Changes in the Neurochemical Composition of Motor Neurons of the Spinal Cord in Mice under Conditions of Space Flight.

Expression of choline acetyltransferase, 200-kDa neurofilament protein, 28-kDa calbindin, neuronal NO synthase, caspase 3, and Ki-67 in the motor neurons of spinal cord segments T3-T5 in male C57Bl/6 mice after 30-day space flight in the Bion-M1 biosatellite was studied by immunohistochemical methods. Under conditions space flight, the size of motoneurons increased, the number of neurons containing choline acetyltransferase and neurofilaments, decreased, and the number of calbindin-positive neurons increased; motoneurons, expressing neuronal NO synthase and caspase 3 appeared, while Ki-67 was not detected. Fragmentation of neurons with the formation structures similar to apoptotic (residual) bodies was observed in individual caspase 3-positive motoneurons.

[CHANGES OF SPINAL MOTOR NEURONS IN MICE AFTER А SPACE FLIGHT].

The expression of choline acetyltransferase (ChAT), neurofilament (NF) protein 200 kDa, calbindin (CAB) 28 kDa, neuronal NO-synthase (nNOS), caspase 3, Ki-67 was studied in motor neurons from TIII­TV segments of the spinal cord in C57/ BL6 male mice by immunohistochemical methods 12 h after a 30 days-long space flight on the Bion-M1 biosatellite. Mice living under standard vivarium conditions served as a control. The motoneurons of experimental animals demonstrated the reactive changes that were manifested by the increase of their size, decrease in the number of subpopulations expressing ChAT and NF, increase of subpopulations containing CAB, appearance of motor neurons expressing nNOS, caspase-3, and the absence of Ki-67. The lack of proliferating gliocytes, the preservation of nucleolar component indicate the absence of necrobiotic changes characteristic of necrosis. The cytoplasmic vacuolization was observed in thionine-stained CAB-immunoreactive neurons. The disintegration of motor neurons into fragments resembling the apoptotic bodies was observed in single cells that contained caspase-3.

Effect of microgravity on glial cell line-derived neurotrophic factor and cerebral dopamine neurotrophic factor gene expression in the mouse brain.

UNLABELLED: Mice were exposed to 1 month of space flight on the Russian biosatellite BION-M1 to determine its effect on the expression of genes involved in the maintenance of the mouse brain dopamine system. The current article focuses on the genes encoding glial cell line-derived neurotrophic factor (GDNF) and cerebral dopamine neurotrophic factor (CDNF). Space flight reduced expression of the GDNF gene in the striatum and hypothalamus but increased it in the frontal cortex and raphe nuclei area. At the same time, actual space flight reduced expression of the gene encoding CDNF in the substantia nigra but increased it in the raphe nuclei area. To separate the effects of space flight from environmental stress contribution, we analyzed expression of the investigated genes in mice housed for 1 month on Earth in the same shuttle cabins that were used for space flight and in mice of the vivarium control group. Shuttle cabin housing failed to alter the expression of the GDNF and CDNF genes in the brain structures investigated. Thus, actual long-term space flight produced dysregulation in genetic control of GDNF and CDNF genes. These changes may be related to downregulation of the dopamine system after space flight, which we have shown earlier. © 2015 Wiley Periodicals, Inc. SIGNIFICANCE: Our results provide the first evidence of microgravity effects on expression of the GDNF and CDNF neurotrophic factor genes. A considerable decrease in mRNA level of GDNF and CDNF in the nigrostriatal dopamine system was found. Because both GDNF and CDNF play a significant role in maintenance and survival of brain dopaminergic neurons, we can assume that this dysregulation in genetic control of GDNF and CDNF genes in substantia nigra could be among the reasons for the deleterious effects of space flight on the dopamine system.

Effect of actual long-term spaceflight on BDNF, TrkB, p75, BAX and BCL-XL genes expression in mouse brain regions.

