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Excessive production and accumulation of β-amyloid peptide (βAP) underlies the pathogenesis of Alzheimer’s disease. Many studies have demonstrated that βAP has a wide spectrum of toxic effects on neuron functions, while the mechanism of action of βAP on other types of excitable cells, particularly skeletal muscle fibers, has received virtually no study. We report here electrophysiological experiments on mouse diaphragm muscle providing the first demonstration that βAP (fragment 25–35, 10−6 M) impairs the processes generating the resting membrane potential in muscle fibers, leading to marked depolarization of fibers via two mechanisms: 1) inhibition of Na+,K+-ATPase, leading to disappearance of the contribution of this pump to forming the resting membrane potential; 2) increases in the cation permeability of membranes due to the formation in them of “amyloid” channels blocked by Zn2+ ions. These data significantly extend our understanding of the mechanisms by which motor impairments and skeletal muscle pathology develop in Alzheimer’s disease, inclusion body myositis, and other diseases associated with βAP accumulation.

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.

Hydrogen sulfide (H2S) is an endogenously synthesized gaseous molecule which, along with nitric oxide and carbon monoxide, induces a number of effects in cardiovascular system under normal and pathological conditions. In the present work, the effects and underlying mechanisms of the H2S donor sodium hydrosulfide (NaHS) on the isometric force of frog myocardium contraction have been studied. NaHS at the concentration of 100 μM induced negative inotropic effect and reduced the maximum velocity of the contraction and relaxation of the isolated ventricle strips. The substrate of H2S synthesis, L-cysteine (200 μM and 1 mM), induced the same effect, while the inhibitors of cystathionin-γ-lyase, the H2S-producing enzyme in heart, β-cyanoalanine (500 μM) and propargylglycine (500 μM), increased the amplitude of contraction. Inhibition of cystathionin-γ-lyase by β-cyanoalanine prevented the negative inotropic effect of L-cysteine. After the inhibition of adenylate cyclase by MDL-12,330A (3 μM) or phosphodiesterases by IBMX (200 μM), the effect of NaHS was less than that in the control. In the presence of membrane-penetrating analogous of cAMP, 8Br-cAMP (100 μM) and pCPT-cAMP (100 μM), the negative inotropic effect of NaHS was completely retained. The effect of NaHS significantly decreased after preliminary application of the NO donor, SNAP (10 μM), and did not change after the inhibition of NO synthases by L-NAME (100 μM). The results suggest the possibility of endogenous synthesis of H2S in frog myocardium and regulation of its contractility by the activation of phosphodiesterases hydrolyzing cAMP, which leads to a decrease in the activation of cAMP-dependent protein kinases and phosphorylation of voltage-dependent L-type Ca channels. As a result, the reduction of calcium entry into cardiomyocytes decreases the contractility of frog myocardium.

Alzheimer's disease (AD) is a devastating and progressive form of dementia that is typically associated with a build-up of amyloid-β plaques and hyperphosphorylated and misfolded tau protein in the brain. Presently, there is no single test that confirms AD; therefore, a definitive diagnosis is only made after a comprehensive medical evaluation, which includes medical history, cognitive tests, and a neurological examination and/or brain imaging. Additionally, the protracted prodromal phase of the disease makes selection of control subjects for clinical trials challenging. In this study we have utilized a gene-expression array to screen blood and skin punch biopsy (fibroblasts, keratinocytes, and endothelial cells) for transcriptional differences that may lead to a greater understanding of AD as well as identify potential biomarkers. Our analysis identified 129 differentially expressed genes from blood of dementia cases when compared to healthy individuals, and four differentially expressed punch biopsy genes between AD subjects and controls. Additionally, we identified a set of genes in both tissue compartments that showed transcriptional variation in AD but were largely stable in controls. The translational products of these variable genes are involved in the maintenance of the Golgi structure, regulation of lipid metabolism, DNA repair, and chromatin remodeling. Our analysis potentially identifies specific genes in both tissue compartments that may ultimately lead to useful biomarkers and may provide new insight into the pathophysiology of AD.

