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© 2016, Springer Science+Business Media New York.Previously, it was shown that both adrenaline and noradrenaline potentiate neuromuscular transmission, but which one of the receptors mediates the facilitating effect of catecholamines is still unclear. In this study, we have investigated the presence of different adrenoceptors at isolated preparations of frog cutaneous pectoris muscle by using methods of immunohistochemistry. The immunopositive reaction was observed while using polyclonal antibodies to α1- (α1B and α1D), α2- (α2A, α2B, and α2C), and β-adrenoceptors (β1, β2 and β3). In all the cases, the immunohistochemical staining of the mentioned proteins was localized in the area of the synaptic contact. Thus, at the neuromuscular junction, a wide range of α1-, α2- and β-adrenoceptors was found. It expands our understanding of the endogenous mechanisms of cholinergic neurotransmission regulation and elucidates the aspects of the mechanisms of action of adrenergic agonists, which are still intensively studied or already used for treatment of neuromuscular disorders with a primary neuro- or myopathology, and neuromuscular diseases characterized by a neuromuscular junction pathology.

© 2016, Pleiades Publishing, Ltd.Using fluorescent microscopy, we found decreased expression of the β1 subunit of Na+/K+-ATPase and subunits of Ca2+-ATPase, increased expression of the α1S subunit of the L-type Ca2+-channel, and no changes in the expression of the α2 subunit of Na+/K+-ATPase in rat postural muscle under the conditions of modeled hypogravity. In the phasic muscle, we observed decreased expression of the β1 subunit, which was similar to that found in the postural muscle, whereas the other studied parameters remained without alterations. However, a decrease in the fluorescence intensity of the β1 subunit was insignificant due to a high variability of data. Thus, hypogravity negatively influenced primarily those skeletal muscles that are responsible for static load.

© 2016, Pleiades Publishing, Ltd.Using fluorescent microscopy, we found decreased expression of the β1 subunit of Na+/K+-ATPase and subunits of Ca2+-ATPase, increased expression of the α1S subunit of the L-type Ca2+-channel, and no changes in the expression of the α2 subunit of Na+/K+-ATPase in rat postural muscle under the conditions of modeled hypogravity. In the phasic muscle, we observed decreased expression of the β1 subunit, which was similar to that found in the postural muscle, whereas the other studied parameters remained without alterations. However, a decrease in the fluorescence intensity of the β1 subunit was insignificant due to a high variability of data. Thus, hypogravity negatively influenced primarily those skeletal muscles that are responsible for static load.

© 2016, Springer Science+Business Media New York.Previously, it was shown that both adrenaline and noradrenaline potentiate neuromuscular transmission, but which one of the receptors mediates the facilitating effect of catecholamines is still unclear. In this study, we have investigated the presence of different adrenoceptors at isolated preparations of frog cutaneous pectoris muscle by using methods of immunohistochemistry. The immunopositive reaction was observed while using polyclonal antibodies to α1- (α1B and α1D), α2- (α2A, α2B, and α2C), and β-adrenoceptors (β1, β2 and β3). In all the cases, the immunohistochemical staining of the mentioned proteins was localized in the area of the synaptic contact. Thus, at the neuromuscular junction, a wide range of α1-, α2- and β-adrenoceptors was found. It expands our understanding of the endogenous mechanisms of cholinergic neurotransmission regulation and elucidates the aspects of the mechanisms of action of adrenergic agonists, which are still intensively studied or already used for treatment of neuromuscular disorders with a primary neuro- or myopathology, and neuromuscular diseases characterized by a neuromuscular junction pathology.

© 2016, Springer Science+Business Media New York.Hibernation is unique mammals’ phenotype demonstrated surviving in seasonal adverse environment conditions. During hibernation, all of systems of organs undergo alterations in their metabolic activity and physiology similar to different physiological conditions associated with human diseases and injuries, which represent a proper model for studying new approaches in clinical treatment. In this study, we have analyzed expression of semaphorins in small hibernator edible dormouse (Glis glis). Semaphorins are factors which involved in a key process of axon guidance and cell–cell communication and can act as tumor suppressor. We found that gene coding Semaphorin-3D (SEMA3D), previously reported to be specific for brain and heart of mammals, represents one of the most upregulated transcripts in the muscles of the hibernating dormice. Furthermore, another member of the same family, Semaphorin-5B (SEMA5B), was strongly induced in the spinal cord of hibernating animals. These observations make semaphorin group, recently attracting more attention due to anti-tumor activity, one of the target for in-depth analysis in relation to the molecular mechanisms of hibernation.