Mice of C57BL/6J strain were exposed to 1-month spaceflight on Russian biosatellite Bion-M1 to determine the effect of long-term actual spaceflight on the expression of genes involved in the processes of neurogenesis and apoptosis. Specifically, we focused on the genes encoding proapoptotic factor BAX, antiapoptotic factor BCL-XL, brain-derived neurotrophic factor (BDNF) and BDNF receptors TrkB and p75. Spaceflight reduced the expression of the antiapoptotic BCL-XL gene in the striatum and hypothalamus, but increased it in the hippocampus. To estimate environmental stress contribution into spaceflight effects we analyzed spaceflight-responsive genes in mice housed for 1 month on Earth in the same shuttle cabins that were used for spaceflight, and in mice of the laboratory control group. It was shown that 1-month shuttle cabin housing decreased BCL-XL gene expression in the striatum but failed to alter BCL-XL mRNA levels in the hippocampus or hypothalamus. Spaceflight failed to alter the expression of the proapoptotic BAX gene in all investigated brain structures, although the insignificant increase of the BAX mRNA level in the hippocampus of spaceflight mice was found. At the same time, shuttle cabin housing produced insignificant decrease in BAX gene expression in the hippocampus. In contrast to the BCL-XL gene, genes encoding BAX, BDNF as well as TrkB and p75 receptors did not respond to 30-day spaceflight. Thus, long-term spaceflight (1) did not affect the expression of genes encoding BDNF as well as TrkB and p75 receptors, (2) produced dysregulation in genetic control of the neuronal apoptosis, (3) implicated BCL-XL as the risk factor for spaceflight-induced behavioral abnormalities.

Risk neurogenes for long-term spaceflight: dopamine and serotonin brain system.

Mice were exposed to 1 month of spaceflight on Russian biosatellite BION-M1 to determine its effect on the expression of key genes in the brain dopamine (DA) and serotonin (5-HT) systems. Spaceflight decreased the expression of crucial genes involved in DA synthesis and degradation, as well as the D1 receptor. However, spaceflight failed to alter the expression of tryptophan hydroxylase-2, 5-HT transporter, 5-HT1A, and 5-HT3 receptor genes, though it reduced 5-HT2A receptor gene expression in the hypothalamus. We revealed risk DA and 5-HT neurogenes for long-term spaceflight for the first time, as well as microgravity-responsive genes (tyrosine hydroxylase, catechol-O-methyltransferase, and D1 receptor in the nigrostriatal system; D1 and 5-HT2A receptors in the hypothalamus; and monoamine oxidase A (MAO A) in the frontal cortex). Decreased genetic control of the DA system may contribute to the spaceflight-induced locomotor impairment and dyskinesia described for both humans and rats.

[Change in formation of the somatosensory cortex ultrastructure in rats due to hypergravity in pre- and postnatal ontogenesis].

Chronic action of gravity force of 2 G on pre- and postnatal rats up to day 75 of postnatal development gave rise to ultrastructural deviations in the somatosensory cortex (SSC) suggesting higher afferentation to SSC as compared with their counterparts at 1 g, more intensive synaptic transmission, afferent input by dendrites and neurons functioning with more frequent interneuronic contacts, glia activation and increased blood supply that altogether pointed to a permanent impact of elevated extero- and proprioceptive input throughout the time of SSC formation.

[Electron microscope analysis of cardiomyocytes in the rat left ventricle under simulation of weightlessness effects and artificial gravitation].

Electron microscopic study of left ventricle cardiomyocytes and quantitative analysis of their mitochondriom was performed in rats exposed to tail-suspension, as a model of weightlessness effects, to artificial gravity produced by intermittent 2G centrifugation and a combination of these effects. It was found that the cardiomyocytes ultrastructure changed slightly after tail-suspension and after intermittent 2G influence, as well as under a combination of these effects. However, the number of intermitochondrial junctions increased significantly in the interfibrillar zone of cardiomyocytes under a combination of tail-suspension and intermittent 2G influence, which agrees with the cell hypertrophy described earlier.

[Ultrastructure of dendritic spines in the somatosensory cortex of rat's brain following 5- and 33-day hypergravity].

Electron microscopy examination of layers III-IV in the somatosensory cortex of rat's brain following 5- and 33-day and repeated 5-day exposure to 2 G revealed ultrastructural changes in dendritic spines, synapses, axon terminals and nervous cell body suggesting their functional activation and increase of the afferent input from hindlimb mechanoreceptors Hypertrophy of the acicular apparatus (AA) due to hypergravity, growth of AA destruction as a result of 33-day exposure and its ample evidence after repeated 5-day exposure lead to the conclusion that AA ultrastructure can be used for assessing afferent input into the brain cortex of animals exposed in a changed field of gravity.

[Purkinje's cells in the vestibular and proprioceptive segments of rat's cerebellum following 14-day space flight].