Alzheimer's dementia (AD) is a degenerative brain disorder characterized mainly by cholinergic failure, but other neuro-transmitters are also deficient especially at late stages of the disease. Misfolded β-amyloid peptide has been identified as a causative agent, however inflammatory changes also play a pivotal role. Even though the most prominent pathology is seen in the cognitive functions, specific abnormalities of the central nervous system (CNS) are also reflected in the periphery, particularly in the immune responses of the body. The aim of this study was to characterize the dopaminergic and serotonergic systems in AD, which are also markedly disrupted along with the hallmark acetyl-choline dysfunction. Peripheral blood mono-nuclear cells (PBMCs) from demented patients were judged against comparison groups including individuals with late-onset depression (LOD), as well as non-demented and non-depressed subjects. Cellular sub-populations were evaluated by mono-clonal antibodies against various cell surface receptors: CD4/CD8 (T-lymphocytes), CD19 (B-lymphocytes), CD14 (monocytes), and CD56 (natural-killer (NK)-cells). The expressions of dopamine D(3) and D(4), as well as serotonin 5-HT(1A), 5-HT(2A), 5-HT(2B) and 5-HT(2C) were also assessed. There were no significant differences among the study groups with respect to the frequency of the cellular sub-types, however a unique profound increase in 5-HT(2C) receptor exclusively in NK-cells was observed in AD. The disease-specific expression of 5-HT(2C), as well as the NK-cell cyto-toxicity, has been linked with cognitive derangement in dementia. These changes not only corroborate the existence of bi-directional communication between the immune system and the CNS, but also elucidate the role of inflammatory activity in AD pathology, and may serve as potential biomarkers for less invasive and early diagnostic purposes as well.

Under the condition of microelectrode recording and fluorescence microscopy with dye FM 1-43 the research of exo-/endocytosis of synaptic vesicles in motor nerve terminals (NT) of frog cutaneous pectoris and white mice diaphragm muscles during high frequency stimulation (20 imp/s) was carried out. A mathematical modeling allowed us to conclude that the obtained experimental data can be explained in the following framework. Three pools of synaptic vesicles are involved in neurotransmitter release in the frog motor NT. Recovery of these pools is provided by endocytosis of two types: fast endocytosis with limited capacity and slow endocytosis. Fast-reconstructing vesicles refill the mobilization pool and slow endocytosis recovers the reserve pool. Our modeling investigation has revealed in frog NT independent recruiting of reserve and mobilization pools to the neurotransmitter secretion, i.e. this pools work concurrently. Experimental data, obtained on mice preparations, are well described with the framework of two-pools model including single type of endocytosis (fast endocytosis).

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.

The transmission of information between neurons inthe brain and between neurons and other excitatory cells(muscle and secretory cells) at the periphery occurs at spe-cialized locations, i.e., synapses. The presynaptic nerveending (NE) in the vast majority of synapses contains largenumbers of synaptic vesicles filled with a chemical trans-mitter (mediator, neurotransmitter). In response to a nerveimpulse, vesicles fuse with the presynaptic membrane (exo-cytosis), releasing a portion (quantum) of transmitter intothe synaptic cleft. Reaching receptors on the postsynapticmembrane, the transmitter activates them, with the resultthat an electrical signal arises in the postsynaptic cell [1, 3].Along with the processes of exocytosis, the intense forma-tion of new vesicles (endocytosis) occur in the NE, whichprevents depletion of vesicles in conditions of high-fre-quency activation of the synapse. After endocytosis, thevesicles are again filled with neurotransmitter, transported,and placed in line for repeated exocytosis. In other words,nerve endings involve a process of circulation (recycling) ofsynaptic vesicles, which is termed the vesicular cycle [1, 2,9, 13]. Naturally, the processes of the vesicular cycle deter-mine the intensity of transmitter secretion and, ultimately,the strength of the synaptic contact.Recent years have seen major scientific advances instudies of the roles of different proteins in protein-proteininteractions in the mechanisms of exoendocytosis and vesi-cle transport (Fig. 1). Only now have investigators started topay detailed attention to the significance of lipids for thefunctioning of nerve endings. This is not surprising, as rad-ical transformations take place in membrane geometry dur-ing the vesicular cycle, along with biochemical and struc-tural “metamorphoses” in their lipids, and changes in thepattern of the activity of many lipid-modifying enzymes,which “prepare” the vesicle membrane and nerve endingsfor exo- and endocytosis. Activation of the key proteins ofexo- and endocytosis and vesicular transport is significant-ly accelerated by particular lipids and their derivatives. It isno less important that the sites of exo- and endocytosis haveunique lipid compositions (and unique protein composi-tions), despite multiple cycles of vesicle fusion and recov