© 2016, Human Stem Cell Institute. All rights reserved.Earlier, by an immunohistochemical method we define that after 30-day space flight in motoneurons of mice lumbar spinal cord immunoexpression of the proteins responsible for synaptic transfer of a nervous impulse and proteins of heat shock proteins decrease. In this research for an assessment of animals recovery process dynamics after space flight we studied an immunoexpression of the proteins participating in synaptic transfer of a nervous impulse (synaptophisyne, and PSD95), neurotrophic factors (a vascular endothelial factor of growth - VEGF and its receptor - Flt-1) and heat shock proteins (Hsp25 and Hsp70) in motoneurons of lumbar spinal cord of a mice after 30-day space flight on the BION-M1 biosatellite and the subsequent week readaptation to conditions of Earth gravitation. In this research by immunohistochemical method determine that after a week of animals staying in the Earth gravitation conditions the immunoexpression of synaptophisyne continued to decrease while the expression of PSD95, Hsp25, Hsp70 and VEGF increased in relation to the animals removed from experiment right after 30-day flight. The obtained data confirm functional plasticity of spinal cord motoneurons in the conditions of gravitation force changing. The fact, which is especially interesting, that "switches on" of neurons protective mechanisms (strengthening of heat shock proteins and neurotrophic factor expression) happens not in response to hypogravitation influence, but only a week after return of animals to conditions of Earth gravitation.

Experiments were conducted on male mice of the C57BL/6J for full genome study of gene expression. The animals were divided into two groups.The 'flight group' mice that had spent 30 days in space and the 'control group' mice that stayed on Earth. The spinal cord of the 'flight' group mice as extracted 14h following the biosatellite's landing. The spinal cord of the control group mice was extracted at the same time.Then the lumbar spinal cord was frozen in liquid nitrogen and kept until the beginning of the study at -80°C. Quality control of the feature was performed using the settings recommended by Agilent Technologies. Signal background was subtracted and the signal intensity of each gene was globally normalized using locally weighted scatterplot smoothing. The analysis suggests that various functional groups of genes in different types of cells in spinal cord may be involved in the development of HLS and some changes in gene expression certainly happened in motor neurons innervating affected skeletal muscles.

It was previously shown that different cell types in vivo and in vitro may die via apoptosis under weightlessness conditions in space as well as in simulated hypogravity on the Earth. We assessed survivability of spinal motoneurons of rats after 35-day antiorthostatic hind limb suspension. Following weight bearing, unloading the total protein content in lumbar spinal cord is dropped by 21%. The electrophysiological studies of m. gastrocnemius revealed an elevated motoneurons' reflex excitability and conduction disturbances in the sciatic nerve axons. The number of myelinated fibers in the ventral root of experimental animals was insignificantly increased by 35-day of antiorthostatic hind limb suspension, although the retrograde axonal transport was significantly decreased during the first week of simulated hypogravity. The results of the immunohistochemical assay with antibodies against proapoptotic protein caspase 9 and cytotoxicity marker neuron specific nitric oxide synthase (nNOS) and the TUNEL staining did not reveal any signs of apoptosis in motoneurons of suspended and control animals. To examine the possible adaptation mechanisms activated in motoneurons in response to simulated hypogravity we investigated immunoexpression of Hsp25 and Hsp70 in lumbar spinal cord of the rats after 35-day antiorthostatic hind limb suspension. Comparative analysis of the immunohistochemical reaction with anti-Hsp25 antibodies revealed differential staining of motoneurons in intact and experimental animals. The density of immunoprecipitate with anti-Hsp25 antibodies was substantially higher in motoneurons of the 35-day suspended than control rats and the more intensive precipitate in this reaction was observed in motoneuron neuritis. Quantitative analysis of Hsp25 expression demonstrated an increase in the Hsp25 level by 95% in experimental rats compared to the control. The immunoexpression of Hsp70 found no qualitative and quantitative differences in control and experimental l

Spontaneous quantal neurotransmitter release and its modulation was studied on neuromuscular preparations of rat soleus from intact animals and from animals in which colchicine had been applied to the sciatic nerve to block the axonal transport. After six days of colchicine application, neither the spontaneous quantal secretion nor its reaction to potassium-induced membrane depolarization or to activation of the presynaptic acetylcholine receptors with carbachol were disturbed in any way. Keeping in mind the relatively short half-life of proteins that take part in exocytosis and its regulation, it may be concluded that the functioning of the terminal neurosecretory apparatus does not depend on the state of axonal transport. These data are consistent with the earlier hypothesis that some proteins performing their functions in nerve terminals are synthesized directly at the site of their operation, rather than in the perikaryon as traditionally assumed. © 2010 Pleiades Publishing, Ltd.

© 2016, Springer Science+Business Media New York.Hibernation is unique mammals’ phenotype demonstrated surviving in seasonal adverse environment conditions. During hibernation, all of systems of organs undergo alterations in their metabolic activity and physiology similar to different physiological conditions associated with human diseases and injuries, which represent a proper model for studying new approaches in clinical treatment. In this study, we have analyzed expression of semaphorins in small hibernator edible dormouse (Glis glis). Semaphorins are factors which involved in a key process of axon guidance and cell–cell communication and can act as tumor suppressor. We found that gene coding Semaphorin-3D (SEMA3D), previously reported to be specific for brain and heart of mammals, represents one of the most upregulated transcripts in the muscles of the hibernating dormice. Furthermore, another member of the same family, Semaphorin-5B (SEMA5B), was strongly induced in the spinal cord of hibernating animals. These observations make semaphorin group, recently attracting more attention due to anti-tumor activity, one of the target for in-depth analysis in relation to the molecular mechanisms of hibernation.