Quantitative cytochemical and morphometric methods were used to investigate cytochrome oxidase activity and sizes of bodies and nuclei of Purkinje's cells in the medical nodulus and upper central lobule of the vermis obtained from rats sacrificed in 5-6 hours of landing after the 14-day SLS-2 mission of NASA space "shuttle" Columbia (STS-58). The reduced cytochrome oxidase activity was explained by suppression of the functional activity of Purkinje's cells in microgravity. Results of the investigations suggest weakening of the regulatory effect of the vermis Purkinje's cells on giant neurons of the dorsocaudal segment of Deiters nucleus. They also strengthen the earlier hypothesis that space flight decays the inhibitory effect of nodulus Purkinje's cells on the medial vestibular nucleus for the reason of change in the "velocity storage" in mammals during and after flight.

[Effect of intermittent hypergravity on cerebellum Purkinje's cells in suspended rats].

The quantitative cytochemical and morphometrical techniques applied in the experiment with 24-d tail-suspension of rats demonstrated a decrease of cytochrome oxidase activity and downsizing of Purkinje's cell bodies in the proprioceptive cerebellum (cerebellar vermis upper central lobule), as well as a decrease of cytochrome oxidase activity in Purkinje's cells in the vestibular cerebellum (nodulus). The observed suppression of the Purkinje's cells functional activity indicates reductions in the proprioceptive and vestibular afferent inputs to these cells during simulation of the microgravity effects in rats. Intermittent exposure to hypergravity (2 G, one hour a day) prevented the decreases in cytochome oxidase activity in Purkinje's cells of both the proprioceptive and vestibular cerebella of tail-suspended rats suggesting a nearly same level of functioning as in the vivarium controls.

[Reaction of the neuronal population of rat's L5 spinal ganglia to primary and repeated modeling of microgravity effects].

Cytochrome oxidase activity as well as neurons' body and nuclei sizes were measured in the spinal L5 ganglia receiving the hindlimb afferent input during primary and repeated 14-d tail-suspension of rats. The measurements were performed using the quantitative cytochemical and morphometric techniques. The animals were suspended 30 days and returned to vivarium. Thirty day later, they were suspended again, together with a group of intact rats, this time for 14 days. Primary 14-day suspension reduced significantly the cytochrome oxidase activity in small, middle and large neurons (cross-section area of bodies less than 800, 800-2000 microm2 and more than 2000 microm2, respectively) and yielded a trend-like diminution of the nerve cells bodies and nuclei. The postponed repeated suspension, unlike the primary 14-d one, resulted in a substantial downsizing of bodies and nuclei of middle neurons suggesting a more dramatic hypofunction and a more dramatic decrease of afferent input. However, the cytochrome oxidase activity was not suppressed considerably in small and middle neurons which might be explained by initiation of the afferent input recovery.

[Influences of primary and repeated modeling of microgravity effects on spinal motoneurons l5 in rats: cytological analysis].

The cytochrome oxidase activity, sizes of bodies, nuclei, and nucleoli of alpha-motoneurons from the L5 anterior horns were investigated in rats once or repeatedly suspended by tails to model the effects of microgravity. Rats were suspended 30 days, then held in vivarium 30 days w/o any behavior restriction and suspended once again 14 days simultaneously with rats that had not been suspended before. The first-time 14-d suspension reduced the cytochrome oxidase activity as well as sizes of bodies and nuclei in alpha-motoneurons which pointed to hypofunctioning of these nervous cells. Repeated (14-d) suspension after the 30-day maintenance in vivarium decreased the alpha-motoneurons parameters much less significantly implying mild hypofunction and a faster adaptation of L5 alpha-motoneurons to microgravity modeled again after a long period of recess.

[Electron microscopy analysis of the structural elements of the vestibular input to nodulus Purkinje's cells in rats exposed to a 9-day space flight].

Electron microscopy was used to study structural elements of the vestibular afferent input to the cerebellar nodulus Purkinje's cells--terminals of mossy fibers and granular cells in the granular layer and parallel fibers and Purkinje's cell dendrites in the molecular layer in rats decapitated in 2-3 hours and 9 days after the 9-day space flight aboard NASA shuttle Columbia (STS 40, SLS-1 mission). Analysis of the revealed ultrastructural changes on the base of morphofunctional correlations leads to the following conclusions: 1) space flight induced a prolonged reduction in vestibular input to most of the mossy fiber terminals and nodulus Purkinje's cells; 2) within the initial hours of recovery the vestibular input to a part of mossy fiber terminals and granular cells was increasing due to elevation of the sensitivity of vestibular receptors in microgravity; 3) regain of the vestibular input to Purkinje's cells after space flight is hampered by structural, as a result of microgravity effects, and also functional, developing shortly after space flight, impediments, and 4) in 9 d after landing the vestibular input to Purkinje's cells was almost normal. The observed reduction in the vestibular input to the nodulus Purkinje's cells during and after the spaceflight microgravity is presumably the key to the mechanism altering the velocity storage in mammals in microgravity and on return from space flight.