Using electrophysiological and optical methods, we studied the effects of sodium hydrosulfide (NaHS), a hydrogen sulfide donor, on the dynamics of transmitter release and exo- and endocytosis of synaptic vesicles in motor nerve endings during long-term high-frequency stimulation (20 Hz) in experiments with the cutaneous pectoris frog muscle. H2S increased the amplitude of endplate currents under conditions of a single stimulation of the motor nerve and slowed down the depression of the end plate currents during high-frequency stimulation (20 Hz, 3 min). Using the endocytic fluorescent dye FM 1–43, we showed that NaHS increased the dye uptake during high-frequency stimulation as compared to the control. However, after termination of the high-frequency stimulation the fluorescence intensity of nerve terminals was lower in the presence of NaHS than in the control experiments. In addition, NaHS slowed the dye release from the nerve terminals that were loaded during 20 Hz stimulation. The results thus obtained indicate that H2S accelerates the synaptic vesicles cycle in frog motor nerve ending by enhancement of exocytosis and fast endocytosis of synaptic vesicles during high-frequency stimulation.

In the muscle wall of the earthworm Lumbricus terrestris, with the aid of fluorescent endocytotic dyes FM1-43, FM2-10, and FM4-64, there are revealed fluorescent spots 1–2 μm in diameter that represent clusters of “synaptic boutons.” Application takes place onto ganglia of the abdominal nerve chain of the Dil membrane probe capable of translocation by axoplasmic transport; the subsequent (next day) staining of nerve structures with the endocytotic marker FM4-64 showed the complete superposition of fluorescence of these dyes fluorescing in different specter areas. The fluorescent marker DiBAC4(3) revealed an enhancement of fluorescence of nerve elements with increase of K+ concentration in the extracellular medium. Use of FM2-10 showed that, the higher the K+ content in solution and, accordingly, the nerve cell depolarization, the faster the release of the marker and, on the contrary, the slower the process in the absence of K+ in the medium. In the Ca2+-free solution and in the presence of the Ca2+ chelator BAPTA or BAPTA-AM, there uptake and release of FM2-10 are blocked, but only after preliminary 40-min incubation in such solution. In clusters of synaptic boutons, exo- and endocytosis processes take place that are also preserved under conditions of rest. This vesicular cycle depends on the membrane potential of nerve structures and on the content of K+ and Ca2+ in the medium, the calcium sensor working most likely by the “all or nothing” principle.

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 the selective β(2)-adrenoreceptor agonist (fenoterol) on the functioning of mouse atrial were studied using both tensometry and fluorescent methods. It has been demonstrated that with the use of a high concentration of fenoterol (in the range of 1-50 µM), there is a more significant positive inotropic effect observed within a shorter period of time. In the case of relatively low doses of fenoterol (1 and 5 µM), its contractility effects are observed 20 min after the application of agonist, whereby in the case of high concentrations (25, 50 and 300 µM), the effects appear within the first minutes. During the first 10-15 min, 5 µM fenoterol causes an increase in the amplitude of Ca-signals in cardiomyocytes (this indicates an increase in the concentration of Ca ions during systole) and the activation of NO synthesis. However, after 20 min, the production of NO decreases; while the amplitude of Ca-signals remains high. The application of 50 µM fenoterol leads to a rapid increase in the amplitude of Ca-signals: at the same time, it causes a decrease in the production of NO, which we found to begin to increase after 10 min of agonist application. It is suggested that the dynamics of the positive inotropic effect occurring under pharmacological stimulation of β(2)-adrenoreceptors depend on the rate of increase in the amplitude of Ca-signals and on the degree of NO synthesis.

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.

Introduction. Alzheimer's β-amyloid peptide (βAP) is known to possess a wide range of toxic effects on neurons in vitro and in vivo; however, there is little information available regarding its impact on other excitable tissues such as skeletal muscles, which, apart from brain cells, are thought to also be targets of βAP. Methods. Utilizing the combination of electrophysiology and myography, we investigated whether βAP also impairs the functioning of myocytes in frogs and mice. Results. Although application of βAP in the range of 10−6 to 10−8 M induced depolarization of muscle fibers in both species, it impaired contractility in frogs but not in mice, by reducing endplate potential amplitude and increasing the threshold potential. Conclusions. Unchanged contractility in the mouse in the presence of βAP is due to a higher safety factor of neuromuscular transmission in mammals compared with amphibians. Possible clinical implications are discussed. Muscle Nerve, 2011.

