders.

Replacement of Cl(-) for Br(-) in bathing solution did not reduce resting potential and had no effect on modulation of transmembrane potential in hyper- and hypoosmotic solutions. Under these conditions, baclofen, an agonist of GABAergic B-type receptors, failed to activate Na(+)/K(+)-pump in earthworm somatic muscle cells. It was hypothesized that the contribution of Cl(-) symport to osmotic homeostasis is not highly selective in respect to replacement of Cl(-) to Br(-) ions, whereas in case of activation of electrogenic ion pumps, this replacement is equivalent to removal of Cl(-) ions from the bathing solution.

Contractile activity of atrial and ventricular myocardial strips isolated from rats of various age was examined under conditions of blockade of non-selective hyperpolarization-activated cation currents. Addition of ZD7288, a blocker of non-selective hyperpolarization-activated cation currents, to the perfusion solution increased the contraction force of atrial and ventricular strips in 1-, 8-, and 20-week rats, but produced an opposite effect on contractile activity of atrial and ventricular strips in 3-week rats.

Carbacholine reduced, while baclofen and norepinephrine increased resting membrane potential in earthworm somatic muscle cells. In the presence of carbacholine, neither norepinephrine, nor baclofen hyperpolarized the membrane. Ouabain decreased resting potential and abolished the effects of carbacholine, norepinephrine, and baclofen on membrane potential. It was hypothesized that carbacholine directly inhibited the ouabain-sensitive component of Na+/K+ pump and abolished the activating effect of norepinephrine and baclofen.

Potential created by electrogenic ionic pumps under conditions of maximum activation in a warm standard ionic medium with K(+) after preincubation in cold potassium-free solution has two components: a higher ouabaine-insensitive "stationary" component, and a lower "regulatory" component sensitive to ouabaine, furosemide, and removal of Cl(-) or Ca(2+) from the medium. Functional heterogeneity of electrogenic ionic pumps is hypothesized implying the existence of two components: "stationary" (not regulated extracellularly) and "regulatory" (controlled and directly related to active Cl(-) transfer).

GABA, baclofen, epinephrine, and norepinephrine hyperpolarized the membrane of earthworm somatic cells. This effect was prevented by furosemide, removal of Cl- from the medium, or activation of Na+/K+ pump by 3-fold increase external potassium concentration. It was hypothesized that GABA, baclofen, epinephrine, and norepinephrine stimulate Na+/K+ transport via specific receptor inputs, but their effect on resting potential can be realized only under conditions of working Cl- symport.

We studied the effects of adenosine and ATP on contractile function of the isolated strip from mouse diaphragm. ATP significantly increased the strength of muscle contraction induced by carbachol. Adenosine had no effect on carbachol-induced muscle contraction. P2 receptor antagonist suramin abolished the effect of ATP. The positive chronotropic effect of ATP was not observed after treatment with specific protein kinase C inhibitor chelerythrine. Our results indicate that the effect of ATP on contractile function of mouse diaphragm is realized via protein kinase C.