© 2016, Human Stem Cell Institute. All rights reserved.Earlier, by an immunohistochemical method we define that after 30-day space flight in motoneurons of mice lumbar spinal cord immunoexpression of the proteins responsible for synaptic transfer of a nervous impulse and proteins of heat shock proteins decrease. In this research for an assessment of animals recovery process dynamics after space flight we studied an immunoexpression of the proteins participating in synaptic transfer of a nervous impulse (synaptophisyne, and PSD95), neurotrophic factors (a vascular endothelial factor of growth - VEGF and its receptor - Flt-1) and heat shock proteins (Hsp25 and Hsp70) in motoneurons of lumbar spinal cord of a mice after 30-day space flight on the BION-M1 biosatellite and the subsequent week readaptation to conditions of Earth gravitation. In this research by immunohistochemical method determine that after a week of animals staying in the Earth gravitation conditions the immunoexpression of synaptophisyne continued to decrease while the expression of PSD95, Hsp25, Hsp70 and VEGF increased in relation to the animals removed from experiment right after 30-day flight. The obtained data confirm functional plasticity of spinal cord motoneurons in the conditions of gravitation force changing. The fact, which is especially interesting, that "switches on" of neurons protective mechanisms (strengthening of heat shock proteins and neurotrophic factor expression) happens not in response to hypogravitation influence, but only a week after return of animals to conditions of Earth gravitation.

Experiments were conducted on male mice of the C57BL/6J for full genome study of gene expression. The animals were divided into two groups.The 'flight group' mice that had spent 30 days in space and the 'control group' mice that stayed on Earth. The spinal cord of the 'flight' group mice as extracted 14h following the biosatellite's landing. The spinal cord of the control group mice was extracted at the same time.Then the lumbar spinal cord was frozen in liquid nitrogen and kept until the beginning of the study at -80°C. Quality control of the feature was performed using the settings recommended by Agilent Technologies. Signal background was subtracted and the signal intensity of each gene was globally normalized using locally weighted scatterplot smoothing. The analysis suggests that various functional groups of genes in different types of cells in spinal cord may be involved in the development of HLS and some changes in gene expression certainly happened in motor neurons innervating affected skeletal muscles.

Spontaneous quantal neurotransmitter release and its modulation was studied on neuromuscular preparations of rat soleus from intact animals and from animals in which colchicine had been applied to the sciatic nerve to block the axonal transport. After six days of colchicine application, neither the spontaneous quantal secretion nor its reaction to potassium-induced membrane depolarization or to activation of the presynaptic acetylcholine receptors with carbachol were disturbed in any way. Keeping in mind the relatively short half-life of proteins that take part in exocytosis and its regulation, it may be concluded that the functioning of the terminal neurosecretory apparatus does not depend on the state of axonal transport. These data are consistent with the earlier hypothesis that some proteins performing their functions in nerve terminals are synthesized directly at the site of their operation, rather than in the perikaryon as traditionally assumed. © 2010 Pleiades Publishing, Ltd.

It was previously shown that different cell types in vivo and in vitro may die via apoptosis under weightlessness conditions in space as well as in simulated hypogravity on the Earth. We assessed survivability of spinal motoneurons of rats after 35-day antiorthostatic hind limb suspension. Following weight bearing, unloading the total protein content in lumbar spinal cord is dropped by 21%. The electrophysiological studies of m. gastrocnemius revealed an elevated motoneurons' reflex excitability and conduction disturbances in the sciatic nerve axons. The number of myelinated fibers in the ventral root of experimental animals was insignificantly increased by 35-day of antiorthostatic hind limb suspension, although the retrograde axonal transport was significantly decreased during the first week of simulated hypogravity. The results of the immunohistochemical assay with antibodies against proapoptotic protein caspase 9 and cytotoxicity marker neuron specific nitric oxide synthase (nNOS) and the TUNEL staining did not reveal any signs of apoptosis in motoneurons of suspended and control animals. To examine the possible adaptation mechanisms activated in motoneurons in response to simulated hypogravity we investigated immunoexpression of Hsp25 and Hsp70 in lumbar spinal cord of the rats after 35-day antiorthostatic hind limb suspension. Comparative analysis of the immunohistochemical reaction with anti-Hsp25 antibodies revealed differential staining of motoneurons in intact and experimental animals. The density of immunoprecipitate with anti-Hsp25 antibodies was substantially higher in motoneurons of the 35-day suspended than control rats and the more intensive precipitate in this reaction was observed in motoneuron neuritis. Quantitative analysis of Hsp25 expression demonstrated an increase in the Hsp25 level by 95% in experimental rats compared to the control. The immunoexpression of Hsp70 found no qualitative and quantitative differences in control and experimental l