In this study, using neuromusclar preparations of frog cutaneous pectoris muscle and mouse diaphragm we have shown that the membranes of nerve terminals (NT) contain nearly two times more cholesterol than the plasma membrane of muscle fibers. Using the fluorescent B subunit of cholera toxin (CT-B), we identified the areas in the NT membranes with high concentrations of GM1 ganglioside, the marker molecule for cholesterol- and sphingolipid-enriched membrane microdomains, so-called lipid rafts. Intense fluorescent spots appear in the NT when lipid rafts were identified during high-frequency stimulation, which cause massive exocytosis of synaptic vesicles followed by endocytosis. The double staining of neuromusclar preparations with CT-B and FM1-43, a dye that is taken up into synaptic vesicles during endocytosis, has demonstrated good colabeling by these dyes. This indicates the presence of large amounts of cholesterol and lipid rafts in membranes of synaptic vesicles. Thus, cholesterol is present in NT membranes at the high concentration required for the organization of lipid rafts, which are common in the plasma membranes of the NT and the membranes of synaptic vesicles. The role of cholesterol and lipid rafts in the processes of exo- and endocytosis is discussed.

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.

The role of cyclic nucleotides in the effects of hydrogen sulfide (H2S) on transmitter release in the frog neuromuscular junction was studied using focal microelectrode recording of synaptic signals. A donor of H2S, sodium hydrogen sulfide (NaHS) (100 µM) reversibly increased the amplitude and quantum content of end-plate currents (EPCs) without changing the nerve ending response. The effect of H2S on evoked transmitter release was decreased by preliminary application of the membrane-permeable cAMP analogue pCPT-cAMP (100 µM). Inhibition of adenylate cyclase by MDL-12330A (0.8 µM) did not alter the effect of NaHS on transmitter release. Membrane-permeable cGMP analogue pCPT-cGMP (50 µM) and inhibitor of guanylate cyclase ODQ (0.1 µM) did not change the effect of NaHS. Therefore, we conclude that the H2S-induced increase in the transmitter release was not a result of guanylate cyclase activity. It was concluded that the H2S effects are related to the functioning of cAMP-dependent mechanisms; however, these effects are not associated with the direct action of H2S on the adenylate cyclase 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.

Skeletal muscles play key roles in the development of various pathologies, including bronchial asthma and several types of auto-immune disorders, e.g. polymyositis. Since most of these maladies have an immunological/allergic element, this paper is devoted to assessing the impact of immunobiological reorganization on the functional properties of isolated skeletal muscles in mice. A combination of two methods (myography and electrophysiology) was used to evaluate extensor digitorum longus (EDL) and diaphragmatic muscle (DM) in this regard. Conventional myographic technique showed that ovalbumin-induced sensitization (OS) produced different changes in the contractile properties of EDL and DM. The amplitudes of carbachol (CCh)-induced contractions increased in DM but decreased in EDL. Those changes were inversely related to OS-mediated changes of non-quantal acetylcholine (ACh) release intensity within the muscle endplate, as shown by the electrophysiologically measured H-effect. These results clearly show that OS-mediated changes of non-quantal ACh release alter the functional properties of postjunctional ACh receptors and therefore contribute to the disturbance of CCh-induced contractility of skeletal muscles. Other mechanisms of OS-mediated changes of skeletal muscle contractility are also proposed and discussed.

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.

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.

An extracellular microelectrode applied to the neuromuscular junction of the frog sternocutaneus muscle was used to study effects of saturated (myristic and arachidic) and unsaturated (arachidonic and oleic) fatty acids on transmitter release and potassium currents in a nerve ending. All these fatty acids decreased evoked transmitter release. Unsaturated fatty acids decreased the amplitude of voltage-dependent and calciumactivated potassium currents in a nerve ending, whereas saturated acids were ineffective. Rhythmic stimulation applied in the presence of arachidonic and oleic acids induced more pronounced facilitation of the transmitter release, whereas the effects of myristic and arachidic acids did not differ from control values. Deoxycholate was not able to reproduce the effects of fatty acids on the transmitter release and potassium current in a nerve ending. It was concluded that fatty acids can modulate transmitter release and synaptic excitatory transmission; these effects can have a significant influence on molecular mechanisms of exocytosis of synaptic vesicles and electrogenesis in a motor nerve ending. The latter effect is characteristic of unsaturated (arachidonic and oleic) acids, whereas the former effect does not depend on the degree of unsaturation at all.

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.