[Impact of 3-month simulation of the microgravity effects on the neuromuscular junction structure in rat's m. soleus].

The neuromuscular junctions were investigated in m. soleus of rats subjected to a 3-month tail suspension simulating the microgravity effects. Electron microscopy analysis revealed some ultrastructural signs of atrophy, degeneration and adipose dystrophy of muscle fibers. The aggregate of these findings points to progressive atrophy in m. soleus, while ultrastructural changes in the neuromuscular synapses testify a reduced functional activity of the synapses and partial denervation of the muscle fibers which, probably, underlay the atrophic process in the muscle. Increases in the number of axon terminals found in some neuromuscular synapses as well as of synaptic vesicles in individual axon terminals are likely to reflect formation of a particularly active pool of spinal motoneurons at L5, possibly associated with the growth in the number of fast fibers resulted from transformation.

[Primary and repeated exposure to hypergravity: ultrastructure of the somatosensory cortex in rats].

Nineteen-day centrifugation of rats at 2 G and ensuing 30-d readaptation to 1 g caused changes in the somatosensory cortex (SSC) ultrastructure pointing to intensification of the afferent input and functional activity of SSC neurons during hypergravity, and recovery of these processes in the course of readaptation. Repeated 5-d centrifugation of the rats at 2 G produced changes in ultrastructure of SSC layers 3 and 4 evidencing a more effective cortex functioning aimed at facilitation of adaptation to hypergravity repeated after the lengthy delay.

Ultrastructure of ependyma in brain third ventricle of the rats exposed to repeated tail-suspension. Scanning electron microscopical study.

By means of scanning electron microscopy the ultrastructure of ependyma was studied in the brain third ventricle of the rats repeatedly exposed to 14-day tail-suspension (TS). Animals were subjected to TS for 30 days, then readapted to horizontal position during 30 days and again, repeatedly subjected to TS for 14 days simultaneously with the rats which were in TS for the first time during 14 days. Repeated TS of rats, inspite of repeated redistribution of body liquid mediums in cranial direction, results in considerably less expressed destructive changes in ultrastructure of ependymocyte cilia, then after primary 14- and 30-day TS, showing much greater cerebrospinal fluid (CSF) outflow from brain ventricles into sagittal venous sinus at postponed for a long time, repeated simulation of weightlessness effects in comparison with CSF outflow at primery one.

[Electron microscopy and hystology of the third ventricle ependyma of rat's brain after repeated simulation of the effects of microgravity].

Scanning electron and light microscopy were applied to study the third ventricle ependyma in rat's brain after 30-d tail-suspension, 30-d readaptation to the horizontal position, and repeated 14-d suspension in parallel with another group of rats exposed to a single 14-d suspension. Despite the repeated blood redistribution toward the head, the second tail-suspension produced significantly less grave destruction of the ependymal cell structure and cilia ultrastructure than the single 14-d or previous 30-d one, which suggests that simulation of microgravity repeated after substantial delay imparts much more persistence to the cerebrospinal fluid outflow from the brain ventricles into the saggital sinus.

[Role of the endocrine glands in divergence of plastic processes and energy metabolism in rats after extended exposure to hypergravity: cytologic investigation].

Cytologic and morphometric investigations of the hypothesis, thyroid, thyroid and pancreas of rats after 19-d centrifugation at 2G revealed suppression of somatotropic hormone production by hypophyseal somatotrophs and insulin by pancreatic beta-cells, and intensification of thyrotrophic hormone production by hypophyseal thyreotrophs, thyroxin by thyreoid parenchyma and glucagon by pancreatic alpha-cells. These change in hormone production are viewed as an endocrine mechanism of the divergence of plastic processes and energy metabolism in animals subjected to chronic exposure to hypergravity.

[Ultrastructure of spindles in m. soleus of rats after repeated simulation of the weightlessness effects].

Postponed for a long time repeated simulation of the weightlessness effects in rats by tail-suspension increases in the amount of intrafusal muscle fibers in m. soleus spindles, reflecting presumably elevation of the sensitivity of mechanoreceptors.