Mental illnesses are frequent co-morbid conditions in chronic systemic diseases. High incidences of depression, anxiety and cognitive impairment complicate cardiovascular and metabolic disorders such as hypertension and diabetes mellitus. Lifestyle changes including regular exercise have been advocated to reduce blood pressure and improve glycaemic control. The purpose of this project was to evaluate the effect of physical training on the most prevalent corollary psychiatric problems in patients with chronic organic ailments. This longitudinal study assessed the mental health of hypertensive (age: 57 ± 8 years) and/or diabetic (age: 53 ± 8 years) patients using mini-mental state examination, Beck’s depression inventory, Beck’s anxiety inventory and self-reporting questionnaire-20 before and after a 3-month supervised resistance and aerobic exercise programme comprising structured physical activity three times a week. Clinically relevant improvement was observed in the Beck’s depression inventory and Beck’s anxiety inventory scores following the 12-week training (61%, p = 0.001, and 53%, p = 0.02, respectively). Even though statistically not significant ( p = 0.398), the cognitive performance of this relatively young patient population also benefited from the programme. These results demonstrate positive effects of active lifestyle on non-psychotic mental disorders in patients with chronic systemic diseases, recommending exercise as an alternative treatment option.

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.

During normal cell metabolism the production of intracellular ATP is associated with the generation of reactive oxygen species (ROS), which appear to be important signalling molecules. Both ATP and ROS can be released extracellularly by skeletal muscle during intense activity. Using voltage clamp recording combined with imaging and biochemical assay of ROS, we tested the hypothesis that at the neuromuscular junction extracellular ATP generates ROS to inhibit transmitter release from motor nerve endings. We found that ATP produced the presynaptic inhibitory action on multiquantal end-plate currents. The inhibitory action of ATP (but not that of adenosine) was significantly reduced by several antioxidants or extracellular catalase, which breaks down H2O2. Consistent with these data, the depressant effect of ATP was dramatically potentiated by the pro-oxidant Fe2+. Exogenous H2O2 reproduced the depressant effects of ATP and showed similar sensitivity to anti- and pro-oxidants. While NO also inhibited synaptic transmission, inhibitors of the NO-producing cascade did not prevent the depressant action of ATP. The ferrous oxidation in xylenol orange assay showed the increase of ROS production by ATP and 2-MeSADP but not by adenosine. Suramin, a non-selective antagonist of P2 receptors, and pertussis toxin prevented the action of ATP on ROS production. Likewise, imaging with the ROS-sensitive dye carboxy-2′,7′-dichlorodihydrofluorescein revealed increased production of ROS in the muscle treated with ATP or ADP while UTP or adenosine had no effect. Thus, generation of ROS contributed to the ATP-mediated negative feedback mechanism controlling quantal secretion of ACh from the motor nerve endings.

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.

Using the technique of extracellular recording from the region of the neuromuscular junction in the cutaneous-sternal muscle in the frog under conditions of a reduced concentration of Ca2+ in the surrounding milieu, we demonstrated that long-lasting (10 min) rhythmic stimulation of the motor nerve with a frequency of 10 sec− 1 leads to a gradual increase in the evoked transmitter release. These changes are accompanied by a decrease in the amplitude of electrical responses of the nerve terminal (NT) and by a retardation of its second phase, as well as by a diminution of the third phase. Under conditions of long-lasting (5 min) stimulation with a frequency of 50 sec−1, we observed a two-phase change in the intensity of transmitter release: on the 2nd min, the initial rise was replaced by inhibition. Modifications of the response of the NT with different stimulation frequencies were qualitatively similar, but with a frequency of 10 sec−1 they were clearly expressed. Mathematical simulation of ion currents in the NT demonstrated that voltage-dependent potassium and sodium channels are inactivated in the course of long-lasting high-frequency excitation; the shape of the action potential is modified with changes in the rate of such inactivation. This leads to either an increase or a decrease of the inward calcium current. We conclude that the change in electrogenesis in the NT with long-lasting high-frequency activation of neuromuscular junctions exerts a significant influence on the dynamics of transmitter release.

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.

Experiments on neuromuscular synapses from frog skin/chest muscle preparations in conditions of extracellular recording addressed changes in the spontaneous and evoked transmitter secretion after long-term (1.5-6 h) maintenance of preparations in calcium-free solution containing EGTA. Use of three microelectrodes for recording of single-quantum postsynaptic signals showed that calcium-free solution altered the characteristic topography of transmitter secretion in nerve terminals, with widening and fusion of groups of transmitter release. These changes persisted after preparations were returned to the initial solution. These data suggest that calcium-free solutions lead to disorganization of the active zones of nerve endings, At initially low extracellular Ca ion concentrations (0.15-0.4 mM), disorganization of active zones induced by prolonged maintenance of preparations in calcium-free solutions led to decreases in the mean amplitude of endplate currents (EPC) because of decreases in their quantum composition, increases in the time course of transmitter secretion, and decreases in the frequency of miniature endplate currents. The relationship between quantum composition of EPC and the extracellular Ca ion concentration showed a sharp displacement towards higher concentrations, without significant changes in the slope of the relationship. At high initial Ca concentrations (1.8 mM), long-term exposure to calcium-free solutions led to a less marked decrease in EPC amplitude. It is suggested that the extra- and intracellular Ca ion concentrations support the maintenance of the characteristic morphofunctional organization of the apparatus responsible for transmitter secretion in frog nerve endings. Disorganization of the active zones leads to disruption of elements involved in transmitter secretion and decreases in the efficiency of secretion.

In experiments on neuromuscular junctions in the frog m. thoraco-cutaneous, we studied changes in the transmitter release and shape of the nerve ending (NE) response related to high-frequency (10 or 50 sec-1) rhythmic stimulation of the motor nerve; an extracellular recording technique was used. At a low extracellular Ca2+ concentration, rhythmic stimulation resulted in a gradual increase in the quantum content of end-plate currents, i.e., in facilitation. Simultaneously, the third (positive) phase of the NE response became smaller, the amplitude of the second (negative) phase of this response also decreased, while the duration of this phase increased. Modifications of the NE response upon stimulation with a 10 sec-1 frequency were more clearly expressed than those at 50 sec-1 stimulation. In Ca2+-free solutions, rhythmic stimulation was accompanied by analogous modifications of the shape of NE responses, and the dynamics of these changes were the same at both the above-mentioned stimulation frequencies. When 0.5-1.0 mM tetraethylammonium was applied, 10 sec-1 stimulation was accompanied by no facilitation of transmitter release; at 50 sec-1 stimulation, this phenomenon was observed but was weaker than in the control, and the shape of NE responses underwent only mild changes. Simulation of electrogenesis in the studied structure showed that modifications of the NE response shape related to rhythmic 10 sec-1 stimulation can develop in the case of a gradual decrease in the voltage-dependent potassium membrane conductivity, which results in prolongation of the de- and repolarization phases of action potentials and increases in the amplitude and duration of the inward calcium current. At higher stimulation frequencies (50 sec-1), this mechanism is accompanied by a gradual increase in the Ca2+-dependent potassium conductivity, due to an increase in the intracellular Ca2+ concentration. These data allow us to conclude that the intensity of facilitation of transmitter release from the frog motor NE is related not only to accumulation of “residual” calcium, but also to changes of presynaptic calcium current due to modification of the kinetics of functioning of the potassium channels.

Extracellular recording experiments using neuromuscular skin/chest muscle preparations from lake frogs were performed at low extracellular Ca2+ ion concentrations to study the effect of L-arginine (the substrate for nitric oxide synthesis) and N(G)-nitro-L-arginine methyl ester (a blocker of NO synthase) on the parameters of evoked transmitter secretion and ion currents in motor nerve endings. L-arginine at a concentration of 100 microM decreased the amplitude of endplate currents as well as their quantum composition, and also increased the amplitude of the third phase of the evoked nerve ending response, which reflects the kinetics of potassium influx currents. N(G)-nitro-L-arginine methyl ester at a condition of 100 microM led to increases in the amplitude and quantum composition of endplate currents and decreased the amplitude of the third phase of the evoked nerve ending response. It is suggested that endogenous nitric oxide is produced in frog neuromuscular synapses, which in normal conditions suppresses transmitter secretion and modulates the function of potassium channels in the nerve ending.

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.

Extracellular recording was used to study the effect of sodium nitroprusside, a donor of NO, on parameters of action potential and ionic currents in single sciatic nerve fibers and unmyelinated nerve terminals in the sternal muscle in frogs. Sodium nitroprusside significantly decreased the duration of action potential in Ranvier node and the amplitude of afterdepolarization. In motor nerve terminals bathed in low Ca2+ saline, sodium nitroprusside increased phase III amplitude of the nerve terminal response corresponding to outward potas-sium currents. Blockade of voltage-dependent potassium channels with 4-aminopyridine abolished the effects of NO. These data indicate that exogenous NO reduced the duration of action potential and afterdepolarization through enhancement of voltage-dependent potassium currents.

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.